Increasing leaf biomass and nitrogen use efficiency by modulation of NTP2

Abstract

Disclosed herein is a mutant, non-naturally occurring or transgenic plant or plant part having reduced or inhibited expression or activity of NtNTP2-T, or reduced or inhibited expression or activity of NtNTP2-T and NtNTP2-S, the NtNTP2-T and NtNTP2-S comprising or consisting of: (i) a polynucleotide sequence of NtNTP2-S comprising at least one of a nucleotide sequence of NtNTP2-S and a nucleotide sequence of NtNTP2-S comprising at least one of a nucleotide sequence of NtNTP2-S, a nucleotide sequence of NtNTP2-S, a nucleotide sequence of NtNTP2-S, and a nucleotide sequence of NtNTP2-S comprising at least one of a nucleotide sequence of NtNTP2-S, and a nucleotide sequence of NtNTP2-S; a nucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 3; or (ii) an NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 11; or (iii) a polypeptide encoded by the polynucleotide shown in (i) or (ii); or (iv) an NtNTP2-S polypeptide having at least 77% sequence identity to SEQ ID NO: 7; or (v) an NtNTP2-T polypeptide having at least 77% sequence identity to SEQ ID NO: 12; wherein the expression or activity of NtNTP2-T or the expression or activity of NtNTP2-T and NtNTP2-S is reduced or inhibited as compared to a control plant.

Description

Technical Field

[0001] The present invention generally relates to mutant, non-naturally occurring or transgenic plant leaves or plant leaf parts with regulated expression or activity of NTP2. In particular, the present invention relates to mutant, non-naturally occurring or transgenic plants or plant parts (such as leaves) with reduced or suppressed expression or activity of NTP2, which impart an increase in biomass and nitrogen use efficiency (NUE) response without reducing nitrate levels in the plant or plant part. Background Art

[0002] Modern industrial agriculture seeks to continuously increase crop yields and reduce costs. Nitrogen fertilizers enable farmers to increase yields. Depending on the crop, fertilizer use accounts for a large portion of production costs and has an associated risk of negative environmental impacts, as 50%-70% of applied nitrogen is lost from the plant-soil system and causes pollution. Improving nitrogen use efficiency (NUE) is important to reduce crop production costs and reduce damage to the environment.

[0003] Reducing tobacco nitrate levels in tobacco plants reduces the accumulation of tobacco-specific nitrosamines (TSNAs) in dried leaves and cigarette smoke (Lu et al. (2016) Plant Biotechnology Journal, 14: 1500–1510 and WO2016046288). TSNAs are a class of compounds produced primarily during tobacco leaf curing, but are additionally formed by high-temperature synthesis during subsequent leaf processing and storage, and possibly during combustion. Two TSNAs found in dried leaves, N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are classified as Class I carcinogens (the highest level) by the International Agency for Research on Cancer. Due to the large amount of evidence suggesting that these compounds are associated with various tobacco-related cancers, the World Health Organization and other experts in the field have recommended the implementation of mandatory enforcement to ensure that future tobacco products have reduced levels of these toxicants. TSNAs have been shown to be strong carcinogens in a large number of animal studies (Hecht (1998) Chem Res Toxicol, Jun;11(6):559-603; Ma et al. (2018) Carcinogenesis, Feb 9;39(2):232-241; Kovi et al. (2018) Toxicol Pathol., Feb;46(2):184-192; Carlson et al. (2018) Chem Res Toxicol., May 21;31(5):358-370).

[0004] While reducing nitrate levels can have a positive impact on reducing TSNA levels in tobacco, growing tobacco under such conditions can have a negative impact on plant biomass and quality and is therefore not a viable option, particularly for commercial tobacco production. There is a general need in the art to develop plants, such as tobacco plants, that have increased yields at lower fertilizer inputs and thus improved NUE. Summary of the invention

[0005] Disclosed herein are two genes (NtNTP2-S and NtNTP2-T) from tobacco (Nicotiana tabacum), which belong to nitrate transporter family 1 NRT1.4 (PTR2 family of peptide transporters) according to a homology search for Arabidopsis thaliana AtNTP2At2g26690. It was found that reducing or inhibiting (e.g., shutting down) the expression or activity of endogenous NtNTP2-T or both NtNTP2-S and NtNTP2-T in tobacco using various different methods conferred a favorable phenotype, which improved agronomic traits compared to control plants grown under the same conditions. In particular: (i) nitrate levels were not reduced; (ii) biomass (e.g., leaf biomass) increased under both standard and nitrogen starvation conditions; and (iii) NUE response (meaning biomass per unit of nitrogen fertilization applied) increased. As used herein, 'unit' in the context of nitrogen fertilizer units applied means 'kg / hectare', as described herein, these results make NtNTP2-T or both NtNTP2-S and NtNTP2-T an excellent target for developing plants with increased yield at lower fertilizer inputs compared to control plants grown under the same conditions. Surprisingly, NtNTP2-T alone, but not NtNTP2-S alone, can confer this favorable phenotype.

[0006] It is also shown herein that reducing or inhibiting the function of NtNTP2 using, for example, mutation or RNAi surprisingly results in changes in (e.g., increases in) root development by producing more and thinner roots compared to wild-type plants grown under the same conditions. Without being bound by any particular theory, this can increase the ability of the plant to absorb nutrients from the soil.

[0007] The favorable phenotypes of the present invention are also surprising in view of the results reported by Chiu et al. (2004) Plant Cell Physiol., 45(9), 1139-1148, which discloses the results of homozygous AtNTP2 insertion mutants in Arabidopsis thaliana. The homozygous AtNTP2 insertion mutants in Arabidopsis thaliana produced (i) 50%-64% lower nitrate levels; (ii) increased plant leaf width due to cell expansion; and (iii) no mention of improved NUE. Therefore, despite the 77% identity between NtNTP2 and AtNTP2, it is surprising and unexpected that NtNtp2 is an excellent target for developing plants with increased yield and higher NUE response (ratio of biomass per unit of nitrogen applied) compared to wild-type plants grown under the same conditions, which was not demonstrated for AtNTP2 in the mentioned publication.

[0008] This favorable phenotype was further surprising in light of the results reported in US2014 / 0201863, where loss of function of Arabidopsis NRT1.7i under nitrogen starvation conditions resulted in growth retardation and a 30% reduction in rosette diameter by a transgenic approach.

[0009] In one aspect, disclosed herein is a mutant, non-naturally occurring or transgenic plant or plant part having reduced or inhibited expression or activity of NtNTP2-T, or reduced or inhibited expression or activity of NtNTP2-T and NtNTP2-S, wherein the NtNTP2-T and NtNTP2-S comprise or consist of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:11; or (iii) a polypeptide encoded by the polynucleotide shown in (i) or (ii); or (iv) a NtNTP2-S polypeptide having at least 77% sequence identity to SEQ ID NO:7; or (v) a NtNTP2-T polypeptide having at least 77% sequence identity to SEQ ID NO:8. NO: 12 has at least 77% sequence identity; wherein the expression or activity of NtNTP2-T or the expression or activity of NtNTP2-T and NtNTP2-S is reduced or inhibited compared to control plants.

[0010] Suitably, the plant or plant part: (i) does not have reduced nitrate levels compared to control plants grown under the same fertilization conditions; and (ii) has increased biomass compared to control plants grown under the same fertilization conditions; and (iii) has an increased NUE response, indicated as biomass per unit of nitrogen applied, compared to control plants grown under the same conditions. The plant or plant part may also have an increase in root development compared to control plants grown under the same fertilization conditions.

[0011] In one embodiment, the plant or plant part (e.g., leaf): (i) does not have reduced nitrate levels compared to a control plant grown under the same conditions; and (ii) has at least a 5% increase in biomass compared to a control plant grown under the same conditions; and (iii) has at least a 5% increase in NUE response compared to a control plant grown under the same conditions. The plant or plant part may also have an increase in root development compared to a control plant grown under the same fertilization conditions.

[0012] In another embodiment, the plant or plant part (e.g., leaf): (i) does not have reduced nitrate levels compared to a control plant grown under the same fertilization conditions; and (ii) has at least a 5% increase in biomass compared to a control plant grown under the same fertilization conditions; and (iii) has at least a 5% increase in NUE response compared to a control plant grown under the same fertilization conditions. The plant or plant part may also have an increase in root development compared to a control plant grown under the same fertilization conditions.

[0013] Mutant, non-naturally occurring or transgenic plants or plant parts can be modified in various ways with the aim of reducing or inhibiting the expression or activity of NtNTP2-T, or reducing or inhibiting the expression or activity of NtNTP2-T and NtNTP2-S. Suitably, the mutant, non-naturally occurring or transgenic plant or plant part in which the expression or activity of NtNTP2-T or the expression or activity of NtNTP2-T and NtNTP2-S is reduced or inhibited comprises: (i) one or more sequence-specific polynucleotides that can interfere with the transcription of NtNTP2-T or NtNTP2-T and NtNTP2-S; (ii) one or more sequence-specific polypeptides that can interfere with the stability of NtNTP2-T or NtNTP2-T and NtNTP2-S; (iii) one or more sequence-specific polynucleotides that can interfere with the enzymatic activity of NtNTP2-T or NtNTP2-T and NtNTP2-S or NtNTP2-T. (iv) genetically edited NtNTP2-T or NtNTP2-T and NtNTP2-S; or (v) at least one genetic change in the NtNTP2-T polynucleotide sequence, or at least one genetic change in the NtNTP2-T polynucleotide and NtNTP2-S polynucleotide sequence, or at least one genetic change in the NtNTP2-T polypeptide sequence, or at least one genetic change in the NtNTP2-T polypeptide sequence and NtNTP2-S polypeptide sequence, suitably, at least one genetic change that causes the encoded polypeptide to terminate or end translation earlier than in the control plant.

[0014] Suitably, NtNTP2-T or NtNTP2-T and NtNTP2-S are gene-edited using the bacterial CRISPR / Cas system.

[0015] Suitably, the at least one genetic alteration is at least one mutation.

[0016] Suitably, the mutant, non-naturally occurring or transgenic plant or plant part comprises at least one nonsense mutation in an NtNTP2-T polynucleotide or an NtNTP2-T polypeptide, or at least one nonsense mutation in an NtNTP2-T polynucleotide or an NtNTP2-T polypeptide, and at least one nonsense mutation in an NtNTP2-S polynucleotide or an NtNTP2-S polypeptide.

[0017] Suitably, the mutant, non-naturally occurring or transgenic plant or plant part comprises a single nucleotide polymorphism in NtNTP2-S at nucleotide position 632 or 633 or 632 and 633 of SEQ ID NO:3, suitably wherein the single nucleotide polymorphism is a 'g' to 'a' mutation at nucleotide position 632 or 633 of SEQ ID NO:3, or a 'g' to 'a' mutation at nucleotide positions 632 and 633 of SEQ ID NO:3.

[0018] Suitably, the mutated NtNTP2-S polynucleotide sequence comprises, consists of or consists essentially of SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6.

[0019] Suitably, the mutant, non-naturally occurring or transgenic plant or plant part comprises a single nucleotide polymorphism in NtNTP2-T at nucleotide position 636 of SEQ ID NO:11, suitably wherein the single nucleotide polymorphism is a 'g' to 'a' mutation at nucleotide position 635 or 636 of SEQ ID NO:11, or a 'g' to 'a' mutation at nucleotide positions 635 and 636 of SEQ ID NO:11.

[0020] Suitably, the mutated NtNTP2-T polynucleotide sequence comprises, consists of or consists essentially of SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15.

[0021] Suitably, the mutant NtNTP2-T polypeptide or the mutant NtNTP2-T polypeptide and the mutant NtNTP2-S polypeptide each have at least one nonsense mutation at positions W212 and W211, respectively.

[0022] Suitably, the mutant NtNTP2-T polypeptide or the mutant NtNTP2-T polypeptide and the mutant NtNTP2-S polypeptide comprise, consist or consist essentially of SEQ ID NO: 16 or SEQ ID NO: 8 and SEQ ID NO: 16, respectively; optionally, wherein the mutant NtNTP2-T polypeptide or the mutant NtNTP2-T polypeptide and the mutant NtNTP2-S polypeptide are truncated.

[0023] Suitably, the plant part is selected from: (i) green leaves or parts thereof; or (ii) dried leaves or parts thereof, suitably, wherein the dried leaves or parts thereof are air-dried, suitably, sun-dried or fire-dried, or roasted; or (iii) dried leaves or parts thereof, suitably, wherein the dried leaves are air-dried, more suitably, the leaves are sun-dried or fire-roasted, or flue-roasted.

[0024] In another aspect, disclosed herein is a method for preparing a plant or plant part, comprising: (a) providing a plant comprising: (i) an NtNTP2-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:3; or (ii) an NtNTP2-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:11; or (iii) a polypeptide encoded by the polynucleotide shown in (i) or (ii); or (iv) an NtNTP2-S polypeptide having at least 77% sequence identity to SEQ ID NO:7; or (v) an NtNTP2-T polypeptide having at least 77% sequence identity to SEQ ID NO:8. NO:12 has at least 77% sequence identity; (b) reducing the expression or activity of NtNTP2-T or a combination of NtNTP2-T and NtNTP2-S in a plant; and (c) obtaining a plant or plant part that: (i) has reduced nitrate levels compared to a control plant grown under the same fertilization conditions; and (ii) has increased biomass compared to a control plant grown under the same fertilization conditions; and (iii) has an increased NUE response compared to a control plant grown under the same fertilization conditions. The plant or plant part may also have an increase in root development compared to a control plant grown under the same fertilization conditions. On the other hand, disclosed herein is a mutant, non-naturally occurring or transgenic plant or part thereof, which is obtained or obtainable by the method of the present invention.

[0025] In another aspect, disclosed herein is a mutant, non-naturally occurring or transgenic plant or part thereof, wherein nitrate levels are not significantly different compared to control plants grown under the same fertilization conditions, wherein biomass yield is higher compared to control plants grown under the same fertilization conditions, and wherein NUE of the plant is higher compared to control plants grown under the same fertilization conditions. The mutant, non-naturally occurring or transgenic plant or part thereof may also have an increase in root development compared to control plants grown under the same fertilization conditions.

[0026] Suitably, such mutant, non-naturally occurring or transgenic plants or plant parts have reduced or inhibited expression or activity of NtNTP2-T, or reduced or inhibited expression or activity of NtNTP2-T and NtNTP2-S, said NtNTP2-T and NtNTP2-S comprising or consisting of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting of or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting of or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:11; or (iii) a polypeptide encoded by the polynucleotide shown in (i) or (ii); or (iv) a NtNTP2-S polypeptide having at least 77% sequence identity to SEQ ID NO:7; or (v) a NtNTP2-T polypeptide having at least 77% sequence identity to SEQ ID NO:8. NO: 12 has at least 77% sequence identity; wherein the expression or activity of NtNTP2-T or the expression or activity of NtNTP2-T and NtNTP2-S is reduced or inhibited compared to control plants.

[0027] In another aspect, disclosed herein is a tobacco product or smoking article comprising a mutant, non-naturally occurring or transgenic plant or plant part according to the present invention.

[0028] In another aspect, the present invention provides a method for improving agronomic traits of a plant, the method comprising reducing or inhibiting the expression or activity of NtNTP2-T or NtNTP2-T and NtNTP2-S, wherein the NtNTP2-T and NtNTP2-S comprise or consist of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:11; or (iii) a polypeptide encoded by the polynucleotide shown in (i) or (ii); or (iv) a NtNTP2-S polypeptide having at least 77% sequence identity to SEQ ID NO:7; or (v) a NtNTP2-T polypeptide having at least 77% sequence identity to SEQ ID NO:8. NO: 12 has at least 77% sequence identity; wherein the expression or activity of NtNTP2-T or the expression or activity of NtNTP2-T and NtNTP2-S is reduced or inhibited compared to control plants.

[0029] Suitably, the agronomic traits are: (i) no reduction in nitrate levels; (ii) increased biomass (eg leaf biomass) under both standard and nitrogen starvation conditions; and (iii) an increase in the NUE response (meaning biomass per unit of nitrogen fertilization applied). BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a photograph showing the morphology of the AA37 ntp2-S W211stop and ntp2-T W212stop double mutant lines in the field at harvest. Representative pictures of two top plants in the field at harvest. Double wild type indicates an example of an out-segregating wild type plant; double mutant indicates an example of a ntp2-SW211stop / ntp2-T W212stop double mutant plant.

[0031] Figure 2 Figure 2 is a graph of nitrate levels in dried leaf material of AA37 ntp2 double stop mutants. Indicated are the average nitrate levels measured in dried leaves (A) and midribs (B) of the middle leaves of plants grown in the burley tobacco protocol. sstt indicates the ntp2 double mutant homozygous genotype, and SSTT indicates the ntp2 double mutant in vitro segregating wild type genotype. Error bars indicate standard errors. For in vitro segregating wild type, N = 8 plots, 10 plants per plot; for double mutants, n = 7 plots, 10 plants per plot.

[0032] Figure 3 Figures showing the morphology of the AA37 ntp2-S W211stop and ntp2-T W212stop double mutant lines in the field. Representative pictures of plants grown in the field 3 months after transplanting. Wild type indicates an example of a 10-plant plot of the wild type in vitro; double mutant indicates an example of a 10-plant plot of the ntp2-SW211stop / ntp2-T W212stop double mutant; AA37 indicates the control AA37 non-mutant figure. The upper figure (Burley tobacco scheme) indicates the part of the field fertilized with 254 units of nitrogen; the lower figure (Virginia tobacco scheme) indicates the part of the field fertilized with 55 units of nitrogen.

[0033] Figure 4 is a graph showing the results of total harvested leaf dry biomass in AA37 ntp2 double stop mutant lines grown under different nitrogen regimes. Indicated are the total harvested leaf dry biomass from the first year of the experiment under standard conditions.

[0034] Total leaf dry biomass (expressed as grams / plant) harvested from plants grown under nitrogen starvation (Burley tobacco protocol) or nitrogen starvation (Virginia tobacco protocol). sstt indicates ntp2 double mutant homozygous genotype, SSTT indicates ntp2 double mutant in vitro segregating wild-type genotype. Error bars indicate 95% confidence intervals. N = 19 plots, 8 plants per plot (two marginal plants were discarded to minimize position effects).

[0035] Figure 5 is a graph showing the segregation of mutant phenotypes. Indicated is the dry biomass of total leaves harvested from plants grown under standard conditions during the second year of the experiment (expressed as grams / plant). SSTT indicates out-segregating wild-type plants, sstt indicates ntp2 double mutant homozygous genotypes, ssTT and SStt indicate homozygous single mutants of -S and -T forms, respectively. Error bars indicate standard errors. N = 8 to 10 plots, 8 plants per plot (two marginal plants were discarded to minimize position effects).

[0036] Figure 6 It is a graph showing the NUE index of AA37 ntp2 double termination mutant strains grown in different nitrogen schemes, which means the biomass per unit of nitrogen fertilization applied. Indicated is the NUE index of total leaves harvested from plants grown under standard conditions (Burley tobacco scheme) or flue-cured nitrogen fertilization (Virginia tobacco scheme) during the 2019 growing season (expressed as kilograms of dried leaf biomass produced per kilogram of N input per hectare, assuming 12,000 plants per hectare). Sstt indicates ntp2 double mutant homozygous genotypes, and SSTT indicates ntp2 double mutant in vitro isolated wild-type genotypes. Error bars indicate 95% confidence intervals. N = 19 plots, 8 plants per plot (two marginal plants were discarded to minimize position effects).

[0037] Figure 7 It is a figure showing the expression level of NtNTP2 gene in tobacco leaves from different varieties before and after harvest. Microarray expression profiles of NtNTP2 during plant growth (green maturity), harvest time (harvest) and early curing (7 hours curing and 15 hours curing) of Swiss Burley (Burley tobacco) and Swiss flue-cured (Virginia tobacco) are shown. Expression levels are indicated as multiple changes in the log2 scale standardized at each total transcription level.

[0038] Figure 8 are two graphs showing how NtNTP2 mutations affect root development. Figure 8 (A) shows the number of lateral roots in seedlings grown on agar plates for 13 days. Figure 8(B) shows the maximum length of submerged roots in 6-week-old seedlings grown under hydroponic conditions. SSTT, sstt, ssTT, and SStt indicate the out-segregating wild type, the homozygous double-S / -T mutant, and the -S and -T single mutants, respectively. Figure 8 Letters at the top of the columns in (B) indicate statistical groups in one-way ANOVA tests. Error bars indicate 95% confidence intervals. p-values ​​are indicated.

[0039] Fig. 9 Two graphs showing the average number of roots per plant for Ntntp2-S W211stop / Ntntp2-T W212stop BC2S2 TN90 (A) and K326 (B) mutant plants and their wild-type segregant controls. SSTT indicates wild-type plants and sstt indicates the double mutant Ntntp2-S W211stop / Ntntp2-T W212stop genetic background. TN90 and K326 BC2S2 plants were grown in a 50% N-fertilized hydroponic solution, in contrast to standard agricultural practices under greenhouse conditions. Between 4 and 6 weeks after transplanting, a minimum of 18 primary roots emerged from the stele for each genotype for the TN90 variety and a minimum of 25 for K326. The statistical validity of the data was tested in a t-student test and p-values ​​are reported. Error bars report 95% confidence intervals. Fig.10 is a graph showing the average root diameter of TN90 BC2S2 plants. SSTT indicates wild-type plants and sstt indicates the double mutant Ntntp2-SW211stop / Ntntp2-T W212stop genetic background. TN90 BC2S2 plants were grown in a hydroponic solution fertilized with 50% N, compared to standard agricultural practices under greenhouse conditions. The diameter of the primary roots sprouting from the central column was measured for a minimum of 655 roots per genotype between 4 and 6 weeks after transplanting. The statistical validity of the data was tested in a t-student test and p-values ​​are reported in the graphs. Error bars indicate 95% confidence intervals.

[0040] Fig.11 is a graph showing the total root diameter distribution of BC2S2 TN90 plants grown in 50% N fertilization. Compared to standard agricultural practices under greenhouse conditions, TN90 BC2S2 plants were grown in a hydroponic solution of 50% N fertilization. Between 4 and 6 weeks after transplanting, the primary roots growing from the central column were counted and their diameters were measured with a thickness gauge. The distribution of root diameters measured for 15 plants of each genotype plotted against the number of roots measured is reported. SSTT indicates wild-type plants and sstt indicates the double mutant Ntntp2-S W211stop / Ntntp2-T W212stop genetic background.

[0041] Fig.12is a diagram showing a synthetic RNAi loop with a 35S CaMV terminator having the DNA sequence shown in SEQ ID NO:45.

[0042] Fig.13 The expression vector is shown to be cloned into a binary vector carrying an MMV promoter and a translation enhancer by HindIII-AvrII. Fig.12 Diagram of the RNAi cycle.

[0043] Fig.14 is a graph showing the average number of roots per plant in TN90 transgenic plants grown under 50% N fertilization. Compared to standard agricultural practices under greenhouse conditions, TN90 transgenic plants were grown in a hydroponic solution with 50% N fertilization. Between 4 and 6 weeks after transplanting, primary roots sprouting from the stele were counted, with a minimum of 24 plants per genotype. CT-T2 indicates control plants transformed with an empty binary vector, and RNAi-T2 indicates plants transformed with an RNAi vector. The statistical validity of the data was tested in a t-student test and the p-values ​​and the percentage increase of the RNAi line compared to the control are reported. The error bars report 95% confidence intervals.

[0044] Fig.15 is a graph of the total root diameter distribution of TN90 transgenic plants grown in 50% N fertilization. In contrast to standard agricultural practices under greenhouse conditions, TN90 transgenic plants were grown in a hydroponic solution of 50% N fertilization. Between 4 and 6 weeks after transplanting, primary roots sprouting from the central column were counted and their diameters were measured. The distribution of root diameters measured for 7 plants of each construct plotted against the number of roots measured is reported. CT-T2 indicates control plants transformed with an empty binary vector, and RNAi-T2 indicates plants transformed with an RNAi vector.

[0045] Some advantages

[0046] Plants with increased yield at lower fertilizer inputs can be obtained and thus improved NUE, meaning biomass per unit of nitrogen fertilizer applied.

[0047] This phenotype can be achieved by developing non-genetically modified (non-GM) plants through non-transgenic methods. This is highly desirable because in many countries (including Europe), the planting and commercialization of GM plants are difficult. Mutant plants with one or more single nucleotide polymorphisms are not considered to be GM plants. For example, in the EU, there are no special regulations for plants derived from mutation breeding. Therefore, in certain embodiments, it is preferred that plants only contain one or more single nucleotide polymorphisms (i.e., one or more mutations) to produce non-genetically modified plants.

[0048] It is possible to grow plants with less nitrogen than normal requirements without causing large biomass losses. Therefore, less nitrogen fertilization can be used, which can achieve a reduction in plant nitrate levels, thereby reducing TSNA, but without reducing yield. This is very advantageous in commercial plant production, such as commercial tobacco plant production.

[0049] Another advantage is that the present invention can be applied to green leaves or dried leaves. When applied to tobacco, this means that green tobacco or dried tobacco can be obtained in higher yields. This can be used for various tobacco applications, including: (i) smoking products; (ii) biofuel production; (iii) production of recombinant products; and (iv) extraction of bioactive compounds. The present invention can also be applied to dried leaves. DETAILED DESCRIPTION

[0050] The section headings used in this disclosure are for organizational purposes and are not intended to be limiting.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. In the event of a conflict, the present document (including definitions) shall prevail. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used to implement or test the present invention. The materials, methods, and examples disclosed herein are merely illustrative and are not intended to be limiting.

[0052] As used herein, the terms "comprises," "including," "having / has," "may," "contain," and variations thereof are intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0053] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0054] The term "and / or" means (a) or (b) or both (a) and (b).

[0055] The present disclosure contemplates other embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0056] For the purpose of describing the numerical ranges herein, each intervening value with the same degree of precision therebetween is expressly contemplated. For example, for the range 6-9, the values ​​7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the values ​​6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are expressly contemplated.

[0057] As used throughout the specification and claims, the following terms have the following meanings:

[0058] "Coding sequence" or "polynucleotide code" refers to the nucleotides (RNA or DNA molecule) comprising a polynucleotide encoding a polypeptide. The coding sequence may also include start and stop signals operably linked to regulatory elements, including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the polynucleotide is administered. The coding sequence may be codon optimized.

[0059] "Complementary" or "complementary" can refer to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs. "Complementarity" refers to a property shared between two polynucleotides such that when they are arranged antiparallel to each other, the nucleotide bases at each position will be complementary.

[0060] "Construct" refers to a double-stranded recombinant polynucleotide fragment comprising one or more polynucleotides. The construct includes a "template strand" that base pairs with a complementary "sense strand or coding strand." A given construct can be inserted into a vector in two possible orientations, the same (or sense) orientation or the opposite (or antisense) orientation with respect to the orientation of a promoter located within a vector (e.g., an expression vector).

[0061] In the context of a control plant or control plant cell, the term "control" refers to a plant or plant cell in which the expression, function or activity of one or more NtNTP2 genes or NtNTP2 polypeptides is not modified (e.g., increased or decreased or inhibited) and thus can be compared with a plant in which the expression, function or activity of the same one or more NtNTP2 genes or NtNTP2 polypeptides has been modified. As used herein, a "control plant" is a plant in which all parameters are substantially equivalent to a test plant or a modified plant except for a test parameter. For example, when referring to a plant in which a polynucleotide has been introduced or a polynucleotide has been modified, a control plant is an equivalent plant in which no such polynucleotide has been introduced or no such polynucleotide has been modified. A control plant can be an equivalent plant in which a control polynucleotide has been introduced. In such cases, a control polynucleotide is a polynucleotide that is expected to produce little or no phenotypic effect on a plant. A control plant can include a blank vector. A control plant can correspond to a wild-type (WT) plant. A control plant can be an empty segregant in which a T1 segregant no longer has a transgenic. For comparative purposes, control plants and plants to be compared to the control plants will be grown under identical conditions.

[0062] "Donor DNA" or "donor template" refers to a double-stranded DNA fragment or molecule that includes at least a portion of a target gene. The donor DNA can encode a fully functional polypeptide or a partially functional polypeptide.

[0063] "Endogenous gene or polypeptide" refers to a gene or polypeptide that originates from the genome of an organism and has not undergone changes (such as loss, gain, or exchange of genetic material). Endogenous genes undergo standard gene transfer and gene expression. Endogenous polypeptides undergo normal expression.

[0064] "Enhancer sequences" refer to sequences that can increase gene expression. These sequences can be located upstream, introns, or downstream of the transcribed region. The transcribed region includes exons and inserted introns from the promoter to the transcription termination region. The enhancement of gene expression can be carried out by a variety of mechanisms, including improving transcription efficiency, stabilizing mature mRNA, and enhancing translation.

[0065] "Expression" refers to the production of a functional product. For example, expression of a polynucleotide fragment can refer to the transcription of the polynucleotide fragment (e.g., transcription to produce mRNA or functional RNA) and / or translation of mRNA into a precursor or mature polypeptide. "Overexpression" refers to the production of a gene product in a transgenic organism that exceeds the level produced by an empty isolate (or non-transgenic) organism from the same experiment.

[0066] "Functional" and "fully functional" describe a polypeptide that has a biological function or activity. "Functional gene" refers to a gene that is transcribed into mRNA, which is translated into a functional or active polypeptide.

[0067] "Gene construct" refers to a DNA or RNA molecule containing a polynucleotide encoding a polypeptide. The coding sequence may include start and stop signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression.

[0068] "Genome editing" refers to changing an endogenous gene encoding an endogenous polypeptide, thereby obtaining a truncated endogenous polypeptide or a polypeptide expression of an endogenous polypeptide with an amino acid substitution. Genome editing may include replacing a region of an endogenous gene to be targeted or an entire endogenous gene with a gene copy having a truncation or amino acid substitution using a repair mechanism such as HDR. Genome editing may also include producing an amino acid substitution in an endogenous gene by generating a double-strand break in the endogenous gene and then repairing it using NHEJ. During the repair period in which an amino acid substitution may be generated, NHEJ may add or delete at least one base pair. Genome editing may also include deleting a gene segment by having two nucleases act simultaneously on the same DNA strand, thereby forming a truncation between the two nuclease target sites and repairing the DNA break by NHEJ.

[0069] "Heterologous" with respect to a sequence refers to a sequence that originates from a foreign species, or, if from the same species, a sequence that has been substantially modified in composition and / or genomic locus from its native form by deliberate human intervention.

[0070] "Homology-mediated repair" or "HDR" refers to a mechanism in cells that repairs double-stranded DNA damage when homologous DNA fragments are present in the nucleus, primarily during the G2 and S phases of the cell cycle. HDR uses donor DNA or a donor template to guide repair and can be used to make specific sequence changes to the genome, including targeted additions of entire genes. If the donor template is provided along with a site-directed nuclease, the cellular machinery will repair the break by homologous recombination, which is enhanced by several orders of magnitude in the presence of DNA cleavage. When homologous DNA fragments are not present, NHEJ can occur instead.

[0071] The term "homology" or "similarity" refers to the degree of sequence similarity between two polypeptides or between two polynucleotide molecules compared by sequence alignment. The degree of homology between two discrete polynucleotides being compared is a function of the number of identical or matching nucleotides at comparable positions.

[0072] In the context of two or more polynucleotides or polypeptides, "same" or "identity" refers to a sequence having a specific percentage of identical residues over a specific region. The percentage can be calculated by optimally aligning the two sequences, comparing a specified region of the two sequences, determining the number of positions where identical residues are present in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to produce the percentage of sequence identity. In the case where the two sequences have different lengths or the alignment produces one or more staggered ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator of the calculation rather than the numerator. The percentage of identity can be determined over the full length of the sequence. When comparing DNA and RNA, thymine (T) and uracil (U) are considered equivalent. Identity can be manually identified or determined by using a computer sequence algorithm such as ClustalW, ClustalX, BLAST, FASTA or Smith-Waterman. The popular multiple alignment program ClustalW (Nucleic Acids Research (1994) 22, 4673-4680; Nucleic Acids Research (1997), 24, 4876-4882) is a suitable means for generating multiple alignments of polypeptides or polynucleotides. Suitable parameters for ClustalW may be as follows: for polynucleotide alignments: gap open penalty = 15.0, gap extension penalty = 6.66, and matrix = identity. For polypeptide alignments: gap open penalty = 10.o, gap extension penalty = 0.2, and matrix = Gonnet. For DNA and protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will appreciate that it may be necessary to change these and other parameters to achieve optimal sequence alignment. The percent identity is then calculated from such an alignment, suitably as (N / T), where N is the number of positions where the sequences share consistent residues and T is the total number of positions compared, including gaps but excluding overhangs.

[0073] The term "increase" or "increased" refers to an increase of about 5% to about 99%, or an increase of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100%, at least about 150%, or at least about 200% or more in quantity or function or activity, such as but not limited to polypeptide function or activity, transcription function or activity and / or polypeptide expression. The term "increased" or the phrase "increased amount" may refer to a quantity or function or activity in a modified plant or a product produced by a modified plant that is greater than the quantity or function or activity found in a product of an unmodified plant or plant of the same variety processed in the same manner. Therefore, in some cases, wild-type plants of the same variety that had been processed in the same manner were used as a control, by which it was measured whether an increase in number was obtained.

[0074] As used herein, the term "reduced" or "reduced" refers to a reduction of about 5% to about 99%, or a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 100%, or at least about 150%, or at least about 200% or more of a quantity or function, such as a polypeptide function, a transcription function, or a gene expression. The term "reduced" or phrase "reduced amount" may refer to a quantity or function in a modified plant or a product produced by a modified plant that is less than a quantity or function found in an unmodified plant or a product from the same plant processed in the same manner. Therefore, in some cases, a wild-type plant of the same species or variety that has been processed in the same manner is used as a control, by which a reduction in quantity is measured.

[0075] The term "inhibit" or "inhibited" refers to a reduction of about 98% to about 100%, or a reduction of at least about 98%, at least about 99%, but particularly about 100% in quantity or function or activity, such as but not limited to polypeptide function or activity, transcriptional function or activity and / or polypeptide expression.

[0076] The term "introducing" refers to providing a polynucleotide (e.g., a construct) or a polypeptide into a cell. Introducing includes reference to the incorporation of a polynucleotide into a eukaryotic cell, where the polynucleotide can be incorporated into the genome of the cell, and includes reference to the transient provision of a polynucleotide or polypeptide to a cell. Introducing includes stable or transient transformation methods, as well as sex crossing. Thus, in the context of inserting a polynucleotide (e.g., a recombinant construct / expression construct) into a cell, "introducing" refers to "transfection" or "transformation" or "transduction", and includes reference to the incorporation of a polynucleotide into a eukaryotic cell, where the polynucleotide can be incorporated into the genome of the cell (e.g., a chromosome, a plasmid, a plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0077] The term "isolated" or "purified" refers to a material that is substantially or essentially free of components that normally accompany it in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A polypeptide that is the main species present in a preparation is substantially purified. In particular, an isolated polynucleotide is separated from an open reading frame that flanks a desired gene and encodes a polypeptide other than the desired polypeptide. As used herein, the term "purified" means that a polynucleotide or polypeptide produces essentially one band in an electrophoretic gel. In particular, it refers to a polynucleotide or polypeptide that has a purity of at least 85%, more preferably at least 95%, and most preferably at least 99%. An isolated polynucleotide can be purified from its naturally occurring host cell. Conventional polynucleotide purification methods known to technicians can be used to obtain isolated polynucleotides. The term also encompasses recombinant polynucleotides and chemically synthesized polynucleotides.

[0078] The term "level" refers to an amount and is used interchangeably with "amount".

[0079] "Modulate" refers to a qualitative or quantitative change, alteration or modification that causes or promotes a process, pathway, function or activity of interest. Without limitation, such a change, alteration or modification may be an increase or decrease in the process, pathway, function or activity of interest. For example, NtNTP2 gene expression or NtNTP2 polypeptide expression or NtNTP2 polypeptide function or activity may be modulated. Typically, the relevant change, alteration or modification will be determined by comparison with a control.

[0080] As used herein, "non-homologous end joining (NHEJ) pathway" refers to a pathway for repairing double-strand breaks in DNA by directly joining the broken ends without the need for a homologous template. The DNA end rejoining by NHEJ, which is independent of the template, is a random error-prone repair method that introduces random micro-insertions and micro-deletions (indels) at DNA breakpoints. This method can be used to intentionally interrupt, delete or change the read range of the targeted gene sequence. NHEJ typically uses a short homologous DNA sequence called microhomology to guide repair. These microhomologies are typically present in single-strand overhangs on the ends of double-strand breaks. When the overhangs are fully compatible, NHEJ typically accurately repairs the break, but there may also be inaccurate repairs that cause nucleotide losses, but more commonly when the overhangs are incompatible.

[0081] The term "non-naturally occurring" describes entities that are not formed in nature or do not exist in nature, such as polynucleotides, genetic mutations, polypeptides, plants, plant cells, and plant materials. Such non-naturally occurring entities or artificial entities can be prepared, synthesized, initiated, modified, intervened, or manipulated by methods described herein or known in the art. Such non-naturally occurring entities or artificial entities can be prepared, synthesized, initiated, modified, intervened, or manipulated by humans. Thus, for example, non-naturally occurring plants, non-naturally occurring plant cells, or non-naturally occurring plant materials can be prepared using traditional plant breeding techniques (e.g., backcrossing) or by genetic manipulation techniques (e.g., antisense RNA, interfering RNA, meganucleases, etc.). By way of further example, a non-naturally occurring plant, non-naturally occurring plant cell or non-naturally occurring plant material can be prepared by introgressing a first plant or plant cell gene into a second plant or plant cell (which itself can be naturally occurring), or by transferring one or more genetic mutations (e.g., one or more polymorphisms) from a first plant or plant cell into a second plant or plant cell, such that the resulting plant, plant cell or plant material or its progeny includes a genetic composition (e.g., a genome, chromosome or segment thereof) that is not naturally occurring or does not exist in nature. The resulting plant, plant cell or plant material is therefore artificial or non-naturally occurring. Accordingly, an artificial or non-naturally occurring plant or plant cell can be prepared by modifying a gene sequence in a first naturally occurring plant or plant cell, even if the resulting gene sequence is naturally present in a second plant or plant cell, the second plant or plant cell comprising a different genetic background from the first plant or plant cell. In certain embodiments, the mutation is not a naturally occurring mutation that occurs naturally in a polynucleotide or polypeptide. Differences in genetic background can be detected by phenotypic differences or by molecular biology techniques known in the art, such as polynucleotide sequencing, the presence or absence of genetic markers (e.g., microsatellite RNA markers).

[0082] "Oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked together. The description of a single strand also defines the sequence of the complementary strand. Therefore, a polynucleotide also encompasses the complementary strand of the depicted single strand. Many variants of a polynucleotide can be used for the same purpose as a given polynucleotide. A polynucleotide also encompasses substantially identical polynucleotides and their complements. A single strand provides a probe that can hybridize with a given sequence under stringent hybridization conditions. Therefore, a polynucleotide also encompasses probes that hybridize under stringent hybridization conditions. A polynucleotide can be single-stranded or double-stranded, or can contain parts of double-stranded and single-stranded sequences. A polynucleotide can be DNA (both genomic DNA and cDNA), RNA or a hybrid, wherein a polynucleotide can include a combination of deoxyribonucleotides and ribonucleotides, and a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. A polynucleotide can be obtained by chemical synthesis methods or by recombinant methods. The specificity of hybridization of single-stranded DNA to complementary fragments is determined by the "stringency" of the reaction conditions (Sambrook et al., Molecular Cloning and Laboratory Manual, 2nd edition, Cold Spring Harbor (1989)). Hybridization stringency increases as the tendency to form DNA duplexes decreases. In polynucleotide hybridization reactions, stringency can be selected to favor specific hybridization (high stringency), which can be used, for example, to identify full-length clones from a library. Low specific hybridization (low stringency) can be used to identify related but imprecise (homologous, but not identical) DNA molecules or segments. DNA duplexes are stabilized according to the following factors:

[0083] (1) The number of complementary base pairs; (2) The type of base pairs; (3) The salt concentration (ionic strength) of the reaction mixture; (4) The reaction temperature; and (5) The presence of certain organic solvents, such as formamide, which reduce the stability of the DNA duplex. In general, the longer the probe, the higher the temperature required for proper annealing. A common approach is to vary the temperature; higher relative temperatures result in more stringent reaction conditions. Hybridization under "stringent conditions" describes a hybridization protocol in which polynucleotides that are at least 60% homologous to each other remain hybridized. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the specific sequence at a specified ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and polynucleotide concentration) at which 50% of the probes complementary to a given sequence hybridize to the given sequence at equilibrium. Since a given sequence is usually present in excess, at the Tm, 50% of the probes are in equilibrium.

[0084] "NtNTP2" is used herein to refer to: (i) NtNTP2-S polynucleotide or NtNTP2-T polynucleotide or a combination of NtNTP2-S and NtNTP2-T polynucleotides; or (ii) NtNTP2-S polypeptide or NtNTP2-T polypeptide or a combination of NtNTP2-S and NtNTP2-T polypeptides.

[0085] "Stringent hybridization conditions" are conditions under which a probe, primer, or oligonucleotide hybridizes only to its specific sequence. Stringent conditions are sequence-dependent and will vary. Stringent conditions typically include: (1) low ionic strength and high temperature washes, e.g., 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate at 50°C; (2) denaturants during hybridization, e.g., 50% (v / v) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (750 mM sodium chloride, 75 mM sodium citrate, pH 6.5) at 42°C; or (3) 50% formamide. Washes also typically include 5xSSC (0.75M NaCl, 75mM sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg / mL), 0.1% SDS, and 10% dextran sulfate at 42° C., and 0.2xSSC (sodium chloride / sodium citrate) at 42° C. and 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1xSSC containing EDTA at 55° C. Suitably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.

[0086] "Medium stringency conditions" use a washing solution and less stringent hybridization conditions so that the polynucleotide will hybridize with the entirety, fragment, derivative or analog of the polynucleotide. An example includes hybridization in 6xSSC, 5x Denport solution, 0.5% SDS and 100 μg / mL denatured salmon sperm DNA at 55°C, followed by one or more washes in 1xSSC, 0.1% SDS at 37°C. Temperature, ionic strength, etc. can be adjusted to accommodate experimental factors, such as probe length. Other medium stringency conditions have been described (see Ausubel et al., Current Protocols in Molecular Biology, Vol. 1-3, John Wiley & Sons, Inc., Hoboken, NJ (1993); Kriegler, Gene Transfer and Expression: A Laboratory Manual, Stockton Press, New York, NY (1990); Perbal, A Practical Guide to Molecular Cloning, 2nd Edition, John Wiley & Sons, New York, NY (1988)).

[0087] "Low stringency conditions" use a wash solution and hybridization conditions that are less stringent than moderate stringency, so that a polynucleotide will hybridize to the entirety, fragment, derivative or analog of a polynucleotide. Non-limiting examples of low stringency hybridization conditions include hybridization in 35% formamide, 5xSSC, 50mM Tris HCl (pH 7.5), 5mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / mL denatured salmon sperm DNA, 10% (weight / volume) dextran sulfate at 40°C, followed by one or more washes in 2xSSC, 25mM Tris HCl (pH 7.4), 5mM EDTA and 0.1% SDS at 50°C. Other conditions of low stringency, such as conditions for cross-species hybridization, have been fully described (see Ausubel et al., 1993; Kriegler, 1990).

[0088] "Operably linked" means that the expression of a gene is under the control of a promoter to which it is spatially linked. A promoter can be located 5' (upstream) or 3' (downstream) of a gene under its control. The distance between a promoter and a gene can be about the same as the distance between a promoter and the gene it controls in the gene that produces the promoter. As is known in the art, changes in this distance can be adjusted without loss of promoter function. "Operably linked" refers to the association of polynucleotide fragments in a single fragment, so that the function of one fragment is regulated by another fragment. For example, a promoter is operably linked to a polynucleotide fragment when it is capable of regulating the transcription of a polynucleotide fragment.

[0089] The term "plant" refers to any plant and its progeny in any stage of its life cycle or growth. The term includes the complete plant, plant organ, plant tissue (such as leaf), plant propagator, plant seed, plant cell and its progeny mentioned. Plant cell includes, but is not limited to, cells from seed, suspension culture, plumule, meristem region, callus, leaf, root, shoot, gametophyte, sporophyte, pollen and pollen grain. Suitable kinds, cultivars, hybrids and varieties of tobacco plants are described herein.

[0090] "Polynucleotide," "polynucleotide sequence," or "polynucleotide fragment" are used interchangeably herein and refer to a polymer of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually in their 5' monophosphate form) are referred to by their single-letter names as follows: "A" for adenylic acid or deoxyadenylic acid (for RNA or DNA, respectively), "C" for cytidylic acid or deoxycytidylic acid, "G" for guanylic acid or deoxyguanylic acid, "U" for uridylic acid, "T" for deoxythymidylic acid, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide. A polynucleotide may be, but is not limited to, genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA, or a fragment thereof. In addition, polynucleotides can be single-stranded or double-stranded, a mixture of single-stranded and double-stranded regions, a hybrid molecule comprising DNA and RNA, or a hybrid molecule or a fragment thereof having a mixture of single-stranded and double-stranded regions. In addition, polynucleotides can be composed of a triple-stranded region comprising DNA, RNA, or both, or a fragment thereof. Polynucleotides can contain one or more modified bases, such as thiophosphates, and can be peptide nucleic acids (PNAs). In general, polynucleotides can be assembled by isolated or cloned cDNA fragments, genomic DNA, oligonucleotides, or individual nucleotides or aforesaid combinations. Although polynucleotides described herein are shown as DNA sequences, polynucleotides include their corresponding RNA sequences and their complementary (e.g., fully complementary) DNA or RNA sequences, including their reverse complements. Polynucleotides of the present disclosure are listed in the attached sequence table.

[0091] "Polypeptide" or "polypeptide sequence" refers to polymers of amino acids in which one or more amino acid residues is an artificial chemical analog of the corresponding naturally occurring amino acid, as well as polymers of naturally occurring amino acids. The term also includes modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. The polypeptides of the present disclosure are listed in the attached sequence listing.

[0092] "Promoter" refers to a molecule of synthetic or natural origin that can confer, activate or enhance the expression of polynucleotides in cells. The term refers to a polynucleotide element / sequence that is usually located upstream of a double-stranded polynucleotide fragment and operably connected thereto. The promoter can be completely derived from the region adjacent to the natural gene of interest, or can be composed of different elements derived from different natural promoters or synthetic polynucleotide fragments. The promoter can include one or more specific transcription regulatory sequences to further improve expression and / or change its spatial expression and / or temporary expression. The promoter can also include terminal enhancers or repressor elements, which can be located up to several thousand base pairs from the transcription start site. The promoter can be derived from sources including viruses, bacteria, fungi, plants, insects and animals. The promoter can be about the cells, tissues or organs where expression occurs or about the developmental stage where expression occurs, or in response to external stimuli (such as physiological pressure, pathogens, metal ions or inducers) to constitutively or differentially regulate the expression of gene components.

[0093] "Tissue-specific promoter" and "tissue-preferred promoter," as used interchangeably herein, refer to promoters that are primarily, but not necessarily exclusively, expressed in one tissue or organ, but may also be expressed in one specific cell. "Developmentally regulated promoter" refers to a promoter whose function is determined by developmental events. "Constitutive promoter" refers to a promoter that causes a gene to be expressed in most cell types at most times. "Inducible promoters" selectively express an operably linked DNA sequence in response to the presence of endogenous or exogenous stimuli, such as by compounds (chemical inducers) or in response to environmental, hormonal, chemical and / or developmental signals. Examples of inducible or regulated promoters include promoters regulated by light, heat, pressure, flooding or drought, pathogens, plant hormones, trauma, or chemicals such as ethanol, jasmonates, salicylic acid, or safeners.

[0094] As used herein, "recombination" refers to the artificial combination of two otherwise isolated sequence fragments, such as polynucleotide fragments isolated by chemical synthesis or by genetic engineering techniques. The term also includes reference to cells or vectors that have been modified by the introduction of heterologous polynucleotides or cells derived from such modified cells, but does not encompass changes to cells or vectors due to naturally occurring events (e.g., spontaneous mutations, natural transformations or transductions or transpositions) such as those that occur without human intervention.

[0095] A "recombinant construct" refers to a combination of polynucleotides that are not normally found together in nature. Thus, a recombinant construct may comprise regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that normally found in nature. A recombinant construct may be a recombinant DNA construct.

[0096] "Regulatory sequence" and "regulatory element" used interchangeably herein refer to polynucleotide sequences located upstream (5' non-coding sequence), within or downstream (3' non-coding sequence) of a coding sequence and affecting the transcription, RNA processing or stability or translation of the associated coding sequence. Regulatory sequences include promoters, translation leader sequences, introns and polyadenylation recognition sequences. The terms "regulatory sequence" and "regulatory element" are used interchangeably herein.

[0097] "Site-specific nuclease" refers to an enzyme that can specifically recognize and cleave a DNA sequence. Site-specific nucleases can be engineered. Examples of engineered site-specific nucleases include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), CRISPR / Cas9-like systems, and meganucleases.

[0098] The term "tobacco" is used to refer to tobacco crops (e.g., various tobacco plants grown in the field rather than the tobacco grown in hydroponics), tobacco plants and parts thereof in a general sense, including but not limited to roots, stems, leaves, flowers and seeds prepared and / or obtained as described herein. It should be understood that "tobacco" includes tobacco plants and parts thereof.

[0099] The term "tobacco product" refers to consumer tobacco products, including but not limited to smoking materials (e.g., cigarettes, cigars and pipe tobacco), snuff, chewing tobacco, chewing gum and lozenges, and components, materials and ingredients for the manufacture of consumer tobacco products. Suitably, these tobacco products are manufactured by leaves and stems of tobacco harvested from tobacco, and are cut, dried, dried and / or fermented according to conventional techniques in tobacco preparation. As described herein, the tobacco in the tobacco product can be combined with a binding agent.

[0100] "Transcription terminator", "termination sequence" or "terminator" refers to a DNA sequence located downstream of a coding sequence, including polyadenylation recognition sequences and other sequences encoding regulatory signals that can affect mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[0101] "Transgenic" refers to any cell, cell line, callus, plant part or plant whose genome is altered by the presence of a heterologous polynucleotide, such as a recombinant construct, including those original transgenic events and those generated from the original transgenic events by sexual crosses or asexual propagation. The term does not include alterations of the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or by naturally occurring events, such as random cross fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[0102] "Transgenic plant" refers to a plant containing one or more heterologous polynucleotides in its genome, that is, a plant containing recombinant genetic material that is not usually found therein and has been introduced into the plant (or introduced into the progenitor cell of the plant) by artificial manipulation. For example, a heterologous polynucleotide can be stably integrated into the genome so that the polynucleotide is transmitted to successive generations. A heterologous polynucleotide can be integrated into the genome alone or as part of a recombinant construct. The commercial development of genetically modified embryos has also developed to the stage of introducing multiple characteristics into crop plants, commonly referred to as gene stacking. In this method, multiple genes that confer different characteristics of concern can be introduced into plants. Gene stacking can be achieved in many ways, including (but not limited to) co-transformation, re-transformation, and crossing with different transgenic lines. Therefore, plants grown from plant cells introduced with recombinant DNA by transformation are transgenic plants, all of which are progeny (sexual or asexual) containing plants that introduce transgenes. It should be understood that the term transgenic plant includes the entire plant or tree and parts of the plant or tree, such as grains, seeds, flowers, leaves, roots, fruits, pollen, stems, etc. Each heterologous polynucleotide can confer a different trait on the transgenic plant.

[0103] "Transcription activator-like effector" or "TALE" refers to a polypeptide structure that recognizes and binds to a specific DNA sequence. "TALE DNA binding domain" refers to a DNA binding domain comprising an array of 33-35 amino acid repeats in series, also known as an RVD module, each of which specifically recognizes a single DNA base pair. The RVD modules can be arranged in any order to assemble an array that recognizes a determined sequence. The binding specificity of the TALE DNA binding domain is determined by a single truncated repeat of 20 amino acids followed by the RVD array. The TALE DNA binding domain may have 12 to 27 RVD modules, each of which contains an RVD and recognizes a single DNA base pair. The specific RVD has identified the recognition of each of the four possible DNA nucleotides (A, T, C, and G). Because the TALE DNA binding domain is a module, the repeat sequences that recognize four different DNA nucleotides can be connected together to recognize any specific DNA sequence. These targeted DNA binding domains can be combined with catalytic domains to form functional enzymes, including artificial transcription factors, methyltransferases, integrases, nucleases, and recombinases.

[0104] "Transcription activator-like effector nucleases" or "TALENs," used interchangeably herein, refer to engineered fusion polypeptides of the catalytic domain of a nuclease (e.g., endonuclease FokI) and a designed TALE DNA binding domain that can target a custom DNA sequence.

[0105] "TALEN monomer" refers to an engineered fusion polypeptide with a catalytic nuclease domain and a designed TALE DNA binding domain. Two TALEN monomers can be designed to target and cleave the TALEN target region.

[0106] "Transgenic" refers to genes or genetic material that contain gene sequences that have been isolated from one organism and introduced into a different organism. This non-natural fragment of DNA may retain the ability to produce RNA or polypeptides in the transgenic organism, or it may alter the normal function of the genetic code of the transgenic organism. The introduction of a transgene has the potential to change the phenotype of an organism.

[0107] "Variant" or "mutant" with respect to a polynucleotide refers to a polynucleotide that differs from a wild-type polynucleotide (such as SEQ ID NO: 3 or SEQ ID NO: 11) by one or more nucleic acid deletions, additions, substitutions or side chain modifications. Exemplary polynucleotide variants or mutants are shown in SEQ ID NO: 4, 5, 6, 13, 14 and 15. "Variant" or "mutant" with respect to a polypeptide refers to a polypeptide that differs in sequence by insertion, deletion or conservative substitution of one or more amino acids. A variant or mutant may retain all or part of or no biological function or activity compared to a polypeptide that does not contain insertion, deletion or conservative substitution of one or more amino acids. Conservative substitutions of amino acids, i.e., substitutions of amino acids with different amino acids that are similar in properties (e.g., hydrophilicity, degree and distribution of charged regions), are generally considered in the art to involve minor changes. Exemplary polypeptide variants or mutants are shown in SEQ ID NO: 8 and 16.

[0108] In the context of plants, the term "variety" refers to a population of plants that share constant characteristics that set them apart from other plants of the same species. Varieties are further characterized by minimal overall variation among individuals within the variety, despite having one or more unique traits. Varieties are typically sold in the marketplace.

[0109] "Vector" refers to a polynucleotide vehicle comprising a combination of polynucleotide components, polynucleotide constructs, and polynucleotide conjugates, etc., for enabling the transport of polynucleotides. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. Suitable vectors include episomes capable of extrachromosomal replication, such as circular double-stranded nucleotide plasmids; linearized double-stranded nucleotide plasmids; and other vectors of any origin. As used herein, "expression vector" is a polynucleotide vehicle comprising a combination of polynucleotide components, polynucleotide constructs, and polynucleotide conjugates, etc., for enabling the expression of polynucleotides. Suitable expression vectors include episomes capable of extrachromosomal replication, such as circular double-stranded nucleotide plasmids; linearized double-stranded nucleotide plasmids; and other functionally equivalent expression vectors of any origin. The expression vector comprises at least one promoter located upstream of the polynucleotide, polynucleotide construct, or polynucleotide conjugate and operably linked thereto, as defined below.

[0110] "Zinc finger" refers to a polypeptide structure that recognizes and binds to a DNA sequence. Zinc finger domains are the most common DNA binding motifs in the human proteome. A single zinc finger contains about 30 amino acids and the domain typically works by binding to 3 consecutive DNA base pairs through the interaction of a single amino acid side chain per base pair.

[0111] "Zinc finger nuclease" or "ZFN" refers to a chimeric polypeptide molecule comprising at least one zinc finger DNA binding domain operably linked to at least one nuclease or portion of a nuclease that, when fully assembled, is capable of cleaving DNA.

[0112] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. For example, any nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and polypeptide and polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definition. In addition, unless the context otherwise requires, singular terms should include plural terms and plural terms should include singular terms.

[0113] In one embodiment, a kind of isolated polynucleotide is provided, it comprises, consists of or consists essentially of a sequence having at least 70% sequence identity with any sequence described herein, said sequence including any polynucleotide shown in the sequence listing. Suitably, the isolated polynucleotide comprises, consists of or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0114] Suitably, the polynucleotides described herein encode active polypeptides having at least about 45%, 50%, 60%, 70%, 80%, 90% 95%, 96%, 97%, 98%, 99%, 100% or more of the function or activity of the polypeptides shown in the sequence listing.

[0115] In another embodiment, an isolated polynucleotide is provided, which comprises, consists of, or essentially consists of a polynucleotide having at least 70% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 (provided that the mutation is maintained), SEQ ID NO:5 (provided that the mutation is maintained), SEQ ID NO:6 (provided that the mutation is maintained), SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13 (provided that the mutation is maintained), SEQ ID NO:14 (provided that the mutation is maintained) or SEQ ID NO:15 (provided that the mutation is maintained). Suitably, the isolated polynucleotide comprises a sequence corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 (provided that the mutation is maintained), SEQ ID NO: 5 (provided that the mutation is maintained), SEQ ID NO: 6 (provided that the mutation is maintained), SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13 (provided that the mutation is maintained), SEQ ID NO: 14 (provided that the mutation is maintained) or SEQ ID NO: 15. NO:15 (provided that the mutation is maintained) has, consists of, or consists essentially of a sequence having at least about 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity.

[0116] In another embodiment, a polynucleotide is provided, which comprises, consists of, or essentially consists of a polynucleotide having substantial homology (i.e., sequence similarity) or substantial identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 (provided that the mutation is maintained), SEQ ID NO: 5 (provided that the mutation is maintained), SEQ ID NO: 6 (provided that the mutation is maintained), SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13 (provided that the mutation is maintained), SEQ ID NO: 14 (provided that the mutation is maintained), or SEQ ID NO: 15 (provided that the mutation is maintained).

[0117] In another embodiment, a fragment of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 (provided that the mutation is maintained), SEQ ID NO:5 (provided that the mutation is maintained), SEQ ID NO:6 (provided that the mutation is maintained), SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13 (provided that the mutation is maintained), SEQ ID NO:14 (provided that the mutation is maintained) or SEQ ID NO:15 (provided that the mutation is maintained) having substantial homology (i.e., sequence similarity) or substantial identity thereto is provided, which is similar to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 (provided that the mutation is maintained), SEQ ID NO:5 (provided that the mutation is maintained), SEQ ID NO:6 (provided that the mutation is maintained), SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13 (provided that the mutation is maintained), SEQ ID NO:14 (provided that the mutation is maintained), SEQ ID NO:15 (provided that the mutation is maintained). ID NO:14 (provided that the mutation is maintained) or SEQ ID NO:15 (provided that the mutation is maintained) The corresponding fragment has at least about 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity.

[0118] In another embodiment, a polynucleotide is provided, which comprises a sufficient or substantial degree of identity or similarity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 (provided that the mutation is maintained), SEQ ID NO: 5 (provided that the mutation is maintained), SEQ ID NO: 6 (provided that the mutation is maintained), SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13 (provided that the mutation is maintained), SEQ ID NO: 14 (provided that the mutation is maintained) or SEQ ID NO: 15 (provided that the mutation is maintained), which encodes a polypeptide that acts as NtNTP2.

[0119] In another embodiment, a polymer of polynucleotides is provided, which comprises, consists of, or consists essentially of a polynucleotide designated herein as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0120] Suitably, the NtNTP2 polynucleotides described herein may encode functional NtNTP2. In an alternative, the NtNTP2 polynucleotides described herein may contain at least one mutation, suitably at least one mutation encoding a stop codon, which causes the encoded polypeptide to terminate or end its translation earlier than expected, which will produce a non-functional fragment of NtNTP2. Such exemplary polynucleotides are disclosed herein.

[0121] As described herein, polynucleotides may comprise nucleotide polymers, which may be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Thus, polynucleotides may be, but are not limited to, genomic DNA, complementary DNA (cDNA), mRNA or antisense RNA or fragments thereof. In addition, polynucleotides may be single-stranded or double-stranded DNA, DNA mixed with single-stranded and double-stranded regions, hybrid molecules including DNA and RNA, or hybrid molecules with a mixture of single-stranded and double-stranded regions or fragments thereof. In addition, polynucleotides may be composed of triple-stranded regions including DNA, RNA or both, or fragments thereof. Polynucleotides may contain one or more modified bases, such as thiophosphates, and may be peptide nucleic acids. In general, polynucleotides may be assembled from isolated or cloned cDNA fragments, genomic DNA, oligonucleotides or individual nucleotides or combinations of the foregoing. Although the polynucleotides described herein are shown as DNA sequences, they include their corresponding RNA sequences and their complementary (e.g., fully complementary) DNA or RNA sequences, including their reverse complements.

[0122] Polynucleotides as described herein will generally contain phosphodiester bonds, although in some cases, polynucleotide analogs are included that may have alternative backbones, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate or O-methylphosphoramidite bonds; and peptide polynucleotide backbones and bonds. Other similar polynucleotides include polynucleotides with positive backbones; non-ionic backbones and non-ribose backbones. Modification of the ribose-phosphate backbone can be done for a variety of reasons, such as increasing the stability and half-life of such molecules in physiological environments, or as probes on biochips. Mixtures of naturally occurring polynucleotides and analogs can be prepared; alternatively, mixtures of different polynucleotide analogs can be prepared, as well as mixtures of naturally occurring polynucleotides and analogs.

[0123] A variety of polynucleotide analogs are known, including for example phosphoramidates, phosphorothioates, phosphorodithioates, O-methylphosphoramidite bonds and peptide polynucleotide backbones and bonds. Other similar polynucleotides include polynucleotides with positive backbones, nonionic backbones and non-ribose backbones. Also included are polynucleotides containing one or more carbocyclic sugars.

[0124] Other analogs include peptide polynucleotides as peptide polynucleotide analogs. These backbones are substantially nonionic under neutral conditions, in contrast to the highly charged phosphodiester backbones of naturally occurring polynucleotides. This can produce advantages. First, the peptide polynucleotide backbone can show improved hybridization kinetics. For mismatched base pairs relative to fully matched base pairs, peptide polynucleotides have greater changes in melting temperature. For internal mismatches, DNA and RNA generally show a 2-4 ° C decrease in melting temperature. In the case of nonionic peptide polynucleotide backbones, the decrease is close to 7-9 ° C. Similarly, due to its nonionic nature, the hybridization of the bases connected to these backbones is relatively insensitive to salt concentration. In addition, peptide polynucleotides may not be degraded by cellular enzymes or are degraded by cellular enzymes to a lesser extent, and therefore may be more stable.

[0125] In the disclosed polynucleotide and its fragment purposes, there is the purpose of fragment as probe in hybridization determination or as primer in amplification determination. Such fragments generally include at least about 10,11,12,13,14,15,16,17,18,19 or 20 or more adjacent nucleotides of DNA sequence. In other embodiments, DNA fragments include at least about 10,15,20,30,40,50 or 60 or more adjacent nucleotides of DNA sequence. Therefore, in one aspect, a method for detecting polynucleotide is also provided, which includes using probe or primer or both. Exemplary primers are described in this article.

[0126] Basic parameters affecting the selection of hybridization conditions and guidance for designing suitable conditions are described by Sambrook, J., EF Fritsch and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Using the knowledge of the genetic code in combination with the polypeptide sequences described herein, degenerate oligonucleotide sets can be prepared. Such oligonucleotides can be used as primers in, for example, polymerase chain reaction (PCR), thereby separating and amplifying DNA fragments. In certain embodiments, degenerate primers can be used as probes for gene libraries. Such libraries include cDNA libraries, genomic libraries, and even electronically expressed sequence tags or DNA libraries. The homologous sequences identified by this method are then used as probes to identify homologs of the sequences identified herein.

[0127] As described herein, polynucleotides and oligonucleotides (e.g., primers or probes) that hybridize to polynucleotides under reduced stringency conditions (generally moderate stringency conditions) and generally highly stringent conditions are also potential uses. Basic parameters that affect the selection of hybridization conditions and guidance for designing appropriate conditions are described by Sambrook, J., EF Fritsch and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and can be readily determined by one of ordinary skill in the art based on, for example, the length or base composition of the polynucleotides.

[0128] One method of achieving moderate and high stringency conditions is defined herein. It should be understood that by applying the basic principles of controlling hybridization reactions and duplex stability, wash temperatures and wash salt concentrations may be adjusted as necessary to achieve the desired degree of stringency (see, e.g., Sambrook, J., EF Fritsch and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, 1996). Harbor, NY). When a polynucleotide hybridizes to a polynucleotide of unknown sequence, the hybrid length is assumed to be the length of the hybridizing polynucleotide. When polynucleotides of known sequence hybridize, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying one or more regions of optimal sequence complementarity. The hybridization temperature for hybrids expected to be less than 50 base pairs in length should be 5°C to 10°C lower than the melting temperature of the hybrid, where the melting temperature is determined according to the following equation. For hybrids less than 18 base pairs in length, the melting temperature (°C) = 2(number of A bases + T bases) + 4(number of G bases + C bases). For hybrids greater than 18 base pairs in length, the melting temperature (°C) = 81. 5+16.6(log10[Na+])+0.41(%G+C)-(600 / N), where N is the number of bases in the hybrid and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] of 1× standard sodium citrate=0.165 M). Typically, each such hybridizing polynucleotide has a length of at least 25% (usually at least 50%, 60%, or 70%, and most usually at least 80%) of the length of the polynucleotide to which it hybridizes, and has at least 60% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) to the polynucleotide to which it hybridizes.

[0129] As will be appreciated by those skilled in the art, linear DNA has two possible orientations: 5'-to-3' direction and 3'-to-5' direction. For example, if the first sequence is positioned in a 5'-to-3' direction, and if the second sequence is positioned in the same polynucleotide molecule / chain in a 5'-to-3' direction, the first sequence and the second sequence are oriented in the same direction, or have the same orientation. Usually, the promoter sequence and the target gene under the regulation of a given promoter are placed in the same orientation. However, for the first sequence positioned in a 5'-to-3' direction, if the second sequence is positioned in the same polynucleotide molecule / chain in a 3'-to-5' direction, the first sequence and the second sequence are oriented in an antisense direction, or have an antisense orientation. If the reverse complementary sequence of the first sequence (5'-to-3' direction) and the first sequence (the first sequence positioned in a 5'-to-3' direction) are positioned in the polynucleotide molecule / chain of the phase, then the two sequences with antisense orientation relative to each other can be alternatively described as having the same orientation. The sequence shown herein is shown in a 5'-to-3' direction.

[0130] Fragments of the polynucleotides described herein are also disclosed and can range from at least about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, about 1100 nucleotides, about 1200 nucleotides, about 1300 nucleotides, or about 1400 nucleotides, and up to the full-length polynucleotide.

[0131] In another aspect, a separated polypeptide is provided, comprising, consisting of or consisting essentially of a polypeptide having at least 77% sequence identity to any polypeptide described herein, including any polypeptide shown in the sequence listing. Suitably, the separated polypeptide comprises, consists of or consists essentially of a sequence having at least 77%, 78%, 79%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity thereto.

[0132] In one embodiment, a polypeptide encoded by SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:16 is provided.

[0133] In another embodiment, an isolated polypeptide is provided which comprises, consists of, or consists essentially of a sequence having at least 77%, 78%, 79%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:12, or SEQ ID NO:16.

[0134] The polypeptide may include a sequence having a sufficient or substantial degree of identity or similarity to SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 16 to function as an NtNTP2. Fragments of the polypeptide generally retain some or all of the NtNTP2 function or activity of the full-length sequence.

[0135] As described herein, a polypeptide can include a mutation, such as a mutation encoding a stop codon, such as SEQ ID NOs: 8 and 16. SEQ ID NO: 8 has a mutation at amino acid position 211 that mutates amino acid W211 of NtNtp2-S to a stop codon. SEQ ID NO: 16 has a mutation at amino acid position 212 that mutates amino acid W212 of NtNtp2-T to a stop codon.

[0136] Although exemplary mutations are disclosed herein, the skilled artisan will appreciate that favorable phenotypes can be obtained by other mutations in the NtNtp2 polynucleotide sequences described herein that cause truncation of the expressed polypeptide. Likewise, other genetic techniques described herein can be used to obtain favorable phenotypes by modulating (e.g., reducing or inhibiting) the expression or activity of NtNtp2.

[0137] Mutants can be produced by introducing any type of changes (e.g., insertion, deletion or substitution of amino acids; changes in glycosylation state; changes affecting refolding or isomerization, three-dimensional structure or self-association state), which can be intentionally engineered or isolated naturally, provided that they still have some or all of their functions or activities. Suitably, such functions or activities are modulated, more suitably, reduced or lost.

[0138] Deletion refers to the removal of one or more amino acids from a polypeptide. Insertion refers to one or more amino acid residues introduced into a predetermined site in a polypeptide. Insertion may comprise intrasequence insertion of a single or multiple amino acids. Substitution refers to the replacement of an amino acid of a polypeptide by another amino acid having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, tendency to form or destroy a-helical structure or beta-sheet structure). Amino acid substitutions are usually single residues, but may be clustered, depending on the functional constraints imposed on the polypeptide, and may range from about 1 to about 10 amino acids. Amino acid substitutions are suitably conservative amino acid substitutions as described below. Amino acid replacements, deletions and / or insertions may be prepared using peptide synthesis techniques such as solid phase peptide synthesis or by recombinant DNA manipulation. Methods for manipulating DNA sequences to produce substitution, insertion or deletion mutants of polypeptides are well known in the art. The mutants may have changes that produce silent changes and produce functionally equivalent polypeptides. Deliberate amino acid substitutions may be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity and amphipathic properties of the residues, as long as the secondary binding of the substance is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with similar hydrophilicity values ​​containing non-charged polar head groups include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine. Conservative substitutions can be made, for example, according to the following table. Amino acids in the same block in the second column and, appropriately, in the same row in the third column can be substituted for each other:

[0139]

[0140] The polypeptide can be a mature polypeptide or an immature polypeptide or a polypeptide derived from an immature polypeptide. The polypeptide can be in linear form or cyclized using known methods. The polypeptide typically comprises at least 10, at least 20, at least 30 or at least 40 contiguous amino acids.

[0141] Fragments of the disclosed polypeptides are also disclosed. The scope of the polypeptide fragments can be at least about 25 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, and up to the full-length polypeptide described herein. The encoded polypeptide fragments can retain all or part of the biological activity of the full-length polypeptide, or do not retain any biological activity.

[0142] Recombinant constructs can be used to transform plants or plant cells to regulate the expression, function or activity of NtNTP2 polypeptides. Recombinant NtNTP2 polynucleotide constructs may include NtNTP2 polynucleotides encoding one or more NtNTP2 polynucleotides as described herein, which are operably linked to a regulatory region suitable for expressing NtNTP2 polypeptides. Therefore, NtNTP2 polynucleotides may include coding sequences encoding NtNTP2 polypeptides as described herein. Plants or plant cells that have regulated the expression, function or activity of NtNTP2 polypeptides may include mutant, non-natural, transgenic, artificial or genetically engineered plants or plant cells. Suitably, transgenic plants or plant cells include genomes that have been changed by stable integration of recombinant DNA. Recombinant DNA includes DNA that has been genetically engineered and constructed outside the cell, and includes DNA containing naturally occurring DNA or cDNA or synthetic DNA. Transgenic plants may include plants regenerated from initially transformed plant cells, as well as later generations or hybridized progeny transgenic plants from transformed plants. Suitably, the transgenic modification alters the expression, function or activity of a NtNTP2 polynucleotide or NtNTP2 polypeptide as described herein, compared to a control plant.

[0143] The NtNTP2 polypeptide encoded by the recombinant NtNTP2 polynucleotide can be a native NtNTP2 polypeptide, or heterologous to the cell. In some cases, the recombinant construct contains an NtNTP2 polynucleotide operably linked to a regulatory region that regulates expression. Examples of suitable regulatory regions are described herein.

[0144] Also provided are vectors containing recombinant polynucleotide constructs, including recombinant NtNTP2 polynucleotide constructs. Suitable vector backbones include, for example, vector backbones conventionally used in the art, such as plasmids, viruses, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or bacteriophage artificial chromosomes. Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophages, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available.

[0145] The vector may include, for example, an origin of replication, a support attachment region, or a marker. The marker gene may confer a selectable phenotype to a plant cell. For example, a marker may confer biocide resistance, such as resistance to antibiotics (e.g., kanamycin, G418, bleomycin, or hygromycin) or herbicides (e.g., glyphosate, chlorsulfuron, or phosphinothricin). In addition, the expression vector may include a tag sequence designed to facilitate manipulation or detection (e.g., purification or location) of the expressed polypeptide. Tag sequences, such as luciferase, β-glucuronidase, green fluorescent polypeptide, glutathione S-transferase, polyhistidine, c-myc, or hemagglutinin sequences are typically expressed as fusions with the encoded polypeptide. Such tags may be inserted anywhere within the polypeptide, including at the carboxyl or amino terminus.

[0146] Plants or plant cells can be transformed by integrating the recombinant NtNTP2 polynucleotide into its genome to become stably transformed. The plants or plant cells described herein can be stably transformed. Stably transformed cells typically retain the introduced NtNTP2 polynucleotides in each cell division. Plants or plant cells can be transiently transformed so that the recombinant NtNTP2 polynucleotides are not integrated into its genome. Transiently transformed cells typically lose all or part of the introduced recombinant NtNTP2 polynucleotides in each cell division, so that after a sufficient number of cell divisions, the introduced recombinant NtNTP2 polynucleotides cannot be detected in daughter cells.

[0147] Many methods in this area can be used for transforming plant cells, including biolistics, gene gun technology, Agrobacterium-mediated transformation, viral vector-mediated transformation, freeze-thaw method, microprojectile bombardment, direct DNA uptake, ultrasonic treatment, microinjection, plant virus-mediated transfer and electroporation. The Agrobacterium system for integrating foreign DNA into plant chromosomes has been widely studied, modified and developed for plant genetic engineering. By conventional methods, naked recombinant DNA molecules are connected to appropriate T-DNA sequences, and the naked recombinant DNA molecules include DNA sequences corresponding to the subject purified polypeptides that are operably connected to regulatory sequences in sense or antisense directions. These are introduced into protoplasts by polyethylene glycol technology or electroporation technology, and both technologies are standard. Alternatively, such vectors containing recombinant DNA molecules encoding purified polypeptides of the present invention are introduced into living Agrobacterium cells and then transferred into plant cells. Transformation by naked DNA without accompanying T-DNA carrier sequences can be completed via fusion of protoplasts with DNA-containing liposomes or via electroporation. Naked DNA without accompanying T-DNA carrier sequences can also be used for transforming cells via inert, high-speed microprojectiles.

[0148] If cells or cultured tissue are used as the recipient tissue for transformation, plants can be regenerated from the transformed culture, if desired, by techniques known to those skilled in the art.

[0149] The selection of regulatory regions to be included in the recombinant construct depends on several factors, including but not limited to efficiency, selectivity, inducibility, desired expression level and cell or tissue preferential expression. By appropriately selecting the regulatory region and placing the regulatory region relative to the coding sequence, regulating the expression of the coding sequence is routine work for those skilled in the art. The transcription of NtNTP2 polynucleotides can be regulated in a similar manner. Some suitable regulatory regions initiate transcription only or predominantly in certain cell types. Methods for identifying and characterizing regulatory regions in plant genomic DNA are known in the art.

[0150] Suitable promoters include tissue-specific promoters recognized by tissue-specific factors, which are present in different tissues or cell types (e.g., root-specific promoters, branch-specific promoters, xylem-specific promoters), or present during different developmental stages, or present in response to different environmental conditions. Suitable promoters include constitutive promoters, which can be activated in most cell types without the need for specific inducing agents. Examples of suitable promoters for controlling RNAi polypeptide production include cauliflower mosaic virus 35S (CaMV / 35S), SSU, OCS, lib4, usp, STLS1, B33, nos, or ubiquitin or phaseolin promoters. Those skilled in the art can produce multiple variants of recombinant promoters.

[0151] Tissue-specific promoters are transcription control elements that are active only at a specific time during plant development in a specific cell or tissue (such as in a vegetative tissue or reproductive tissue). For example, when the expression of a polynucleotide in certain tissues is preferred, tissue-specific expression can be advantageous. Examples of tissue-specific promoters under developmental control include promoters that can only (or mainly only) initiate transcription in certain tissues, such as vegetative tissues (e.g., roots or leaves) or reproductive tissues (such as fruit, ovules, seeds, pollen, pistils, flowers, or any embryonic tissues). Reproductive tissue-specific promoters can be, for example, anther-specific, ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed and seed coat-specific, pollen-specific, petal-specific, sepal-specific, or a combination thereof.

[0152] Suitable leaf-specific promoters include the pyruvate orthophosphate dikinase (PPDK) promoter from a C4 plant (maize), the cab-m1Ca+2 promoter from maize, the Arabidopsis thaliana myb-related gene promoter (Atmyb5), the ribulose bisphosphate carboxylase (RBCS) promoter (e.g., the tomato RBCS1, RBCS2, and RBCS3A genes expressed in leaves and light-grown seedlings, RBCS1 and RBCS2 expressed in developing tomato fruit, or the ribulose bisphosphate carboxylase promoter expressed almost exclusively at high levels in mesophyll cells of leaves and sheaths).

[0153] Suitable senescence-specific promoters include tomato promoters active during fruit ripening, leaf wilting and abscission, maize promoters of genes encoding cysteine ​​proteases, promoters of 82E4, and promoters of SAG genes. Suitable anther-specific promoters may be used. Suitable root-preferred promoters known to those skilled in the art may be selected. Suitable seed-preferred promoters include seed-specific promoters (those active during seed development, such as promoters of seed storage polypeptides) and seed germination promoters (those active during seed germination). Such seed-preferred promoters include Cim1 (cytokinin-induced messenger); cZ19B1 (maize 19 kDa zein); milps (inositol-1-phosphate synthase); mZE40-2, also known as Zm-40; nuclc; and celA (cellulose synthase). γ-zein is an endosperm-specific promoter. Glob-1 is an embryo-specific promoter. For dicotyledons, seed-specific promoters include bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include the maize 15 kDa zein promoter, the 22 kDa zein promoter, the 27 kDa zein promoter, the g-zein promoter, the 27 kDa γ-zein promoter (such as the gzw64A promoter, see Genbank Accession No. S78780), the waxy promoter, the shrunken 1 promoter, the shrunken 2 promoter, the globulin 1 promoter (see Genbank Accession No. L22344), the Itp2 promoter, the cim1 promoter, the maize end1 and end2 promoters, the nuc1 promoter, the Zm40 promoter, eep1 and eep2; lec1, the thioredoxin H promoter; the mlip15 promoter, the PCNA2 promoter; and the shrunken-2 promoter.

[0154] Examples of inducible promoters include promoters that respond to pathogen attack, anaerobic conditions, high temperature, light, drought, cold temperature or high salt concentration. Pathogen-inducible promoters include promoters from polypeptides associated with pathogenesis (PR polypeptides), which are induced after infection by pathogens (e.g., PR polypeptides, SAR polypeptides, β-1,3-glucanase, chitinase).

[0155] In addition to plant promoters, other suitable promoters may be derived from bacterial sources, for example, the octopine synthase promoter, the nopaline synthase promoter, and other promoters from the Ti plasmid, or may be derived from viral promoters (e.g., the 35S and 19S RNA promoters of the cauliflower mosaic virus (CaMV), the constitutive promoter of the tobacco mosaic virus, the cauliflower mosaic virus (CaMV) 19S and 35S promoters, or the figwort mosaic virus 35S promoter).

[0156] Suitable methods for introducing polynucleotides into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al., Biotechniques 4:320-334 (1986)), electroporation (Riggs et al., Proc. Natl. Acad. Sci. USA 83:5602-5606 (1986)), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,981,840 and 5,563,055), direct gene transfer (Paszkowski et al., EMBO J. 3:2717-2722 (1984)), and ballistic particle acceleration (see, e.g., U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al., Plant Cell, Tissue, and Organ Culture: Fundamental Biology, 1996). Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin) (1995); and McCabe et al., Biotechnology 6:923-926 (1988)).

[0157] Disclosed are plants or plant cells comprising mutations in one or more NtNTP2 polynucleotides or NtNTP2 polypeptides as described herein, wherein the mutations result in a modulated function or activity of NtNTP2 (including a complete loss of function or activity). Suitably, the mutations reduce or inhibit the expression or activity of NtNTP2. In addition to the specific mutations described herein, the mutant plants or plant cells may have one or more other mutations in the same NtNTP2 polynucleotides or NtNTP2 polypeptides as described herein or in one or more other polynucleotides or polypeptides within the genome. In addition to mutations, the skilled person may also utilize other techniques to modulate the function or activity of NtNTP2, as described in detail herein.

[0158] Also provided is a method for modulating the level of NtNTP2 polypeptides in a plant or plant material, the method comprising introducing one or more mutations into the genome of the plant that modulate the expression of at least one NtNTP2 gene, wherein the at least one NtNTP2 gene is selected from the sequences according to the present disclosure.

[0159] Also provided is a method for identifying a plant that does not have reduced nitrate levels compared to a control plant grown under the same fertilization conditions; and has increased biomass compared to a control plant grown under the same fertilization conditions; and has an increased NUE response compared to a control plant grown under the same fertilization conditions, the method comprising screening a polynucleotide sample of a plant of interest for the presence of one or more mutations in the NtNTP2 sequence described herein. The plant may also have an increase in root development compared to a control plant grown under the same fertilization conditions.

[0160] Also disclosed are plants or plant cells that are heterozygous or homozygous for one or more mutations in one or more NtNTP2 genes according to the disclosure, wherein the mutations result in modulated expression of the NtNTP2 gene or function or activity of the NtNTP2 polypeptide encoded thereby.

[0161] A large number of methods can be used to combine mutations in a plant, including sexual crossing. Plants having one or more favorable heterozygous or homozygous mutations in the NtNTP2 gene (which modulate the expression of the NtNTP2 gene or the function or activity of the NtNTP2 polypeptide encoded therefrom) can be crossed with plants having one or more favorable heterozygous or homozygous mutations in one or more other genes (which modulate the expression of the gene or the function or activity of the polypeptide encoded therefrom). In one embodiment, crossing is performed to introduce one or more favorable heterozygous or homozygous mutations in the NtNTP2 gene of the same plant.

[0162] A function or activity is increased or decreased if the function or activity of one or more NtNTP2 polypeptides of the present disclosure in a plant is lower or higher than the function or activity of the same polypeptide in a plant that has not been modified to modulate the function or activity of the polypeptide and has been grown, harvested and optionally dried using the same protocol.

[0163] In some embodiments, mutations are introduced into plants or plant cells using a mutagenesis method, and the introduced mutations are identified or selected using methods known to those skilled in the art such as Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis. Mutations that affect NtNTP2 gene expression or interfere with the function of the encoded NtNTP2 polypeptide can be determined using methods well known in the art. Insertion mutations in NtNTP2 gene exons typically result in null mutations. Mutations in conserved residues can be particularly effective in reducing or inhibiting the metabolic function of the encoded NtNTP2 polypeptide. For example, it should be understood that mutations in one or more highly conserved regions may change the function of the NtNTP2 polypeptide, while mutations outside those highly conserved regions may have little or no effect on the function of the NtNTP2 polypeptide. In addition, mutations in single nucleotides can produce stop codons, which will result in truncated NtNTP2 polypeptides, and depending on the degree of truncation, reduced or completely lost function. Suitably, the mutation results in reduced expression of NtNTP2 and reduced biological function of NtNTP2, more suitably, the mutation results in complete loss of expression of NtNTP2 and complete loss of biological function of NtNTP2.

[0164] Also disclosed are methods for obtaining mutant NtNTP2 polynucleotides and mutant NtNTP2 polypeptides. Any target plant, including plant cells or plant materials or plant leaves, can be genetically modified by a variety of known methods of inducing mutagenesis, including site-directed mutagenesis, oligonucleotide-directed mutagenesis, chemically induced mutagenesis, radiation-induced mutagenesis, mutagenesis using modified bases, mutagenesis using gapped duplex DNA, double-strand break mutagenesis, mutagenesis using repair-deficient host strains, mutagenesis by total gene synthesis, DNA shuffling and other equivalent methods.

[0165] The mutant NtNTP2 polypeptide can be used to prepare a mutant, non-naturally occurring or transgenic plant (e.g., a mutant, non-naturally occurring, transgenic, artificial or genetically engineered plant) comprising one or more mutant polypeptides. Suitably, the mutant NtNTP2 polypeptide can retain all, part or no function of the unmutated polypeptide. Thus, the function of the mutant NtNTP2 polypeptide may be higher, lower or substantially the same as that of the unmutated polypeptide. In certain embodiments, it is preferred that the mutant NtNTP2 polypeptide has reduced function or does not retain any function.

[0166] Mutations in NtNTP2 polynucleotides and NtNTP2 polypeptides may include artificial mutations or synthetic mutations or genetically engineered mutations. Mutations in NtNTP2 polynucleotides and NtNTP2 polypeptides described herein may be mutations obtained or obtainable by a process including in vitro or in vivo manipulation steps. Mutations in NtNTP2 polynucleotides and NtNTP2 polypeptides described herein may be mutations obtained or obtainable by a process including human intervention.

[0167] Methods for randomly introducing mutations in polynucleotides may include chemical mutagenesis and radiomutagenesis. Chemical mutagenesis involves the use of exogenously added chemicals (such as mutagenic, teratogenic or carcinogenic organic compounds) to induce mutations. Mutagens (including chemical mutagens or radiation) that primarily produce point mutations and short deletions, insertions, missense mutations, simple sequence repeats, transversions and / or conversions can be used to produce mutations. Mutagens include ethyl methanesulfonate, methyl methanesulfonate, N-ethyl-N-nitrosourea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12-dimethyl-benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, busulfan, diepoxyalkanes (diepoxyoctane, diepoxybutane, etc.), 2-methoxy-6-chloro-9-[3-(ethyl-2-chloro-ethyl)aminopropylamino]acridine dihydrochloride and formaldehyde.

[0168] It is also envisioned that spontaneous mutations in the locus may not be directly caused by a mutagen, as long as they produce a desired phenotype. Suitable mutagenic agents may also include, for example, ionizing radiation, such as X-rays, gamma rays, fast neutron irradiation, and UV radiation. For each type of plant tissue, the dosage of the mutagenic chemical or radiation is determined experimentally so as to obtain a mutation frequency below a threshold level characterized by lethality or reproductive sterility. Any plant polynucleotide preparation method known to those skilled in the art can be used to prepare plant polynucleotides for mutation screening.

[0169] The mutation process may include one or more plant crossing steps.

[0170] After mutation, screening can be performed to identify mutations that produce premature stop codons or non-functional genes. After mutation, screening can be performed to identify mutations that produce functional genes that can be expressed at increased or reduced levels. The screening of mutants can be carried out by sequencing or by using one or more probes or primers specific to the NtNTP2 gene or NtNTP2 polypeptide. Specific mutations can also be produced in NtNTP2 polynucleotides, which can lead to regulated NtNTP2 gene expression, regulated mRNA stability or regulated polypeptide stability. Such plants are referred to as "non-naturally occurring" or "mutant" plants in this article. Typically, mutant or non-naturally occurring plants will include at least a portion (e.g., DNA or RNA) of foreign or synthetic or artificial nucleotides that did not exist in the plant before being manipulated. The foreign nucleotides can be a single nucleotide, two or more nucleotides, two or more consecutive nucleotides, or two or more non-contiguous nucleotides, for example at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 or more consecutive or non-contiguous nucleotides.

[0171] In addition to mutagenesis, compositions that can modulate the expression or function or activity of one or more NtNTP2 polynucleotides or NtNTP2 polypeptides include sequence-specific polynucleotides that can interfere with the transcription of one or more endogenous genes; sequence-specific polynucleotides that can interfere with the translation of RNA transcripts (e.g., double-stranded RNA, siRNA, ribozymes); sequence-specific polypeptides that can interfere with the stability of one or more polypeptides; sequence-specific polynucleotides that can interfere with the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides relative to substrates or regulatory polypeptides; antibodies that show specificity for one or more polypeptides; small molecule compounds that can interfere with the stability of one or more polypeptides or the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides; zinc finger polypeptides that bind to one or more polynucleotides; and large-range nucleases that are functional to one or more polynucleotides. Gene editing technology, genetic editing technology, and genome editing technology are well known in the art.

[0172] Zinc finger polypeptides can be used to regulate the expression or function or activity of one or more NtNTP2 polynucleotides described herein. In multiple embodiments, a genomic DNA sequence comprising a portion or all of the NtNTP2 polynucleotide coding sequence is modified by zinc finger nuclease-mediated mutagenesis. Unique sites for zinc finger polypeptide binding are searched in the genomic DNA sequence. Alternatively, two unique sites for zinc finger polypeptide binding are searched in the genomic DNA sequence, wherein the two sites are on opposite chains and close together, for example, 1, 2, 3, 4, 5, 6 or more base pairs apart. Therefore, zinc finger polypeptides that bind to NtNTP2 polynucleotides are provided. Zinc finger polypeptides can be engineered to recognize selected target sites in genes. Zinc finger polypeptides can include any combination of motifs derived from natural zinc finger DNA binding domains and non-natural zinc finger DNA binding domains by truncating or extending or site-directed mutagenesis processes in conjunction with selection methods, such as, but not limited to, phage display selection, bacterial two-hybrid selection, or bacterial single-hybrid selection. The term "non-natural zinc finger DNA binding domain" refers to a zinc finger DNA binding domain that binds to a three base pair sequence within a polynucleotide target and is not present in the cell or organism containing the polynucleotide to be modified. Methods for designing zinc finger polypeptides that bind to specific polynucleotides unique to a target gene are known in the art.

[0173] In other embodiments, zinc finger polypeptides can be selected to bind to regulatory sequences of NtNTP2 polynucleotides. More specifically, regulatory sequences can include transcription start sites, start codons, exon regions, exon-intron boundaries, terminators, or stop codons. Accordingly, the present disclosure provides mutants, non-naturally occurring, or transgenic plants or plant cells produced by zinc finger nuclease-mediated mutagenesis near or within one or more NtNTP2 polynucleotides described herein, and methods for preparing such plants or plant cells by zinc finger nuclease-mediated mutagenesis. The methods for delivering zinc finger polypeptides and zinc finger nucleases to plants are similar to the methods described below for delivering large-range nucleases.

[0174] In another aspect, methods for producing mutant, non-naturally occurring or transgenic or otherwise genetically modified plants using meganucleases such as I-Crel are described. Naturally occurring meganucleases as well as recombinant meganucleases can be used to specifically cause double-strand breaks at a single site or a relatively small number of sites in plant genomic DNA to allow destruction of one or more NtNTP2 polynucleotides described herein. The meganuclease can be an engineered meganuclease with altered DNA recognition properties. The meganuclease polypeptide can be delivered to plant cells by a variety of different mechanisms known in the art.

[0175] The present invention encompasses the use of meganucleases to inactivate one or more NtNTP2 polynucleotides described herein (or any combination thereof as described herein) in a plant cell or plant. Specifically, the present disclosure provides a method for inactivating NtNTP2 polynucleotides in a plant using a meganuclease, comprising: a) providing a plant cell comprising an NtNTP2 polynucleotide as described herein; (b) introducing a meganuclease or a construct encoding a meganuclease into the plant cell; and (c) allowing the meganuclease to substantially inactivate the NtNTP2 polynucleotide.

[0176] Meganuclease can be used to cut the meganuclease recognition site in the coding region of NtNTP2 polynucleotide. This type of cutting usually results in DNA deletion at the meganuclease recognition site after the mutagenic DNA repair connected by non-homologous ends. This type of mutation in the gene coding sequence is usually enough to inactivate the gene. This method of modifying plant cells first involves using a suitable transformation method to deliver the meganuclease expression cassette to the plant cell. For maximum efficiency, it is expected that the meganuclease expression cassette will be connected to a selectable marker, and the cells successfully transformed are selected in the presence of a selection agent. This method allows the meganuclease expression cassette to be integrated into the genome, but if plant may need regulatory approval, this may be undesirable. In this case, using conventional breeding techniques, the meganuclease expression cassette (and the selectable marker gene connected) can be separated in subsequent plant generations.

[0177] After delivery of the meganuclease expression cassette, the plant cells are initially grown under conditions typical for the specific transformation procedure used. This may mean growing the transformed cells on culture medium at a temperature below 26°C, usually in the dark. Such standard conditions may be used for a period of time, suitably 1-4 days, to allow the plant cells to recover from the transformation process. At any time point after this initial recovery period, the growth temperature may be raised to stimulate the function of the engineered meganuclease cleavage and mutation meganuclease recognition sites.

[0178] A gene editing method involves the use of transcription activator-like effector nucleases (TALENs), which induce double-strand breaks to which cells can repair mechanisms. NHEJ reconnects DNA from either side of the double-strand break, with little or no sequence overlap for annealing. This repair mechanism induces errors in the genome via insertion or deletion, or chromosome rearrangement. Any such error can render the gene product encoded at the position non-functional. For some applications, it may be necessary to precisely remove NtNTP2 polynucleotides from the plant genome. Such applications may use a pair of engineered meganucleases that each cut a meganuclease recognition site on either side of the expected deletion. TALENs that can recognize and bind genes and introduce double-strand breaks into the genome can also be used. Therefore, on the other hand, methods for producing mutant, non-naturally occurring, or transgenic or otherwise genetically modified plants as described herein are contemplated.

[0179] Another gene editing approach involves the use of bacterial CRISPR / Cas systems. Bacteria and archaea display chromosomal elements called clustered regularly interspaced short palindromic repeats (CRISPR), which are part of the adaptive immune system and protect against invading viruses and plasmid DNA. In the type II CRISPR system, CRISPR RNA (crRNA) works together with trans-activating crRNA (tracrRNA) and CRISPR-associated (Cas) polypeptides to introduce double-strand breaks in the target DNA. Target cleavage by Cas9 requires base pairing between crRNA and tracrRNA, as well as base pairing between crRNA and target DNA. Target recognition is facilitated by the presence of a short motif called a protospacer-adjacent motif (PAM), which conforms to the sequence NGG. This system can be used for genome editing. Cas9 usually works by programming with a dual RNA consisting of a crRNA and a tracrRNA. However, the core components of these RNAs can be combined into a single hybrid 'guide RNA' for Cas9 targeting. The use of non-coding RNA guides for site-specific cleavage of target DNA is expected to be significantly more straightforward than existing technologies (such as TALEN). Using the CRISPR / Cas strategy, re-targeting the nuclease complex only requires the introduction of a new RNA sequence without redesigning the specificity of the polypeptide transcription factor. CRISPR / Cas technology is implemented in plants by the method of international application WO 2015 / 189693 A1, which discloses a virus-mediated genome editing platform that is widely applicable to plant species. The RNA2 genome of tobacco rattle virus (TRV) is designed to carry guide RNA and deliver it to Nicotiana benthamiana plants that overexpress Cas9 endonuclease. In the context of the present disclosure, the guide RNA can be derived from any sequence disclosed herein, and the teachings of WO 2015 / 189693A1 are applicable to editing the genome of plant cells and obtaining desired mutant plants. The rapid development of this technology has resulted in a variety of protocols with broad applicability in plants, which have been well catalogued in many recent scientific review articles (e.g., Schiml et al., Plant Methods 2016 12:8; and Khatodia et al., Front Plant Sci. 2016;7:506).Bortesi and Fischer (Biotechnology Advances (2015) 33, 1, 41-52) give a review of the CRISPR / Cas system, with a particular focus on applications in plants. Bortesi and Fischer also made a comparison between CRISPR / Cas technology, zinc finger nucleases, and TALENs. Liu et al. (2017) Acta Pharmaceutica Sinica B 7, 3, 292-302 and Curr. Op. in Plant Biol discuss recent advances in the use of CRISPR / Cas for manipulating plant genomes. (2017) 36, 1–8 discusses updated advances in the use of CRISPR / Cas for manipulating plant genomes. CRISPR / Cas9 plasmids for plants are listed in the nonprofit plasmid repository (addgene.org) “addgene”, and CRISPR / Cas plasmids are commercially available.

[0180] Antisense technology is another well-known method that can be used to regulate the expression or activity of NtNTP2 polypeptides. The polynucleotide of the gene to be inhibited is cloned and operably linked to the regulatory region and the transcription termination sequence so that the antisense strand of RNA is transcribed. The recombinant construct is then transformed into plant cells and the antisense strand of RNA is produced. The polynucleotide need not be the entire sequence of the gene to be inhibited, but is generally substantially complementary to at least a portion of the sense strand of the gene to be inhibited.

[0181] Polynucleotides can be transcribed into ribozymes, or catalytic RNAs, which affect the expression of mRNA. Ribozymes can be designed to specifically pair with almost any target RNA, and cut the phosphodiester backbone at a specific position, thereby inactivating the target RNA function. Heterologous polynucleotides can encode ribozymes designed to cut specific mRNA transcripts, thereby preventing the expression of polypeptides. Hammerhead ribozymes can be used to destroy specific mRNAs, but multiple ribozymes that cut mRNA at site-specific recognition sequences can be used. Hammerhead ribozymes cut mRNA at the position indicated by the flanking region, and the flanking region forms a complementary base pair with the target mRNA. The only requirement is that the target RNA contains 5'-UG-3' polynucleotides. The construction and generation of hammerhead ribozymes are known in the art. The hammerhead ribozyme sequence can be embedded in a stable RNA such as a transfer RNA (tRNA) to increase the efficiency of in vivo cutting.

[0182] In one embodiment, the sequence-specific polynucleotide that can interfere with the translation of RNA transcripts is an interfering RNA. RNA interference or RNA silencing is an evolutionarily conservative process, and specific mRNA can be targeted for enzymatic degradation by it. A double-stranded RNA (double-stranded RNA) is introduced or produced by a cell (such as a double-stranded RNA virus or an interfering RNA polynucleotide) to start an interfering RNA approach. Double-stranded RNA can be converted into multiple small interfering RNA (siRNA) duplexes of 21-24bp in length by RNase III, and RNase III is a double-stranded RNA specific endonuclease. siRNA can be subsequently recognized by the RNA-induced silencing complex, and the silencing complex promotes the unwinding of siRNA by an ATP-dependent process. The unwrapped antisense strand of siRNA guides the activated RNA-induced silencing complex to the target mRNA, and the target mRNA comprises a sequence complementary to the siRNA antisense strand. Targeted mRNA and antisense strand can form an A-shaped spiral, and the major groove of the A-shaped spiral can be recognized by the activated RNA-induced silencing complex. Target mRNA can be cut by the RNA-induced silencing complex at a single site defined by the binding site at the 5' end of the siRNA chain. The activated RNA-induced silencing complex can be recycled to catalyze another cleavage event.

[0183] Interfering RNA expression vectors may include interfering RNA constructs encoding interfering RNA polynucleotides, which exhibit RNA interference by reducing the expression level of mRNA, pre-mRNA or related RNA variants. The expression vector may include a promoter placed upstream of the interfering RNA construct and operably connected to the interfering RNA construct, as further described herein. The interfering RNA expression vector may include a suitable minimal core promoter, an interfering RNA construct of interest, an upstream (5') regulatory region, a downstream (3') regulatory region, including transcription termination and polyadenylation signals, and other sequences known to those skilled in the art, such as a variety of selection markers.

[0184] Double-stranded RNA molecules may include siRNA molecules assembled from a single oligonucleotide in a stem-loop structure, wherein the self-complementary sense and antisense regions of the siRNA molecule are connected by a polynucleotide-based or non-polynucleotide-based linker, and circular single-stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands, wherein the circular RNA can be processed in vivo or in vitro to produce active siRNA molecules capable of mediating RNA interference.

[0185] The use of small hairpin RNA molecules is also contemplated. Except for the reverse complementary (meaning) sequence, they also include specific antisense sequences, which are usually separated by spacers or loop sequences. The cutting of spacers or loops provides single-stranded RNA molecules and their reverse complementary sequences so that they can anneal to form double-stranded RNA molecules (optionally with additional processing steps, which can result in the addition or removal of one, two, three or more nucleotides from the 3' end or 5' end of any one or two chains). Spacers can have sufficient lengths to allow antisense and sense sequences to anneal and form double-stranded structures (or stems) before spacer cutting (and optionally, the subsequent processing steps of the addition or removal of one, two, three, four or more nucleotides from the 3' end or 5' end of any one or two chains). Spacer sequences are typically unrelated polynucleotides located between two complementary polynucleotide regions, and when annealed to double-stranded polynucleotides, the two complementary polynucleotide regions comprise small hairpin RNAs. Spacer sequences generally include about 3 to about 100 nucleotides.

[0186] Can be by selecting suitable sequence composition, ring size and stem length for producing hairpin duplex, produce any purpose RNA polynucleotide.The suitable range for designing the stem length of hairpin duplex comprises the stem length of at least about 10,11,12,13,14,15,16,17,18,19 or 20 nucleotide, such as about 14-30 nucleotide, about 30-50 nucleotide, about 50-100 nucleotide, about 100-150 nucleotide, about 150-200 nucleotide, about 200-300 nucleotide, about 300-400 nucleotide, about 400-500 nucleotide, about 500-600 nucleotide and about 600-700 nucleotide.The suitable range for designing the ring length of hairpin duplex comprises about 4-25 nucleotide, about 25-50 nucleotide or if the stem length of hairpin duplex is quite large, longer ring length so. In certain embodiments, the double-stranded RNA or ssRNA molecule length is between about 15 and about 40 nucleotides. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule with a length between about 15 and about 35 nucleotides. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule with a length between about 17 and about 30 nucleotides. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule with a length between about 19 and about 25 nucleotides. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule with a length between about 21 to about 23 nucleotides. In certain embodiments, the hairpin structure with a duplex region greater than 21 nucleotides can promote effective siRNA directional silencing, regardless of the loop sequence and length. The exemplary sequences of RNA interference are described herein.

[0187] The target mRNA sequence is usually between about 14 and about 50 nucleotides in length. Therefore, the target mRNA can be scanned for a region between about 14 and about 50 nucleotides in length, which region suitably meets one or more of the following criteria: the ratio of A+T / G+C is between about 2:1 and about 1:2; AA dinucleotides or CA dinucleotides at the 5' end; a sequence of at least 10 consecutive nucleotides that is unique to the target mRNA (i.e., the sequence is not present in other mRNA sequences from the same plant); and there is no "run" of more than three consecutive guanine (G) nucleotides or more than three consecutive cytosine (C) nucleotides. These criteria can be evaluated using various techniques known in the art, for example, computer programs such as BLAST can be used to search publicly available databases to determine whether the selected sequence is unique to the target mRNA. Alternatively, commercially available computer software (e.g., commercially available OligoEngine, Target Finder and siRNA design tools) can be used to select sequences (and design siRNA sequences).

[0188] In one embodiment, select a target mRNA sequence that meets one or more of the above criteria and has a length between about 14 and about 30 nucleotides. In another embodiment, select a sequence that meets one or more of the above criteria and has a length between about 16 and about 30 nucleotides. In yet another embodiment, select a sequence that meets one or more of the above criteria and has a length between about 19 and about 30 nucleotides. In another embodiment, select a sequence that meets one or more of the above criteria and has a length between about 19 and about 25 nucleotides.

[0189] In an exemplary embodiment, the siRNA molecule comprises a specific antisense sequence that is complementary to at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides of any one of the polynucleotides described herein.

[0190] The specific antisense sequence included in the siRNA molecule can be identical or substantially identical to the complement. In one embodiment, the specific antisense sequence included in the siRNA molecule is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the complement of the target mRNA sequence. Methods for determining sequence identity are known in the art and can be determined, for example, by using the BLASTN program provided on the University of Wisconsin Computer Group (GCG) software or the NCBI website.

[0191] One method of inducing double-stranded RNA silencing in plants is to transform with a gene construct that produces hairpin RNA (see Nature (2000) 407, 319-320). This type of construct includes a reverse region of the target gene sequence separated by an appropriate spacer. Due to the production of intron splicing hairpin RNA, the insertion of functional plant intron regions as spacer fragments also improves the efficiency of gene silencing induction (Plant J. (2001), 27, 581-590). Suitably, the stem length is about 50 nucleotides to about 1 kilobase length. The method for producing intron splicing hairpin RNA is fully described in the art (see, for example, Bioscience, Biotechnology, and Biochemistry (2008) 72, 2, 615-617).

[0192] Interfering RNA molecules with duplex or double-stranded structures, such as double-stranded RNA or small hairpin RNA, may have blunt ends, or may have 3' or 5' overhangs. As used herein, "overhangs" refer to one or more unpaired nucleotides that protrude from the duplex structure when the 3' end of one RNA chain extends beyond the 5' end of the other chain (3' overhang), or vice versa (5' overhang). The nucleotides comprising the overhangs may be ribonucleotides, deoxyribonucleotides, or modified forms thereof. In one embodiment, at least one chain of the interfering RNA molecule has a 3' overhang of about 1 to about 6 nucleotides in length. In other embodiments, the 3' overhang length is about 1 to about 5 nucleotides, about 1 to about 3 nucleotides, and about 2 to about 4 nucleotides.

[0193] When the interfering RNA molecule includes a 3' overhang at one end of the molecule, the other end can be blunt-ended or also have an overhang (5' or 3'). When the interfering RNA molecule includes an overhang at both ends of the molecule, the length of the overhang can be the same or different. In one embodiment, the interfering RNA molecule includes a 3' overhang of about 1 to about 3 nucleotides at both ends of the molecule. In another embodiment, the interfering RNA molecule is a double-stranded RNA with a 3' overhang of 2 nucleotides at both ends of the molecule. In another embodiment, the nucleotides constituting the overhang of the interfering RNA are TT dinucleotides or UU dinucleotides.

[0194] Interfering RNA molecules may include one or more 5' or 3' cap structures.The term "cap structure" refers to a chemical modification incorporated at either end of an oligonucleotide that protects the molecule from exonuclease degradation and may also facilitate delivery or localization within a cell.

[0195] Another modification suitable for interfering RNA molecules is to chemically connect one or more parts or conjugates to the interfering RNA molecules, which enhance the function, cellular distribution, cellular uptake, bioavailability or stability of the interfering RNA molecules. Polynucleotides can be synthesized or modified by methods fully determined in the art. Chemical modifications include 2' modifications, introduction of non-natural bases, covalent attachment to ligands, and replacement of phosphate bonds with thiophosphate bonds. In this embodiment, the integrity of the duplex structure is strengthened by at least one, and typically two chemical bonds.

[0196] The nucleotides of one or both of the two single strands can be modified to regulate the activation of cellular enzymes, such as but not limited to certain nucleases. Techniques for reducing or inhibiting the activation of cellular enzymes are known in the art, including but not limited to 2'-amino modifications, 2'-fluoro modifications, 2'-alkyl modifications, uncharged backbone modifications, morpholino modifications, 2'-O-methyl modifications, and phosphoramidates.

[0197] The ligand can be bound to the interfering RNA molecule, for example to enhance its cellular uptake. In certain embodiments, a hydrophobic ligand is bound to the molecule to facilitate direct penetration of the cell membrane. In some cases, the binding of a cationic ligand to an oligonucleotide generally results in improved resistance to nucleases.

[0198] "Targeted Induced Local Lesions in the Genome" (TILLING) is another mutagenesis technique that can be used to generate and / or identify NtNTP2 polynucleotides encoding NtNTP2 polypeptides with modified expression, function or activity. TILLING also allows the selection of plants carrying such mutants. TILLING combines high-density mutagenesis with high-throughput screening methods. Methods for TILLING are well known in the art (see McCallum et al., (2000) Nat Biotechnol 18:455-457 and Stemple (2004) Nat Rev Genet 5(2):145-50).

[0199] Various embodiments relate to expression vectors comprising one or more NtNTP2 polynucleotides described herein or interfering RNA constructs comprising one or more NtNTP2 polynucleotides.

[0200] Various embodiments relate to expression vectors comprising one or more of the NtNTP2 polynucleotides described herein or one or more interfering RNA constructs.

[0201] Multiple embodiments relate to expression vectors comprising one or more NtNTP2 polynucleotides described herein or one or more interfering RNA constructs encoding one or more interfering RNA polynucleotides capable of self-annealing to form a hairpin structure, wherein the construct comprises (a) one or more of the NtNTP2 polynucleotides described herein; (b) a second sequence encoding a spacer element that forms a loop of the hairpin structure; and (c) a third sequence disposed in the same orientation as the first sequence and comprising a reverse complement of the first sequence, wherein the second sequence is disposed between the first sequence and the third sequence, and the second sequence is operably linked to the first sequence and the third sequence.

[0202] The disclosed sequences can be used to construct a variety of NtNTP2 polynucleotides that do not form hairpin structures. For example, double-stranded RNA can be formed by (1) transcribing a first strand of DNA by being operably linked to a first promoter, and (2) transcribing the reverse complement of the first strand of a DNA fragment by being operably linked to a second promoter. Each strand of the polynucleotide can be transcribed by the same expression vector or by different expression vectors. RNA duplexes with RNA interference can be enzymatically converted into siRNA to modulate RNA levels.

[0203] Thus, various embodiments relate to expression vectors comprising one or more NtNTP2 polynucleotides described herein or an interfering RNA construct encoding an interfering RNA polynucleotide capable of self-annealing, wherein the construct comprises (a) one or more of the NtNTP2 polynucleotides described herein; and (b) a second sequence comprising a complementary (e.g., reverse complementary) sequence of the first sequence, placed in the same orientation as the first sequence.

[0204] Provided are various compositions and methods for modulating the endogenous expression level of one or more of the NtNTP2 polypeptides described herein (or any combination thereof as described herein) by promoting co-suppression of gene expression.

[0205] Various compositions and methods are provided for regulating the expression level of endogenous genes by regulating the translation of mRNA. A host plant cell can be transformed with an expression vector comprising: a promoter operably linked to an NtNTP2 polynucleotide, the polynucleotide being placed in an antisense orientation with respect to the promoter to allow expression of an RNA polynucleotide having sequence complementarity to a portion of the mRNA.

[0206] A variety of expression vectors for regulating mRNA translation can include: a promoter operably linked to an NtNTP2 polynucleotide, wherein the sequence is placed in an antisense orientation with respect to the promoter. The length of the antisense RNA polynucleotide can vary and can be about 15-20 nucleotides, about 20-30 nucleotides, about 30-50 nucleotides, about 50-75 nucleotides, about 75-100 nucleotides, about 100-150 nucleotides, about 150-200 nucleotides, and about 200-300 nucleotides.

[0207] Alternatively, the targeted gene can be inactivated by introducing a transposon (e.g., IS element) into the genome of the target plant. These mobile genetic factors can be introduced by sexual cross fertilization, and insertion mutants can be screened for the loss of polypeptide function. By, for example, sexual cross fertilization, by hybridizing the parent plant with a plant that does not implement transposon-induced mutagenesis, the disrupted gene in the parent plant can be introduced into other plants. Any standard breeding techniques known to those skilled in the art can be utilized. In one embodiment, one or more genes can be inactivated by inserting one or more transposons. Mutation may result in homozygous destruction of one or more genes, heterozygous destruction of one or more genes, or if the destruction exceeds one gene, a combination of homozygous and heterozygous destruction may result. Suitable transposable elements include retrotransposons, retrotransposons, and SINE-like elements. Such methods are known to those skilled in the art.

[0208] Alternatively, targeted genes can be inactivated by introducing ribozymes derived from many small circular RNAs into plants, which are capable of self-cutting and replication. These RNAs can replicate alone (viroid RNA) or with helper viruses (satellite RNA). Examples of suitable RNAs include those derived from avocado day spot viroids, and satellite RNAs derived from tobacco ringspot virus, alfalfa transient stripe virus, velvety tobacco mottle virus, scopolamine mottle virus, and underground clover mottle virus. A variety of target RNA-specific ribozymes are known to those skilled in the art.

[0209] A mutant or non-naturally occurring plant or plant cell may have any combination of one or more mutations in one or more genes that result in regulation of the expression or function or activity of those genes or their products. For example, a mutant or non-naturally occurring plant or plant cell may have a single mutation in a single gene; multiple mutations in a single gene; a single mutation in two or more or three or more or four or more genes; or multiple mutations in two or more or three or more or four or more genes. Examples of these mutations are described herein. As another example, a mutant or non-naturally occurring plant or plant cell may have one or more mutations in a specific portion of a NtNTP2 gene (such as in a region of a gene encoding an active site of a NtNTP2 polypeptide or a portion thereof). As another example, a mutant or non-naturally occurring plant or plant cell may have one or more mutations in a region outside of one or more NtNTP2 genes (such as in an upstream or downstream region of a gene that it regulates), provided that they regulate the function or expression of the NtNTP2 gene. Upstream elements may include promoters, enhancers, or transcription factors. Some elements, such as enhancers, may be placed upstream or downstream of the gene that it regulates. An element need not be located close to the gene it regulates, as some elements have been found hundreds of thousands of base pairs upstream or downstream of the gene it regulates. A mutant or non-naturally occurring plant or plant cell can have one or more mutations located within the first 100 nucleotides of a gene, within the first 200 nucleotides of a gene, within the first 300 nucleotides of a gene, within the first 400 nucleotides of a gene, within the first 500 nucleotides of a gene, within the first 600 nucleotides of a gene, within the first 700 nucleotides of a gene, within the first 800 nucleotides of a gene, within the first 900 nucleotides of a gene, within the first 1000 nucleotides of a gene, within the first 1100 nucleotides of a gene, within the first 1200 nucleotides of a gene, within the first 1300 nucleotides of a gene, within the first 1400 nucleotides of a gene, or within the first 1500 nucleotides of a gene. The mutant or non-naturally occurring plant or plant cell can have one or more mutations located within the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or fifteenth set of 100 nucleotides of the gene, or a combination thereof. A mutant or non-naturally occurring plant or plant cell comprising a mutant NtNTP2 polypeptide is disclosed (e.g., a mutant, non-naturally occurring or transgenic plant or plant cell as described herein, etc.).

[0210] In one embodiment, the seeds from the plant are induced to mutagenesis and subsequently grown into first generation mutant plants. The first generation plants are then self-pollinated, and the seeds from the first generation plants are grown into second generation plants, which are subsequently screened for mutations in their loci. Although the mutagenized plant material can be screened for mutations, the advantage of screening the second generation plants is that all somatic mutations correspond to germline mutations. It will be appreciated by those skilled in the art that a variety of plant materials including but not limited to seeds, pollen, plant tissues or plant cells can be induced to produce mutant plants. However, when screening for mutations in plant polynucleotides, the type of mutagenized plant material may have an impact. For example, when mutagenesis is implemented to pollen before pollination of non-mutagenized plants, the seeds obtained by pollination are grown into first generation plants. Each cell of the first generation plant will contain the mutations produced in the pollen; therefore, these first generation plants can be screened for mutations subsequently, rather than waiting until the second generation.

[0211] NtNTP2 polynucleotides prepared from individual plants, plant cells or plant materials can be optionally combined to accelerate screening for mutations in plant populations derived from mutagenized plant tissues, cells or materials. One or more subsequent generations of plants, plant cells or plant materials can be screened. The size of the optionally combined group depends on the sensitivity of the screening method used.

[0212] After the optional merging of samples, NtNTP2 polynucleotide specific amplification techniques, such as PCR, can be performed on them. Any one or more primers or probes specific to the sequence of the NtNTP2 gene or the NtNTP2 gene next door can be used to amplify the sequence in the sample optionally merged. Suitably, one or more primers or probes are designed to amplify the region of the locus where useful mutations are most likely to occur. Most suitably, primers are designed to detect mutations in the NtNTP2 polynucleotide region. In addition, primers and probes preferably avoid known polymorphic sites, so that point mutations can be easily screened. For the convenience of detection of amplified products, any conventional labeling method can be used to mark one or more primers or probes. Primers or probes can be designed based on the sequence described herein using methods fully understood in the art.

[0213] To facilitate detection of the amplified product, any conventional labeling method can be used to label the primer or probe. Using methods well understood in the art, these primers or probes can be designed based on the sequences described herein.

[0214] Polymorphisms can be identified by methods known in the art and some have been described in the literature.

[0215] In some embodiments, plants can be regenerated or grown from plants, plant tissues or plant cells. Any suitable method for regenerating or growing plants from plant cells or plant tissues can be used, such as (but not limited to) from protoplast tissue culture or regeneration. Suitably, plants can be regenerated by growing transformed plant cells on callus induction medium, shoot induction medium and / or root induction medium. See, for example, McCormick et al. (1986) Plant Cell Reports 5: 81-84. These plants can then be grown and pollinated with the same transformed strain or different strains, and the resulting hybrids with the desired phenotypic characteristic expression are identified. Two or more generations can be grown to ensure that the expression of the desired phenotypic characteristics is stably maintained and inherited, and seeds are picked to ensure that the expression of the desired phenotypic characteristics is obtained. Therefore, as used herein, "transformed seeds" refer to seeds containing nucleotide constructs that are stably integrated into the plant genome.

[0216] Therefore, in another aspect, a method for preparing a mutant plant is provided. The method involves providing at least one cell of a plant comprising an NtNTP2 gene encoding a functional NtNTP2 polypeptide. Next, at least one cell of the plant is treated under conditions effective to regulate (reduce) the function of the NtNTP2 polynucleotides described herein. The at least one mutant plant cell is then propagated into a mutant plant, wherein the mutant plant has a regulated (reduced or inhibited) level of NtNTP2 polypeptide compared to a control plant. In one embodiment of this method for preparing a mutant plant, the treatment step involves subjecting at least one cell to a chemical mutagen as described above under conditions effective to obtain at least one mutant plant cell. In another embodiment of this method, the treatment step involves subjecting at least one cell to a radiation source under conditions effective to obtain at least one mutant plant cell. The term "mutant plant" includes mutant plants in which the genotype is modified (suitably, by means other than genetic engineering or genetic modification) compared to a control plant.

[0217] In certain embodiments, mutant plants, mutant plant cells or mutant plant materials may include one or more mutations, which occur naturally in another plant, plant cell or plant material, and confer desired traits. The mutation may be introduced (e.g., gene infiltration) into another plant, plant cell or plant material (e.g., a plant, plant cell or plant material having a genetic background different from that of the plant from which the mutation originates), to confer the trait. Therefore, for example, a mutation naturally occurring in a first plant may be introduced into a second plant, such as a second plant having a genetic background different from that of the first plant. Technicians are therefore able to search for and identify plants that naturally carry one or more mutant alleles of a gene described herein in the genome, which confer desired traits. Naturally occurring mutant alleles may be transferred to a second plant by a variety of methods (comprising breeding, backcrossing and gene infiltration) to produce strains, varieties or hybrids having one or more mutations in a gene described herein. The same technique may also be applied to the infiltration of one or more non-natural mutations from a first plant to a second plant. Plants displaying desired traits may be screened in a library of mutant plants. Suitably, the selection is performed using knowledge of NtNTP2 polynucleotides as described herein. Thus, genetic traits can be screened compared to controls. Such screening methods may involve the application of conventional amplification and / or hybridization techniques as discussed herein. Therefore, another aspect of the present disclosure relates to a method for identifying mutant plants, the method comprising the following steps: (a) providing a sample comprising NtNTP2 polynucleotides from a plant; (b) determining the sequence of the NtNTP2 polynucleotide, wherein the difference in the sequence of the NtNTP2 polynucleotide compared to the NtNTP2 polynucleotide of a control plant indicates that the plant is a mutant plant. In another aspect, a method is provided for identifying mutant plants that: (i) do not have reduced nitrate levels compared to control plants grown under the same fertilization conditions; and (ii) have increased biomass compared to control plants grown under the same fertilization conditions; and (iii) have an increased NUE response compared to control plants grown under the same fertilization conditions, the method comprising: (a) providing a sample from a plant to be screened; (b) determining whether the sample contains one or more mutations in one or more NtNTP2 polynucleotides described herein; and (c) determining nitrate levels, biomass, and NUE responses compared to control plants grown under the same fertilization conditions.

[0218] In another aspect, a method for preparing a mutant plant is provided, which mutant plant: (i) does not have reduced nitrate levels compared to a control plant grown under the same fertilization conditions; and (ii) has increased biomass compared to a control plant grown under the same fertilization conditions; and (iii) has an increased NUE response compared to a control plant grown under the same fertilization conditions, the method comprising: (a) providing a sample from a first plant; (b) determining whether the sample contains one or more mutations in one or more NtNTP2 polynucleotides described herein, which mutations result in: (i) reduced nitrate levels compared to a control plant grown under the same fertilization conditions; and (ii) increased biomass compared to a control plant grown under the same fertilization conditions; and (iii) increased NUE response compared to a control plant grown under the same fertilization conditions; and (c) transferring one or more mutations to a second plant. The mutant plant may also have an increase in root development compared to a control plant grown under the same fertilization conditions.

[0219] The mutation can be transferred to the second plant using a variety of methods known in the art, such as by genetic engineering, genetic manipulation, introgression, plant breeding, backcrossing, etc. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background than the first plant.

[0220] On the other hand, a method for preparing a mutant plant is provided, which mutant plant: (i) has a reduced nitrate level compared to a control plant grown under the same fertilization conditions; and (ii) has an increased biomass compared to a control plant grown under the same fertilization conditions; and (iii) has an increased NUE response compared to a control plant grown under the same fertilization conditions, the method comprising: (a) providing a sample from a first plant; (b) determining whether the sample contains one or more mutations in one or more NtNTP2 polynucleotides described herein, which mutations result in reduced nitrate levels, increased biomass, and increased NUE response compared to a control plant grown under the same fertilization conditions; and (c) introgressing one or more mutations from the first plant into a second plant. Compared to a control plant grown under the same fertilization conditions, the mutant plant may also have an increase in root development. In one embodiment, the gene introgression step includes plant breeding, optionally including backcrossing, etc. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a genetic background different from that of the first plant. In one embodiment, the first plant is not a cultivar or an elite cultivar. In one embodiment, the second plant is a cultivar or an elite cultivar.

[0221] Another aspect relates to mutant plants (including cultivars or elite cultivars mutant plants) obtained or obtainable by the methods described herein. In certain embodiments, a "mutant plant" may have one or more mutations localized only to a specific region of the plant, such as within the sequence of one or more NtNTP2 polynucleotides described herein. According to this embodiment, the remaining genomic sequence of the mutant plant will be identical or substantially identical to that of the plant before mutagenesis.

[0222] In certain embodiments, the mutant plant may have one or more mutations located in more than one genomic region of the plant, such as within the sequence of one or more NtNTP2 polynucleotides described herein and within one or more other regions of the genome. According to this embodiment, the remaining genomic sequence of the mutant plant will be different or substantially different from that of the plant before mutagenesis. In certain embodiments, the mutant plant may not have one or more mutations in one or more, two or more, three or more, four or more, or five or more exons of the polynucleotides described herein; or may not have one or more mutations in one or more, two or more, three or more, four or more, or five or more introns of the polynucleotides described herein; or may not have one or more mutations in the promoter of the polynucleotides described herein; or may not have one or more mutations in the 3' untranslated region of the polynucleotides described herein; or may not have one or more mutations in the 5' untranslated region of the polynucleotides described herein; or may not have one or more mutations in the coding region of the polynucleotides described herein; or may not have one or more mutations in the non-coding region of the polynucleotides described herein; or any combination of two or more, three or more, four or more, five or more; or six or more thereof in a portion thereof.

[0223] In another aspect, a method for identifying a plant, plant cell or plant material comprising a mutation in a gene encoding a NtNTP2 polynucleotide as described herein is provided, the method comprising: (a) mutagenizing a plant, plant cell or plant material; (b) obtaining a sample from the plant, plant cell or plant material or its progeny; (c) determining the polynucleotide sequence of the NtNTP2 gene or its variant or fragment, wherein the difference in the sequence indicates one or more mutations therein. The method also allows the selection of plants with mutations that occur in genomic regions that affect the expression of the NtNTP2 gene in plant cells, such as transcription start sites, start codons, intronic regions, exon-intron boundaries, terminators or stop codons.

[0224] The mutant, non-naturally occurring or transgenic plants or parts thereof according to the present disclosure have a favorable phenotype, wherein the nitrate levels are not significantly different compared to control plants grown under the same fertilization conditions, the leaf biomass yield is higher compared to control plants grown under the same fertilization conditions, and the NUE of the plants is higher compared to control plants grown under the same fertilization conditions. The morphological difference observed between the mutant, non-naturally occurring or transgenic plants compared to the control is an increase in root development compared to control plants grown under the same fertilization conditions.

[0225] No significant differences in nitrate levels were observed between non-naturally occurring or transgenic plants of the -S and -T mutants compared to controls (in stem leaves and midribs of mid-stem leaves). Therefore, reducing the activity of NtNTP2 in the -S and -T forms is not expected to have a significant effect on nitrate levels.

[0226] Compared with control, according to the disclosed mutant, non-natural or transgenic plant or its part produce more biomass (for example, leaf biomass).Suitably, leaf biomass is green leaf or dry leaf or dried leaf, more suitably, green leaf.Compared with the control of growing under the same conditions, the increase of biomass can be at least about 5%, at least about 9%, at least about 10%, at least about 13%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29% or at least about 30% or at least about 31% or at least about 34% or at least about 35% or at least about 36% or at least about 37% or more.Compared with standard fertilization scheme, the biomass produced in the nitrogen starvation scheme is higher. For example, in a standard fertilization scheme or in a nitrogen starvation scheme, the biomass increase can be at least about 5% or more compared to a control grown under the same conditions. In one embodiment, the biomass increase under nitrogen starvation conditions is at least about 28% compared to a control grown under the same conditions. Therefore, the loss of NtNTP2 activity results in an increase in yield in different nitrogen schemes.

[0227] The NUE index (i.e., the units of biomass produced (expressed as kg / hectare, assuming 12,000 plants per hectare) per unit of nitrogen fertilizer input (expressed as kg nitrogen per hectare) measured for a mutant, non-naturally occurring or transgenic plant or part thereof according to the disclosure when grown under nitrogen starvation conditions can be increased by at least about 5%, at least about 9%, at least about 10%, at least about 13%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, or at least about 22% or more compared to a control in a standard fertilization regimen. Compared to a control grown under the same conditions, the NUE index can be increased by at least about 5%, at least about 9%, at least about 10%, at least about 13%, or at least about 14%, at least about 20%, at least about 25%, or at least about 28% or more. In one embodiment, the NUE index under nitrogen starvation conditions is increased by at least about 28% compared to a control grown under the same conditions. Thus, the loss of NtNTP2 activity results in an increase in NUE in different nitrogen regimes. Thus, the impairment of NTP2 protein activity can increase the ability of plants to adapt to nitrogen starvation, thereby increasing the NUE of plants, meaning the biomass per unit of nitrogen fertilization applied.

[0228] Thus, mutant, non-naturally occurring or transgenic plants or parts thereof according to the present disclosure have a favorable phenotype wherein nitrate levels are not significantly different, biomass yield is increased by at least about 5%, at least about 9%, at least about 10%, at least about 13%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about The mutant, non-naturally occurring or transgenic plant or part thereof can have an increase in root development compared to a control plant grown under the same fertilization conditions.

[0229] In one embodiment, under standard fertilization conditions, the plant or plant part (e.g., leaf): (i) does not have reduced nitrate levels compared to a control plant grown under the same fertilization conditions; and (ii) has at least a 5% increase in biomass compared to a control plant grown under the same fertilization conditions; and (iii) has at least a 5% increase in NUE response compared to a control plant grown under the same fertilization conditions. The plant or part thereof may have an increase in root development compared to a control plant grown under the same fertilization conditions.

[0230] In another embodiment, under nitrogen starvation conditions, the plant or plant part (e.g., leaf): (i) does not have reduced nitrate levels compared to a control plant grown under the same fertilization conditions; and (ii) has at least a 5% increase in biomass compared to a control plant grown under the same fertilization conditions; and (iii) has at least a 5% increase in NUE response compared to a control plant grown under the same fertilization conditions. The plant or part thereof may have an increase in root development compared to a control plant grown under the same fertilization conditions.

[0231] In an embodiment, the phenotype differs according to the fertilization input. Thus, in one embodiment, under standard fertilization conditions, the plant or plant part (e.g., leaf): (i) does not have reduced nitrate levels compared to control plants; and (ii) has at least a 5% increase in biomass compared to control plants; and (iii) has at least a 5% increase in NUE response compared to control plants. In another embodiment, under nitrogen starvation conditions, the plant or plant part (e.g., leaf): (i) does not have reduced nitrate levels compared to control plants; and (ii) has at least a 5% increase in biomass compared to control plants; and (iii) has at least a 5% increase in NUE response compared to control plants. The plant or part thereof may have an increase in root development compared to a control plant grown under the same fertilization conditions.

[0232] Thus, the plants of the present disclosure have increased yield at lower fertilizer inputs, and therefore have improved NUE.Lower fertilizer inputs can achieve a reduction in TSNA.

[0233] According to the present disclosure, 'standard conditions' are 254 nitrogen units (one unit is expressed as kg / hectare), while 'nitrogen starvation conditions' are 55 nitrogen units (one unit is expressed as kg / hectare).

[0234] Plants according to the present disclosure include, but are not limited to, monocots and dicots and plant cell systems, including species from one of the following families: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae, Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae, Dioscoreaceae, Ephedraceae, The present invention relates to a plant family consisting of: Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae, Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae, Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae, Theaceae, or Vitaceae.

[0235] Suitable species may include members of the following genera: Abelmoschus, Abies, Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula , Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura Datura, Dianthus, Digitalis, Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus, Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hippophae rhamnoides, yoscyamus), Jatropha, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa, Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum,Petunia, Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus, Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale ale, Solanum, Sorghum, Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale, Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.

[0236] Suitable species may include: Panicum spp., Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Andropogon gerardii, Pennisetum purpureum, Phalarisarundinacea, Cynodon dactylon, Festuca arundinacea, Spartina pectinata, Medicago sativa, Arundo donax, Secalecereale, Salix spp., Eucalyptus spp.), triticale (Triticosecale), bamboo, sunflower (Helianthus annuus), safflower (Carthamus tinctorius), jatropha (Jatropha curcas), castor (Ricinus communis), oil palm (Elaeis guineensis), flax (Linum usitatissimum), mustard (Brassica juncea), sugar beet (Beta vulgaris), cassava (Manihot esculenta), tomato (Lycopersicon esculentum), lettuce (Lactuca sativa), banana (Musyclise alca), potato (Solanum tuberosum), cabbage (broccoli, cauliflower, Brussels sprouts) (Brassica oleracea), camellia (Camellia sinensis), strawberry (Fragaria ananassa), cocoa (Theobroma cacao), coffee (Coffeaycliseca), grapes (Vitis vinifera), pineapple (Ananas comosus), pepper (Capsicum annum), onion (Allium cepa), cantaloupe (Cucumis melo), cucumber (Cucumis sativus), winter squash (Cucurbita maxima), pumpkin (Cucurbita moschata), spinach (Spinacea oleracea), watermelon (Citrullus lanatus), okra (Abelmoschus esculentus), eggplant (Solanum melongena), rose (Rosas pp.), carnation (Dianthus caryophyllus), petunia (Petunia spp.), poinsettia (Poinsettia pulcherrima), white lupin (Lupinus albus), oats (Uniola paniculata), bentgrass (Agrostis spp.), poplar (Populus tremuloides), pine (Pinus spp.), fir (Abies spp.), maple (Acer spp.), barley (Hordeum vulgare), bluegrass (Poa pratensis), Lolium spp.) and cattail grass (Phleum pratense), switchgrass (Panicum virgatum), sudangrass (Sorghuycliseor) 4747 or (sorghum, sudangrass), giant silvergrass (Miscanthus giganteus), sugarcane (Saccharum sp.), poplar (Populus balsamifera), corn (Zea mays), soybean (Glycine max), rapeseed (Brassica napus), wheat (Triticum aestivum), upland cotton (Gossypium hirsutum), rice (Oryza sativa), sunflower (Helianthus annuus), alfalfa (Medicago sativa), sugar beet (Betavulgaris) or pearl millet (Pennisetum glaucum). .

[0237] Suitable species can include species of the genus Nicotiana, including N. rustica and N. tabacum (e.g., LA B21, LN KY171, TI 1406, Basma, Galpao, Perique, Beinhart 1000-1, and Petico). Other Nicotiana species include N. acaulis, N. acuminata, N. africana, N. alata, N. ameghinoi, N. amplexicaulis, N. arentsii, N. attenuata, N. azambujae, N. benavidesii, N. benthamiana, N. bigelovii, N. bonariensis, N. cavicolola, N. clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N. excelsior, N. forge etiana), N. fragrans, N. glauca, N. glutinosa, N. goodspeedii, N. gossei, N. hybrid, N. ingulba, N. kawakamii, N. knightiana, N. langsdorffii, N. linearis, N. longiflora, N. maritima, N. megalosiphon, N. miersii, N. noctiflora, N. nudicaulis, N. obtusifolia, N. occidentalis, N. occidentalis subsp. hesperis), ear-shaped tobacco (N.otophora), round tobacco (N. paniculata), few-flowered tobacco (N. pauciflora), petunia-shaped tobacco (N. petunioides), blue jasmine tobacco (N.plumbaginifolia), N. quadrivalvis, N. raimondii, N. repanda, N. rosulata, N. rosulata subspecies subsp. ingulba), round-leaf tobacco (N. rotundifolia), setter's tobacco (N. setchellii), pseudo-tobacco (N. simulans), front-leaf tobacco (N. solanifolia), Spegazzinii, Stocktonii, sweet tobacco (N. suaveolens), beautiful flower tobacco (N. sylvestris), pseudo-spicate tobacco (N. thyrsiflora), hairy tobacco (N. tomentosa), hairy tobacco (N. tomentosiformis), trigonal tobacco (N. trigonophylla), shade tobacco (N. umbratica), wave-leaf tobacco (N. undulata), trembling tobacco (N. velutina), sequence leaf tobacco (N. wigandioides) and flower tobacco (Nx sanderae). In one embodiment, the plant is tobacco. .

[0238] This paper also contains and uses tobacco cultivars and fine tobacco cultivars.Therefore, transgenic, non-natural existence or mutant plant can be tobacco cultivars or fine tobacco cultivars, and it comprises one or more transgenic or one or more gene mutations or its combination.Gene mutation (for example, one or more polymorphisms) can be the sudden change that non-natural exists in individual tobacco cultivars or tobacco cultivars (for example, fine tobacco cultivars), or can be the sudden change that naturally exists, and condition is that described sudden change is not naturally present in individual tobacco cultivars or tobacco cultivars (for example, fine tobacco cultivars).

[0239] Particularly useful common tobacco varieties include Burley, Black, Flue-cured, and Oriental types. Non-limiting examples of varieties or cultivars are: BD 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF911, DT 538 LC Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04PLC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501LC, K 149, K 326, K 346, K 358, K394, K399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171, KY 907, KY907LC, KY14xL8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14×L8, narrow leaf Madole, narrow leaf Madole LC, NBH 98, N-126, N-777LC, N-7371LC, NC 100, NC 102, NC 2000, NC291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH129, NC 2002, Neal Smith Madole,OXFORD 207,PD 7302LC, PD 7309LC, PD 7312LC, 'Perique' tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG81, RG H51, RGH 4, RGH 51, RS1410, Speight 168, Speight 172, Speight 179, Speight210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN97, TN97LC, TN D94, TND950, TR (Tom Rosson) Madole, VA 309, VA359, AA 37-1, B13P, Xanthi (Mitchell-Mor), Bel-W3, 79-615, Samsun Holmes NN, KTRDC No. 2 Hybrid 49, White Burley 21, KY8959, KY9, MD 609, PG01, PG04, PO1, PO2, PO3, RG11, RG 8, VA509, AS44, Banket A1, Basma Drama B84 / 31, Basma IZichna ZP4 / B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 347, Criollo Misionero, Delcrest, Djebel 81, DVH 405, Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsage E1, LA BU 21, NC 2326, NC 297, PVH 2110, RedRussian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156 / 1, Prilep P12-2 / 1, Yaka JK-48, Yaka JB 125 / 3, TI-1068, KDH-960, TI-1070, TW136, Basma, TKF 4028, L8, TKF 2002, GR141, Basma xanthi, GR149, GR153, Petit Havana. The above low conversion subvariants are also contemplated even if not specifically stated herein.

[0240] Embodiments also relate to compositions and methods for producing mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants that have been modified to modulate the expression or function of the NtNTP2 polynucleotides described herein (or any combination thereof as described herein). Advantageously, the mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants obtained can be similar or substantially identical to control plants in overall appearance. Various phenotypic characteristics, such as degree of maturity, number of leaves per plant, stalk height, leaf insertion angle, leaf size (width and length), internode distance, and leaf-midrib ratio can be evaluated by field observations.

[0241] One aspect relates to the seed of mutant plant, non-natural plant, hybrid plant or transgenic plant as described herein. Another aspect relates to the pollen or ovule of mutant plant, non-natural plant, hybrid plant or transgenic plant as described herein. In addition, mutant plant, non-natural plant, hybrid plant or transgenic plant as described herein are provided, which also comprises polynucleotides that confer male sterility. Tissue culture of regenerable cells of mutant plant, non-natural plant, hybrid plant or transgenic plant or a part thereof as described herein is also provided, wherein the culture regenerates plants that can express all morphological and physiological characteristics of the parent. Regenerable cells include cells from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and parts thereof, ovules, buds, stems, handles, pith and capsules or callus or protoplasts derived from them.

[0242] The mutant, non-naturally occurring or transgenic plant leaves or plant leaf parts obtained according to the present disclosure can be similar or substantially the same in visual appearance to corresponding control plant leaves or plant leaf parts. In one embodiment, the number of leaves is substantially the same as the control. In another embodiment, the chlorophyll content is substantially the same as the control plant grown under the same fertilization conditions. In other embodiments, the size or form or number or color of the leaves is substantially the same as the control plant grown under the same fertilization conditions.

[0243] The polynucleotides and recombinant constructs described herein can be used to modulate the expression or function or activity of the NtNTP2 polynucleotides or NtNTP2 polypeptides described herein.

[0244] Plants carrying mutant alleles of one or more NtNTP2 polynucleotides described herein (or any combination thereof described herein) can be used in plant breeding programs to produce useful lines, varieties and hybrids containing leaves of desired genotypes and phenotypes. In particular, the mutant alleles can be introgressed into the above-mentioned commercially important varieties. Therefore, a method for plant breeding is provided, which includes hybridizing mutant plants, non-naturally occurring plants or transgenic plants as described herein with plants containing different genetic consistencies. The method can also include hybridizing progeny plants with another plant, and optionally repeating the hybridization until a progeny with the desired genotype and phenotype is obtained. One purpose of this type of breeding method is to introduce desired genetic traits into other varieties, breeding lines, hybrids or cultivars, especially those with commercial interests. Another purpose is to facilitate the genetic modification of different genes in a single plant variety, line, hybrid or cultivar. Consider intraspecific and interspecific mating. Progeny plants derived from this type of hybridization, also referred to as breeding lines, are examples of non-naturally occurring plants of the present disclosure.

[0245] In one embodiment, a method for producing a non-natural plant is provided, the method comprising: (a) hybridizing a mutant or transgenic plant with a second plant to produce progeny tobacco seeds; (b) growing progeny tobacco seeds under plant growth conditions to produce non-natural plants. The method may also include: (c) hybridizing a non-natural plant of the previous generation with itself or another plant to obtain progeny tobacco seeds; (d) growing the progeny tobacco seeds of step (c) under plant growth conditions to obtain other non-natural plants; and repeating the hybridization step and growth step of (c) and (d) for many times, to produce further progeny of non-natural plants. The method may optionally include providing a step of a parent plant before step (a), the parent plant comprising a genetic consistency characterized and different from a mutant or transgenic plant. In some embodiments, depending on the breeding plan, the hybridization and growth steps are repeated 0 to 2 times, 0 to 3 times, 0 to 4 times, 0 to 5 times, 0 to 6 times, 0 to 7 times, 0 to 8 times, 0 to 9 times or 0 to 10 times, so as to produce the generation of non-natural plants. Backcrossing is an example of such a method, wherein a progeny is crossed with one of its parents or another plant that is genetically similar to its parent, so as to obtain a progeny plant with a genetic identity closer to one of the parents in the next generation. Techniques for plant breeding, particularly plant breeding, are well known and can be used in the methods of the present disclosure. The present disclosure also provides non-naturally occurring plants produced by these methods. Certain embodiments do not include the step of selecting a plant. Suitably, leaves or a portion of leaves are harvested from the produced plant.

[0246] In some embodiments of the methods described herein, standard field procedures are used to evaluate the strains derived from breeding and screening variant NtNTP2 genes in the field. The control genotype comprising the original non-mutagenized parent is included, and the entry is arranged in the field by randomized complete block design or other appropriate field design. For tobacco, standard agronomic practices are used, such as tobacco harvested, weighed and sampled, for chemical and other common tests before and during drying. Statistical analysis of the data is performed to confirm the similarity between the selected strain and the parental strain. Cytogenetics analysis of the selected plant is optionally performed to confirm the chromosome set and chromosome pairing relationship.

[0247] DNA fingerprinting, single nucleotide polymorphism, microsatellite markers or similar techniques can be used in the breeding program of marker-assisted selection (MAS) to transfer or cultivate the mutant allele of NtNTP2 gene into other tobacco as described herein. For example, breeders can produce isolated populations by hybridization of genotypes containing the mutant NtNTP2 alleles of concern and the genotypes of agronomy expectations. One of the techniques listed herein can be used to screen the plants or backcross generations in F2 using the markers developed from the genome sequence or its fragments. Plants identified as having mutant NtNTP2 alleles can be backcrossed or self-pollinated to produce a second population to be screened. Depends on the expected genetic pattern, it is necessary to self-pollinate the selected plant before each round of backcrossing to help identify desired individual plants. Backcrossing or other breeding operations can be repeated until the desired phenotype of the recurrent parent is recovered.

[0248] According to the present disclosure, in a breeding program, successful hybridization obtains fertile F1 plants. The selected F1 plant can be hybridized with one of the parents, and the first backcross generation plant is self-pollinated to produce a population that screens for variant NtNTP2 gene expression (e.g., an invalid version of the gene) again. The process of backcrossing, self-pollination and screening is repeated, for example, at least 4 times, until the final screening produces fertile and recurrent parent-like plants. If necessary, this plant is self-pollinated, and the progeny is subsequently screened again to confirm that the plant exhibits variant NtNTP2 gene expression. In some embodiments, the variant NtNTP2 gene expression of the plant population in the F2 generation is screened, for example, according to standard methods, plants that cannot express NtNTP2 polypeptides due to the lack of NtNTP2 genes are identified, for example, by using a PCR method, wherein the primers are based on the polynucleotide sequence information of the NtNTP2 polynucleotides described herein (or any combination thereof as described herein).

[0249] Hybrid tobacco varieties can be produced in the following manner: stop the self-pollination of the female parent plant (that is, seed parent) of the first variety, allow the pollen from the male parent plant of the second variety to fertilize the female parent plant, and allow F1 hybrid seeds to form on female plants. The self-pollination of female plants can be stopped by emasculating flowers in the early stages of flower development. Alternatively, the form of male sterility can be used to stop the formation of pollen on female parent plants. For example, male sterility can be produced by cytoplasmic male sterility (CMS) or transgenic male sterility, wherein transgenic suppresses microspores and / or pollen formation or self-incompatibility. The female parent plant containing CMS is particularly useful. In the embodiment where the female parent plant is CMS, pollen is gathered in the crops from male fertile plants and is manually applied to the stigma of the CMS female parent plant, and the resulting F1 seed is gathered in the crops.

[0250] Kind described herein and strain can be used for forming single cross tobacco F1 hybrid.In this class embodiment, the plant of parental variety can be grown into basically homogeneous adjacent population, so that male parent plant and the natural cross pollination of female parent plant.Selectively gather in the crops the F1 seed formed on female parent plant by conventional means.Also can plant two parental plant varieties in large quantities, and gather in the crops the admixture of the F1 hybrid seed formed on female parent due to self-pollination and the seed formed on male parent.Or, can carry out three-line hybridization, wherein single cross F1 hybrid is used as female parent, and hybridizes with different male parents.As another alternative, can produce double cross hybrid, wherein the F1 progeny of two different single crosses carries out self-crossing.

[0251] Those population members having the desired trait or phenotype can be screened or selected in a mutant, non-naturally occurring or transgenic plant population. For example, those plants having the desired expression level or the function of the NtNTP2 polypeptide encoded by it can be screened in the progeny population of a single transformation event. Physical and biochemical methods can be used to identify the expression or activity level. These methods include Southern analysis or PCR amplification for detecting polynucleotides; Northern blots, S1 RNase protection, primer extension or RT-PCR amplification for detecting RNA transcripts; enzyme analysis for detecting enzyme or ribozyme function of polypeptides and polynucleotides; and polypeptide gel electrophoresis, Western blots, immunoprecipitation and enzyme-linked immunosorbent assays for detecting polypeptides. Other techniques such as in situ hybridization, enzyme staining, immunostaining and enzyme assays can also be used to detect the presence or expression, function or activity of NtNTP2 polypeptides or NtNTP2 polynucleotides.

[0252] Mutant, non-naturally occurring or transgenic plant cells and plants as described herein include one or more recombinant NtNTP2 polynucleotides, one or more NtNTP2 polynucleotide constructs, one or more double-stranded RNAs, one or more combinations, or one or more vectors / expression vectors.

[0253] One or more of the following further genetic modifications may be present in the mutant, non-naturally occurring or transgenic plant leaf or plant leaf part.

[0254] One or more genes involved in the conversion of nitrogen metabolic intermediates can be modified to reduce the level of at least one tobacco-specific nitrosamine (TSNA). Non-limiting examples of such genes include those encoding nicotine demethylases (such as CYP82E4, CYP82E5 and CYP82E10 described in WO2006 / 091194, WO2008 / 070274, WO2009 / 064771 and WO2011 / 088180), and nitrate reductases, such as described in WO2016046288.

[0255] One or more genes involved in heavy metal absorption or heavy metal transport can be modified to reduce heavy metal content. Non-limiting examples include genes in: multidrug resistance-related polypeptide family, cation diffusion facilitating factor (CDF) family, Zrt-Irt-like polypeptide (ZIP) family, cation exchanger (CAX) family, copper transporter (COPT) family, heavy metal ATPase family (e.g., HMA, as described in WO2009 / 074325 and WO2017 / 129739), natural resistance-related macrophage polypeptide (NRAMP) homolog family and other members of the ATP binding cassette (ABC) transporter family (e.g., MRP), as described in WO2012 / 028309, which participate in the transport of heavy metals such as cadmium.

[0256] Other exemplary modifications may produce plants with modulated expression or function of isopropylmalate synthase, which results in changes in sucrose ester composition, which can be used to alter the preference profile (see WO2013029799).

[0257] Other exemplary modifications may produce plants with modulated expression or function of threonine synthase, wherein the level of methionine may be modulated (see WO2013029800).

[0258] Other exemplary modifications may produce plants with modulated expression or function of one or more of neoxanthin synthase, lycopene β cyclase and 9-cis-epoxycarotenoid dioxygenase to modulate β-damascenone content to alter flavor characteristics (see WO2013064499).

[0259] Other exemplary modifications may produce plants having a CLC chloride channel family member with modulated expression or function to regulate nitrate levels therein (see WO2014096283 and WO2015197727).

[0260] Other exemplary modifications may produce plants with modulated expression or function of one or more asparagine synthetases to modulate the levels of asparagine in the leaves and to modulate the levels of acrylamide in the aerosols produced when the leaves are heated or burned (see WO2017042162).

[0261] Other examples of modifications include adjusting herbicide tolerance, for example, glyphosate is the active ingredient of many broad-spectrum herbicides. Glyphosate-resistant transgenic plants have been developed by transferring the aroA gene (glyphosate EPSP synthase from Salmonella typhimurium and E. coli). Sulfonylurea-resistant plants have been produced by transforming the mutant ALS (acetolactate synthase) gene from Arabidopsis thaliana. The OB polypeptide from the photosystem II of mutant Amaranthus hybridus has been transferred into plants to produce atrazine-resistant transgenic plants; and bromoxynil-resistant transgenic plants have been produced by incorporating the bxn gene from the bacterium Klebsiella pneumoniae.

[0262] Another exemplary modification results in a plant that is resistant to insects. Bacillus thuringiensis (Bt) toxins can provide an effective way to delay the emergence of Bt-resistant pests, as recently demonstrated in cauliflower, where the pyramidal cry1Ac and cry1C Bt genes controlled diamondback moths that were resistant to either single polypeptide and significantly delayed the evolution of resistant insects.

[0263] Another exemplary modification produces plants that are resistant to diseases caused by pathogens (e.g., viruses, bacteria, fungi). Plants expressing the Xa21 gene (resistant to bacterial blight) and plants expressing a Bt fusion gene and a chitinase gene (resistant to yellow stem borer and resistant to sheath) have been designed.

[0264] Another exemplary modification results in altered reproductive capacity, such as male sterility.

[0265] Another exemplary modification generates plants tolerant to abiotic stress (e.g., drought, temperature, salinity), and by transferring acylglycerolphosphatase from Arabidopsis, tolerant transgenic plants have been generated; genes encoding mannitol dehydrogenase and sorbitol dehydrogenase, which are involved in the synthesis of mannitol and sorbitol, improve drought resistance.

[0266] Another exemplary modification produces a plant in which the activity of one or more endogenous glycosyltransferases, such as N-acetylglucosaminyltransferase, β(1,2)-xylosyltransferase, and α(1,3)-fucosyltransferase, is modulated (see WO / 2011 / 117249).

[0267] Another exemplary modification produces a plant in which the activity of one or more nicotine N-demethylases is modulated, such that the levels of nornicotine and nornicotine metabolites formed during curing can be modulated (see WO2015169927).

[0268] Other exemplary modifications can produce plants with improved storage polypeptides and oils, plants with enhanced photosynthetic efficiency, plants with extended shelf life, plants with enhanced carbohydrate content, and plants resistant to fungi. Transgenic plants in which the expression of S-adenosyl-L-methionine (SAM) and / or cystathionine gamma-synthase (CGS) has been modulated are also contemplated.

[0269] One or more genes involved in the nicotine synthesis pathway can be modified to produce plants or plant parts that produce regulated levels of nicotine when dried. The nicotine synthesis gene can be selected from the group consisting of: A622, BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE 2, MPO1, MPO2, MYC2a, MYC2b, NBB1, nic1, nic2, NUP1, NUP2, PMT1, PMT2, PMT3, PMT4 and QPT or a combination of one or more thereof.

[0270] One or more genes involved in controlling the amount of one or more alkaloids can be modified to obtain plants or plant parts that produce regulated levels of alkaloids. The alkaloid level control gene can be selected from the group consisting of: BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE 2, MYC2a, MYC2b, nic1, nic2, NUP1 and NUP2 or a combination of one or more thereof.

[0271] Leaf material (such as blade and midrib) can be incorporated into or used to prepare various consumables, including but not limited to aerosol forming materials, aerosol forming devices, smoking articles, smokeable products, smokeless products, medicine or beauty products, intravenous preparations, tablets, powders and tobacco products. The example of aerosol forming materials includes tobacco composition, tobacco, tobacco extract, tobacco shreds, cut fillers, dried tobacco, expanded tobacco, homogenized tobacco, reconstituted tobacco and pipe tobacco. Smoking articles and smokeable products are the types of aerosol forming devices. The example of smoking articles or smokeable products includes cigarettes, cigarillos and cigars. The example of smokeless products includes chewing tobacco and snuff. In some aerosol forming devices instead of burning, tobacco composition or another aerosol forming material is heated by one or more electric heating elements to produce aerosol. In another type of heated aerosol forming device, aerosol is produced by transferring heat from combustible fuel element or heat source to physically separated aerosol forming materials, and the aerosol forming material can be located in the heat source, around the heat source or downstream of the heat source. Smokeless tobacco products and various tobacco-containing aerosol-forming materials may contain tobacco in any form, including dry particles, fragments, small particles, powders or slurries deposited on, mixed in, surrounded by or otherwise combined with other ingredients, which may take any form, such as flakes, films, tabs, foams or beads. As used herein, the term "smoke" is used to describe a type of aerosol produced by a smoking article such as a cigarette or by burning an aerosol-forming material.

[0272] In one example, leaf material (such as leaf blades and midribs) can be processed according to the method described in US20190142058A1, wherein a cast sheet of homogenized tobacco material is prepared by: pulping cellulose fibers with water; grinding a tobacco mixture of one or more tobacco types into tobacco particles; combining the pulped cellulose fibers with the tobacco particles and a binder to form a slurry; homogenizing the slurry; casting the slurry to form a cast sheet of homogenized tobacco material from the slurry; discarding unwanted portions of the cast sheet; and introducing the discarded unwanted portions of the cast sheet into the slurry. Therefore, leaf material (such as blade and midrib) can be combined with a binding agent, such as natural pectin, such as fruit, citrus or tobacco pectin; guar gum, such as hydroxyethyl guar gum and hydroxypropyl guar gum; locust bean gum, such as hydroxyethyl and hydroxypropyl locust bean gum; alginate; starch, such as modified or derived starch; cellulose, such as methyl, ethyl, ethyl hydroxymethyl and carboxymethyl cellulose; tamarind gum; dextran; pullulan (pullalon); konjac flour; xanthan gum etc. Therefore, tobacco material can comprise leaf material and binding agent as described herein. In one embodiment, dried plant leaf material is also provided. The process of drying green tobacco leaf is known to those skilled in the art, and includes but is not limited to airing, fire-cured drying, flue drying and sun drying as described herein.

[0273] In another embodiment, a tobacco product comprising a tobacco-containing aerosol-forming material comprising plant leaf material, suitably dried leaves, is described.The tobacco product described herein may be a blended tobacco product, which may also include unmodified tobacco.

[0274] Mutant, non-naturally occurring or transgenic plant leaves or plant leaf parts can have other uses, for example, in agriculture. For example, the mutant, non-naturally occurring or transgenic plant leaves or plant leaf parts described herein can be used to make animal feed and human food.

[0275] The disclosure also provides a method for producing seed, which comprises cultivating mutant plants as described herein, non-naturally occurring plants or transgenic plants, and collecting seeds from the cultivated plants. The seeds from plants described herein can be conditioned by means known in the art, and packaged in packaging materials to form manufactured articles. Packaging materials such as paper and cloth are well known in the art. The packaging of seeds can carry a mark describing the properties of seeds therein, such as a label or mark fixed to the packaging material, a mark printed on the packaging.

[0276] Compositions, methods and kits for genotyping plants for identification, selection or breeding may include means for detecting the presence of NtNTP2 polynucleotides (or any combination thereof as described herein) in a polynucleotide sample. Thus, a composition is described comprising one or more primers for specifically amplifying at least a portion of one or more NtNTP2 polynucleotides, and optionally one or more probes and optionally one or more reagents for performing amplification or detection.

[0277] Accordingly, NtNTP2 gene-specific oligonucleotide primers or probes are disclosed, which contain about 10 or more adjacent polynucleotides corresponding to the NtNTP2 polynucleotides described herein. The primer or probe may include or consist of about 15, 20, 25, 30, 40, 45 or 50 or more adjacent polynucleotides, and the primer or probe hybridizes (e.g., specifically hybridizes) with the polynucleotides described herein. In some embodiments, the primer or probe may include or consist of about 10 to 50 consecutive nucleotides, about 10 to 40 consecutive nucleotides, about 10 to 30 consecutive nucleotides, or about 15 to 30 consecutive nucleotides, which can be used for sequence-dependent methods of NtNTP2 gene identification (e.g., Southern hybridization) or separation (e.g., in situ hybridization of bacterial colonies or plaques) or NtNTP2 gene detection (e.g., as one or more amplification primers in amplification or detection). One or more specific primers or probes can be designed and used to amplify or detect part or all of a polynucleotide. As a specific example, two primers can be used in a PCR scheme to amplify the NtNTP2 polynucleotide fragment. PCR can also be carried out using a primer derived from the NtNTP2 polynucleotide sequence and a second primer hybridized with the polynucleotide sequence upstream or downstream sequence, the polynucleotide sequence such as a promoter sequence, the 3' end of the mRNA precursor or a sequence derived from a carrier. Examples of heat and isothermal techniques for in vitro amplification of polynucleotides are well known in the art. The sample can be or can be derived from a plant, a plant cell or a plant material, or a tobacco product prepared or derived from a plant, a plant cell or a plant material as described herein.

[0278] On the other hand, a method for detecting the NtNTP2 polynucleotide described herein (or any combination thereof as described herein) in a sample is also provided, the method comprising the following steps: (a) providing a sample containing or suspected of containing an NtNTP2 polynucleotide; (b) contacting the sample with one or more primers or one or more probes to specifically detect at least a portion of the NtNTP2 polynucleotide; and (c) detecting the presence of an amplification product, wherein the presence of the amplification product indicates the presence of the NtNTP2 polynucleotide in the sample.

[0279] In another aspect, the use of one or more primers or probes for specifically detecting at least a portion of an NtNTP2 polynucleotide is also provided. A kit for detecting at least a portion of an NtNTP2 polynucleotide is also provided, comprising one or more primers or probes for specifically detecting at least a portion of an NtNTP2 polynucleotide. The kit may comprise reagents for NtNTP2 polynucleotide amplification (such as PCR), or reagents for probe hybridization detection techniques (such as Southern blotting, Northern blotting, in situ hybridization or microarray). The kit may include reagents for antibody binding detection techniques (such as protein blotting, ELISA, SELDI mass spectrometry or test strips). The kit may include reagents for DNA sequencing. The kit may include reagents and instructions for use.

[0280] In some embodiments, the kit may include instructions for one or more of the methods. The kit may be used for genetic identity determination, phylogenetic studies, genotyping, haplotype typing, pedigree analysis, or plant breeding, particularly co-dominance scoring.

[0281] The present disclosure also provides a method for genotyping plants, plant cells or plant materials comprising NtNTP2 polynucleotides as described herein. Genotyping provides a means for distinguishing homologs of chromosome pairs, and can be used to distinguish segregants in plant populations. Molecular marker methods can be used for systematic studies, characterizing genetic relationships between crop varieties, identifying hybrids or somatic hybrids, locating chromosome segments that affect single gene traits, map cloning and quantitative genetic studies. The specific method of genotyping can adopt any number of molecular marker analysis techniques, including amplified fragment length polymorphism (AFLP). AFLP is the product of allelic differences between amplified fragments caused by polynucleotide variability. Therefore, the present disclosure further provides a method for the separation of chromosome sequences connected to one or more NtNTP2 genes or polynucleotides and these NtNTP2 genes or polynucleotide genes using techniques such as AFLP analysis.

[0282] The present disclosure also provides a method for improving agronomic traits of a plant by reducing or inhibiting the expression or activity of NtNTP2-T or NtNTP2-T and NtNTP2-S, wherein the NtNTP2-T and NtNTP2-S comprise or consist of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:11; or (iii) a polypeptide encoded by the polynucleotide shown in (i) or (ii); or (iv) a NtNTP2-S polypeptide having at least 77% sequence identity to SEQ ID NO:7; or (v) a NtNTP2-T polypeptide having at least 77% sequence identity to SEQ ID NO:8. NO:12 has at least 77% sequence identity; wherein the expression or activity of NtNTP2-T or the expression or activity of NtNTP2-T and NtNTP2-S is reduced or inhibited compared to control plants. Suitably, the agronomic traits are: (i) nitrate levels are not reduced; (ii) biomass (e.g., leaf biomass) is increased under both standard and nitrogen starvation conditions; and (iii) NUE response (meaning biomass per unit of nitrogen fertilization applied) is increased. Any method described herein can be used to reduce or inhibit the expression or activity of NtNTP2-T or NtNTP2-T and NtNTP2-S. As an example, one or more sequence-specific polynucleotides that can interfere with the transcription of NtNTP2-T or NtNTP2-T and NtNTP2-S can be used. As a further example, one or more sequence-specific polypeptides that can interfere with the stability of NtNTP2-T or NtNTP2-T and NtNTP2-S can be used. As an example, one or more sequence-specific polynucleotides that can interfere with the enzymatic activity of NtNTP2-T or NtNTP2-T and NtNTP2-S or the binding activity of NtNTP2-T or NtNTP2-T and NtNTP2-S with respect to substrates or regulatory proteins. As a further example, gene-edited NtNTP2-T or NtNTP2-T and NtNTP2-S can be used. Suitably, NtNTP2-T or NtNTP2-T and NtNTP2-S are gene-edited using a bacterial CRISPR / Cas system. As a further example, at least one genetic change in the NtNTP2-T polynucleotide sequence or at least one genetic change in the NtNTP2-T polynucleotide and NtNTP2-S polynucleotide sequence or at least one genetic change in the NtNTP2-T polypeptide sequence or at least one genetic change in the NtNTP2-T polypeptide sequence and NtNTP2-S polypeptide sequence can be used.Suitably, at least one genetic change may be at least one genetic change that causes the encoded polypeptide to terminate or end translation earlier than in a control plant. As a further example, at least one genetic change may reduce or inhibit the expression or activity of NtNTP2-T or NtNTP2-T and NtNTP2-S. At least one genetic change may comprise at least one nonsense mutation in a NtNTP2-T polynucleotide or NtNTP2-T polypeptide or at least one nonsense mutation in a NtNTP2-T polynucleotide or NtNTP2-T polypeptide and at least one nonsense mutation in a NtNTP2-S polynucleotide or NtNTP2-S polypeptide. For example, the mutation may be a single nucleotide polymorphism in NtNTP2-S at nucleotide position 632 or 633 or 632 and 633 of SEQ ID NO: 3, suitably, wherein the single nucleotide polymorphism is a 'g' to 'a' mutation at nucleotide position 632 or 633 of SEQ ID NO: 3, or a 'g' to 'a' mutation at nucleotide positions 632 and 633 of SEQ ID NO: 3. The mutated NtNTP2-S polynucleotide sequence may comprise, consist of or consist essentially of SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6. Suitably, the mutation may be a single nucleotide polymorphism in NtNTP2-T at nucleotide position 636 of SEQ ID NO: 11, wherein the single nucleotide polymorphism is a 'g' to 'a' mutation at nucleotide position 635 or 636 of SEQ ID NO: 11, or a 'g' to 'a' mutation at nucleotide positions 635 and 636 of SEQ ID NO: 11. The mutated NtNTP2-T polynucleotide sequence may comprise, consist of or consist essentially of SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15. The mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and the mutated NtNTP2-S polypeptide may have at least one nonsense mutation at position W212 or W212 and W211, respectively. The mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and the mutated NtNTP2-S polypeptide may comprise, consist of or consist essentially of SEQ ID NO: 16 or SEQ ID NO: 8 and SEQ ID NO: 16, respectively. The sequences deposited in the database are described herein and may change over time. Suitably, according to the current version of the sequence database. Alternatively, according to the disclaimer in effect on the filing date. As known to the skilled person, the accession number may be a version / date accession number. The citable accession number of the current database entry is the same as here, but the decimal point and any subsequent digits are omitted.GenBank is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences (National Center for Biotechnology Information, US National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA; Nucleic Acids Research, 2013 Jan; 41 (D1): D36-42), unless otherwise stated, the accession numbers provided are related thereto. Suitably, according to the current version. More suitably, according to the version available on the effective filing date. Most suitably, the GenBank database version referred to is NCBI-GenBank version 241: December 15, 2020. Suitably, according to the current version of the sequence database. Alternatively, according to the disclaimer effective on the filing date. For the avoidance of doubt, according to the UniProt Knowledge Base (UniProtKB) 2021_01 version released on February 10, 2021 by the UniProt Consortium European Bioinformatics Institute (EBI), SIB Swiss Bioinformatics and Protein Information Resource Institute (PIR). UniProt (Universal Protein Resource) is a comprehensive protein information directory ("UniProt: the universal protein knowledgebase" Nucleic Acids Res. 45: D158-D169 (2017)).

[0283] The invention is further described in the following examples, which are provided to describe the invention in more detail. These examples set forth preferred modes presently contemplated for carrying out the invention and are intended to illustrate but not to limit the invention.

[0284] Example

[0285] Example 1 - EMS for breeding of ntp2 mutation and ntp2-S W211stop / ntp2-T W212stop double mutation Mutant population screening

[0286] To find specific mutations in the EMS-generated AA37 mutant population, primers covering parts of the NtNtp2-S and NtNtp2-T gene sequences were designed. The resulting amplified fragments were sequenced. The primer pairs used to identify ntp2 termination mutants are reported in Table 1.

[0287] The point mutation that causes the stop codon and therefore causes the non-functional version of the truncation of protein is considered to be interested.The stop mutant list that identifies is reported in Table 2.This table reports the details of the stop codon mutation identified during the EMS screening.F seq and R seq hurdles indicate SNP (single nucleotide polymorphism) mutation (respectively at 5 ' and 3 ' direction of mutation).Original wild-type codon and corresponding amino acid (codon ori and AS ori hurdles) and corresponding mutation pattern (codon mut and AS mut hurdles) are also shown.

[0288] TaqMan analysis was used to screen for different ntp2 mutations and genotypes. Table 3 reports the details of the primers and probes used to genotype the AA37 ntp2W211stop and W212stop double mutant lines. The identity column indicates the gene and phenotype identified (wt indicates the wild-type genotype, mut indicates the mutant genotype; tAg and tgA indicate two different mutations identified in the NtNtp2-S gene). The F primer and R primer columns indicate the primer sequences of the forward primer and reverse primer, respectively. The probe column reports the sequence of the probe used (underlined bases indicate the difference between wt and different mutations).

[0289] The double mutant Ntntp2-S W211stop / Ntntp2-T W212stop in the AA37 background was generated by crossing a single mutant plant Ntntp2-S W211stop (tgA mutation) with a single mutant plant Ntntp2-T W212stop.

[0290] Example 2 - Field trial design and execution

[0291] Field trials were held for two seasons. Seed breeding, sawing and seedling growth were carried out in a greenhouse according to ID-160: Burley and Dark Tobacco Production Guide, 2021-2022 (uky.edu) Burley and Dark Tobacco, 2022 Flue-cured Tobacco Guide | North Carolina Virginia Tobacco Standards Extension Publishing Company (ncsu.edu). Ten plant plots per genotype were transplanted in a randomly distributed field design with 7 to 19 replicates per genotype, depending on the growing season. The first field trial was conducted under burley conditions with a total input of 250 nitrogen units applied, one unit expressed as kg / hectare. Plants were topped before flowering and at harvest, 5 mid-stem leaves per plant were harvested and hung to air dry for curing. Two identical randomly distributed fields were implemented under different nitrogen input conditions: one under burley tobacco conditions (254 nitrogen units, expressed as units of kg / ha) and one under nitrogen starved Virginia tobacco conditions (55 nitrogen units, i.e. 25% of the nitrogen input compared to burley tobacco conditions). For each genotype, 19 replicates were grown per field. Plants were topped before flowering and stems were dried after a few weeks.

[0292] Example 3 - Analysis of Dried Materials

[0293] After curing, the number of plants and the weight of dried leaf material in each plot were recorded for biomass detection. Standard drying procedures are described in ID-160: Guide to Burley and Dark Tobacco Production, 2021-2022 (uky.edu) Burley and Dark Tobacco, 2022 Flue-cured Tobacco Guide | North Carolina Virginia Tobacco Extension Publishing Company (ncsu.edu). All leaves are separated from the stem and weighed, removing the material of the marginal plants to avoid position effects. Representative leaf samples are collected from 10 to 15 middle stem leaves in each plot for determination of nitrate levels. The samples are freeze-dried and crushed into powder. Nitrite-nitrate content (expressed as nitrate levels in this report) was measured using the Cayman Chemical Nitrate / Nitrite Colorimetric Assay Kit (Item No. 780001) according to the supplier's instructions and as described in FASEB Journal (1992) 6, 3051-3064; Anal. Biochem. (1982) 126, 131-138; and Methods (1995) 7, 48-54.

[0294] Example 4 - NtNtp2 gene and protein sequences

[0295] Two NtNtp2 genes are present in the tobacco genome, one on chromosome 8 (-S forming Ntab-TN90_AYMYSS948 in the publicly available TN90 genome) and the other on chromosome 21 (-T forming Ntab-TN90_AYMYSS1317). The genomic, transcript, gene and protein sequences of the NtNtp2 genes are shown in the accompanying sequence listing.

[0296] The NtNTP2-T putative protein sequence was BLASTed against protein databases from different plant species (Eggplant, Arabidopsis, Nicotiana tabacum and Solanum lycopersicum) and the highest homology is reported in Table 4. Homologous proteins (predicted proteins) and the degree of identity expressed as the percentage of identical residues are reported. Table 5 reports the degree of identity to the respective predicted coding regions (CDS) expressed as the percentage of identical residues.

[0297] Example 5 - Plant phenotype and nitrate levels in the ntp2-S W211stop / ntp2-T W212stop double mutant flat

[0298] AA37 tobacco plants were grown in the field under the burley tobacco regimen (254 nitrogen units, as described above) during two consecutive growing seasons. At harvest, by observing the plants in the field, there were no obvious morphological differences between the double mutant and wild-type out-segregating genotypes, as shown in Table 1. Figure 1 as shown in .

[0299] Nitrate levels in dried leaves and midribs of mid-stem leaves were measured as averages per plot (n = 7 to 8 plots, 10 plants per plot) and recorded in Figure 2 No significant differences in nitrate levels were detected between the out-segregating wt and double ntp2 mutant genotypes (p-value = 0.21361 for leaf data and p-value = 0.8485846 for midrib data in a two-tailed Student's test).

[0300] In contrast, in Arabidopsis, ntp2 insertion mutants had 50-64% lower nitrate levels in petioles and midribs than wild type and 13% higher nitrate levels in leaves than wild type, which was statistically significant (Chiu et al. (2004) supra).

[0301] From these results, there were no statistically significant differences in leaf nitrate content between homozygous mutants and wild-type plants, but not in the midrib.

[0302] In conclusion, attenuation of the -S and -T forms of NtNTP2 protein activity did not significantly affect nitrate levels in lamina and midribs of cured leaves of AA37 tobacco from plants grown under Burley conditions.

[0303] Example 6 - Yield effect of Ntp2-S W211stop / ntp2-T W212stop mutants.

[0304] Two parallel fields were run under Burley and Virginia tobacco fertilization conditions (254 N units and 55 N units, i.e., 100% and 25% N inputs, respectively). The double mutant and segregating wild-type plants grown under Burley tobacco conditions showed no obvious phenotypic differences (see Figure 3 , upper panel). When grown under nitrogen starvation conditions (Virginia tobacco protocol, 25% nitrogen units compared to Burley tobacco conditions), ntp2 double mutant plants appeared slightly larger than their segregating wild-type counterparts, with more developed leaves (see Figure 3 , figure below).

[0305] Plants were topped and stemmed, and leaf dry biomass was recorded for all commercially valuable leaf stages. Figure 4 The yield data reported in report the yield phenotype of the ntp2 double stop mutant in different nitrogen regimes. In the standard burley tobacco fertilization regime, the ntp2 double stop mutant plants produced 16.2% more leaf biomass than their segregating wild type (p value = 0.00654 in a two-tailed Student's test). In the nitrogen starvation regime, the increase in biomass was even higher, reaching 28.3% (p value = 0.003184) compared to the wild type.

[0306] It was concluded that the characteristic phenotype of loss of NtNTP2 activity is increased yield under the different nitrogen regimes tested.

[0307] In Chiu et al. (2004), supra, the authors demonstrated that Arabidopsis homozygous Atntp2 insertion mutants developed wider and longer leaves compared to wild-type plants due to increased cell proliferation. Interestingly, the current field results show that in tobacco AA37 plants, loss of NtNTP2 protein function not only resulted in higher yields under standard fertilization regimes but also limited biomass losses due to growth under nitrogen starvation. In fact, as Figure 4 As shown in , the ntp2 double-stop mutant plants had reduced yield differences under standard and nitrogen starvation conditions compared to the segregating wild-type genotype (30.8% vs. 37.3%), and the mutants had statistically significantly higher dry biomass compared to wild-type plants under all conditions (see Figure 4 ).

[0308] During the second experiment, segregation tests were performed to determine the contribution of the -S and -T mutations to the yield phenotype. Figure 5Total dry biomass data in grams per plant are reported for different segregant phenotypes of the Ntntp2 mutation. Ntntp2-S W211stop and Ntntp2-T W212stop double mutant plants (sstt) showed an average total dry leaf biomass increase of 9.4% per plant compared to the segregant wild type (SSTT). In the case of the ntp2-S W211stop single mutant ( Figure 5 In experiments with ssTT in the culture medium, no biomass effect of ntp2 loss of function was detected.

[0309] Therefore, loss of function of the NTP2-S protein is not sufficient to obtain this phenotype. Loss of function of the NTP2-T protein alone is sufficient to produce a yield-enhanced phenotype in AA37 tobacco plants (SStt), with a 13.9% increase in yield compared to the double segregating wild type (SSTT), p value = 0.04545133.

[0310] Example 7 - Ntp2-S W211stop / ntp2-T W212stop mutant NUE

[0311] from Figure 4 From the dry biomass data reported in , the difference in biomass between standard fertilization and nitrogen starvation conditions (25% N units compared to standard conditions) was reduced for the ntp2 double stop mutant plants compared to the out-isolated wild type (30.8% vs. 37.3%, respectively). Therefore, we calculated the NUE index, i.e., the units of biomass (expressed as kg / ha, assuming 12,000 plants per ha) produced per unit of nitrogen fertilization input (expressed as kg of nitrogen per ha), as indicated in Table 6. The total dry biomass harvested per ha was reported for each ntp2 genotype (Genotype), expressed as kg (biomass) considering 12,000 plants per ha, the available nitrogen units in the different fertilization schemes were expressed as kg of nitrogen per ha (N units), and the NUE index was calculated as biomass per N unit applied.

[0312] As expected, when grown under nitrogen starvation conditions, both genotypes increased their NUE index, meaning the biomass per unit of nitrogen fertilization applied.

[0313] As shown in Table 6 and Figure 6 As shown in, under nitrogen starvation conditions, the NUE index of the ntp2 double-terminator mutant plants was 16% higher than that of the segregating wild-type genotype (3.97 vs. 4.61), and under normal nitrogen conditions, the NUE index of the ntp2 double-terminator mutant plants was 28% higher than that of the segregating wild-type genotype (11.49 vs. 14.75).

[0314] Therefore, impairment of NTP2 protein activity in tobacco increases the ability of plants to adapt to nitrogen starvation, thereby increasing plant NUE.

[0315] Example 8 - Screening

[0316] To select potential candidate targets for increasing NUE in tobacco, microarray data were obtained for the identification of gene candidates that were differentially expressed in green and mature leaves of different tobacco varieties. The expression dynamics of Swiss Burley tobacco material (known to exhibit low NUE) during air-curing were compared with the corresponding features of other tobacco varieties with higher nitrogen use efficiency (such as Virginia-type tobacco) during maturity and respective early curing processes. The selection of candidate genes was based on literature search and their significance in nitrogen assimilation pathways and transport. Genes that were differentially expressed between Virginia and Burley tobacco were the focus. The NtNtp2 gene was identified as being highly expressed in Burley tobacco green mature leaves and at harvest; in Virginia tobacco, the expression level increased during curing, reaching Burley tobacco levels, such as Figure 7 as shown in .

[0317] Example 9 - Root Phenotype

[0318] To further understand the mechanism of increased NUE in the NtNTP2 mutant, root development was monitored in seedlings grown on agar plates and in solution culture. Figure 8 In both cases, NtNTP2-T homozygous mutants and NtNTP2-S and NtNTP2-T double mutant plants showed an increase in root development, expressed as the number of lateral roots (A) or the maximum length of aquatic roots (B). Impairment of NtNTP2 activity therefore leads to an increase in root development.

[0319] Example 10 - Mutant Ntntp2-S W211stop / Ntntp2-T in greenhouse experiments W212stop root phenotype

[0320] Ntntp2-S W211stop / Ntntp2-T W212stop BC2S2 TN90 and K326 mutant plants and their wild-type segregant controls were grown in a greenhouse in a hydroponic solution with standard 50% nitrogen fertilizer (N50%). The number and average diameter of primary roots emerging from the stele were recorded for each plant at 4 to 6 weeks after transplanting. Fig. 9 As shown in , TN90 (Burley type) and K326 (Virginia type) mutant plants displayed a statistically validated higher number of primary lateral roots compared to their respective out-segregating controls (+39.7% for TN90, p-value 0.00083, and +47.7% for K326, p-value 0.000587).

[0321] For TN90 plants, the diameter of the main lateral roots of plants grown in N50% was measured using a thickness meter (Mitutoyo ABSOLUTE, Mitutoyo Europe GmbH, Borsigstraβe 8-10 D-41469 Neuss). Fig.10 As shown in , mutant plants produced primary roots that were 20% thinner than the abnormal wild type.

[0322] Surprisingly, impaired NtNTP2 activity resulted in changes in root development, producing more numerous and finer roots compared to wild-type plants grown under the same conditions, as Fig.11 This can increase the plant's ability to absorb nutrients from the soil.

[0323] Example 11 - Transgenic constructs for RNAi plants

[0324] A fragment of the gene coding region of NtNtp2-T was selected for designing RNA interference (RNAi) constructs. Fig.12 The RNAi loop was synthesized as shown in . The DNA sequence is shown in SEQ ID NO: 45. The RNAi loop was cloned into a binary vector carrying an MMV promoter and a translation enhancer (see WO2012 / 098111) via HindIII-AvrII restriction sites to obtain Fig.12 Constructs as indicated.

[0325] Tobacco TN90 cells were transformed with Agrobacterium tumefaciens carrying a binary vector of RNAi of NtNtp2 and plants were regenerated on kanamycin. Transgenic TN90 T2 seedlings for RNAi constructs (RNAi-T2) and empty vectors (control plants, CT-T2) were selected on kanamycin and transplanted into a greenhouse to grow in a hydroponic solution fertilized with 50% nitrogen, compared to standard practice (N50%). At 4 to 6 weeks after transplantation, the number of primary roots growing from the pith of each plant and the average diameter were recorded. Fig.14 As shown in , transgenic plants of the RNAi constructs showed a statistically validated higher number of primary lateral roots (+39.7% for TN90, p-value 0.00083, and +47.7% for K326, p-value 0.000587) compared to their respective out-segregating controls.

[0326] The diameters of primary lateral roots of RNAi and control plants grown in N50% were measured using a thickness meter (Mitutoyo ABSOLUTE, Mitutoyo Europe GmbH, Borsigstraβe 8-10 D-41469 Neuss). Fig.15As indicated in , RNAi plants produced thinner primary roots compared to the out-isolated wild type.

[0327] Example 12 - Conclusion

[0328] Impairment of NtNTP2 protein activity did not determine a decrease in nitrate levels in dried leaves or midribs of AA37 tobacco plants grown under Burley conditions, nor did it determine major changes in the amounts of other chemical compounds measured.

[0329] The phenotype of the Ntntp2 double-stop mutation or the Ntntp2-T homozygous mutation in tobacco is an increase in dry leaf biomass under standard and nitrogen starvation conditions when compared to out-isolated wild-type plants.

[0330] Another phenotype of the Ntntp2 double stop mutation or Ntntp2-T homozygous mutation in tobacco is an increase in the plant's NUE response when expressed as kg of dry leaf biomass produced per kg of nitrogen input per hectare. This makes the NtNtp2 gene a good target for NUE solutions.

[0331] The phenotype of RNAi-engineered NtNtp2-T tobacco plants with impaired NtNTP2 expression and / or activity is a change in root development, producing more and thinner roots compared to wild-type plants grown under the same conditions. This could increase the plant's ability to absorb nutrients from the soil.

[0332] Any publication cited or described in this article provides relevant information disclosed before the filing date of this application. Statements herein should not be interpreted as admitting that the inventor loses the qualification prior to such disclosure. All publications mentioned in the above specification are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the present invention will be apparent to those skilled in the art. Although the present invention has been described in conjunction with specific preferred embodiments, the claimed present invention should not be unduly limited to such specific embodiments. In fact, different improvements to the described modes for realizing the present invention that are apparent to those skilled in the art of cell biology, molecular biology and plant biology or related fields are intended to be within the scope of the following claims.

[0333] Table 1

[0334] Primers used to screen the AA37 EMS-mutated tobacco population for the ntp2 mutation search.

[0335] Gene Forward primer (5' to 3') Reverse primer (5' to 3') NtNtp2-S GAATAGCAGTAAATCTGGTGACG CACACCATGTTAAGAGTATTTTAAG NtNtp2-T TGAGGCCGTCAATATTAATAATGT ACACCATGTTAAGAGTATCTTCGA

[0336] Table 2

[0337] Exemplary mutations resulting in stop codons and truncated NTP2-T and -S proteins.

[0338]

[0339] Table 3

[0340] TaqMan primers and probes validated for use in developing AA37 ntp2 W211stop and W212stop double mutant lines.

[0341]

[0342] Table 4

[0343] BLAST results of NtNTP2 protein

[0344] Plant species Predicted proteins Identity (%) Arabidopsis AT2G26690.1 76 Roundleaf tobacco Niben261Chr10g0848012.1 88 Roundleaf tobacco Niben261Chr04g0558005.1 70 Roundleaf tobacco Niben261Chr01g1573001.1 74 tomato SOLYC07G032490.3.1 84 tomato SOLYC01G096880.4.1 79

[0345] Table 5

[0346] BLAST results of NtNtp2 CDS

[0347] Plant species Predicted CDS Identity (%) Arabidopsis AT2G26690.1 69 Roundleaf tobacco Niben261Chr10g0848012.1 88 Roundleaf tobacco Niben261Chr04g0558005.1 81 Roundleaf tobacco Niben261Chr01g1573001.1 76 tomato SOLYC07G032490.3.1 85 tomato SOLYC01G096880.4.1 78

[0348] Table 6

[0349] Calculation of NUE index for field results.

[0350]

[0351] sequence

[0352] SEQ ID NO:1-NtNtp2-S genome sequence

[0353]

[0354] SEQ ID NO:2—NtNtp2-S transcript sequence (start and stop codons are shown in bold)

[0355]

[0356]

[0357] SEQ ID NO:3—NtNtp2-S gene sequence

[0358]

[0359] SEQ ID NO:4-SEQ ID NO:3 NtNtp2-S gene sequence, g to a mutation is shown in bold (nucleotide 632

[0360] g to a mutation)

[0361]

[0362]

[0363] SEQ ID NO:5 - NtNtp2-S gene sequence of SEQ ID NO:3, with the g to a mutation shown in bold (g to a mutation at nucleotide 633)

[0364]

[0365] SEQ ID NO:6 - NtNtp2-S gene sequence of SEQ ID NO:3, with the g to a mutation shown in bold (g to a mutation at nucleotides 632 and 633)

[0366]

[0367]

[0368] SEQ ID NO:7—NtNtp2-S polypeptide sequence, encoded by SEQ ID NO:3

[0369] MSWTVSDAVDYKGSPADKSKTGGWVPAALILGIEIVERLSTMGIAVNLVTYLGGTMHLPSSTSANIVTDFMGTSFLLCLLGGFLADSFLGRYKTIVIFALIQTLGTGMLTLAVSLPQLRPPLCHRHDVNCQPAKGSQMGILYMALYLIALGTGGLKSSVSGFGTDQFDEKDDKEKAQMAYFFNRFFFFISIGTLTAVTVLVYIQDEVGRSWAYGVCSVSMLIAILIFLLGTRRYRYKKSSGSPIVHIFQVIFAAIRKRKMDLPYDVGMLYETTNSEATRIQHTQEFRVLDKAAIVAEGDFEDQYGSCSAPNPWKLSTVTRVEEVKMMARLLPIWATTIIFWTTYAQMITFSVEQASTMERSIGNFQIPAGSLTVFFVAAILITLAVYDRIIMPLWKKWKGKPGFSSLQKISIGLVLSIIGMAAAALAEKKRLTVAKSVGRHSSSTNLPISVFFLIPQFFLVGAGEAFIYTGQLDFFITQSPKGMKTMSTGLFLTTLSLGFFISSFLVSVIKKVTGGNGTDEGWLADNINNGRLDLFYWLLAVLGVINFVIYLICATWYKPRKAKSAIQMENPHTKNAAEEKC*

[0370] SEQ ID NO:8 - NtNtp2 - S truncated polypeptide, encoded by SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6; stop codon at position W211 of SEQ ID NO:7

[0371] MSWTVSDAVDYKGSPADKSKTGGWVPAALILGIEIVERLSTMGIAVNLVTYLGGTMHLPSSTSANIVTDFMGTSFLLCLLGGFLADSFLGRYKTIVIFALIQTLGTGMLTLAVSLPQLRPPLCHRHDVNCQPAKGSQMGILYMALYLIALGTGGLKSSVSGFGTDQFDEKDDKEKAQMAYFFNRFFFFISIGTLTAVTVLVYIQDEVGRS -

[0372] SEQ ID NO:9 - NtNtp2 - T genomic sequence

[0373]

[0374] SEQ ID NO:10—NtNtp2-T transcript sequence (start and stop codons are shown in bold)

[0375]

[0376] SEQ ID NO:11-NtNtp2-T gene sequence

[0377]

[0378] SEQ ID NO:12—NtNtp2-T polypeptide sequence, encoded by SEQ ID NO:11

[0379] *

[0380] SEQ ID NO: 13 - NtNtp2-T gene sequence of SEQ ID NO: 11, with the g to a mutation shown in bold (g to a mutation at nucleotide 636 resulting in a tga stop codon)

[0381]

[0382] NtNtp2-T gene sequence of SEQ ID NO:14 to SEQ ID NO:11, with the g to a mutation shown in bold (g to a mutation at nucleotide 635 resulting in a tag stop codon)

[0383]

[0384]

[0385] NtNtp2-T gene sequence of SEQ ID NO:15 to SEQ ID NO:11, with the gg to aa mutation shown in bold (g to a mutation at nucleotide 635 and g to a mutation at nucleotide 636 resulting in a taa stop codon)

[0386]

[0387] SEQ ID NO:16—NtNtp2-T truncated polypeptide sequence encoded by SEQ ID NO:13 or SEQ ID NO:14 or SEQ ID NO:15; stop codon at position W212 of SEQ ID NO:12

[0388] MMSWTVSDAVDYKGSPADKSKTGGWVPAALILGIEIVERLSTMGIAVNLVTYLGGTMHLPSSTSANIVTDFMGTSFLLCLLGGFLADSFLGRYKTIAIFALIQTLG TGMLTLAVSLPQLRPPPCHRHDVSCQPAKGSQMGILYMALYLIALGTGGLKSSVSGFGTDQFDEKDDKEKAQMAYFFNRFFFFISIGTLTAVTVLVYIQDEVGRS-

[0389] SEQ ID NO: 17—NtNtp2-S forward primer

[0390] gaatagcagtaaatctggtgacg

[0391] SEQ ID NO: 18—NtNtp2-S reverse primer

[0392] cacaccatgttaagagtattttaag

[0393] SEQ ID NO:19—NtNtp2-T forward primer

[0394] tgaggccgtcaatattaataatgt

[0395] SEQ ID NO:20—NtNtp2-T reverse primer

[0396] acaccatgttaagagtatcttcga

[0397] SEQ ID NO:21-ntp2-T Sequence in the 5' position of the W212stop SNP mutation

[0398] acggagctg

[0399] SEQ ID NO:22-ntp2-T Sequence in the 3' position of the W212stop SNP mutation

[0400] gcttggtg

[0401] SEQ ID NO:23 - Sequence in the 5' position of ntp2-T W212stop SNP mutation

[0402] acggagct

[0403] SEQ ID NO:24—Sequence in the 3' position of ntp2-T W212stop SNP mutation

[0404] ggcttggtg

[0405] SEQ ID NO:26 NtNTP2-S wt reverse primer

[0406] cggagcttttcttgtatcggtacc

[0407] SEQ ID NO:27-NtNTP2-S wt forward primer

[0408] agcatagggactttgacagcagtt

[0409] SEQ ID NO:28 NtNTP2-S wt probe

[0410] agctgggcttacgg

[0411] SEQ ID NO:29 NtNTP2-S mut tAg forward primer

[0412] agcatagggactttgacagcagtt

[0413] SEQ ID NO:30 NtNTP2-S mut tAg reverse primer

[0414] cggagcttttcttgtatcggtacc

[0415] SEQ ID NO:31 NtNTP2-S mut tAg probe

[0416] agctaggcttacggtgt

[0417] SEQ ID NO:32 NtNTP2-S mut tgA forward primer

[0418] agcatagggactttgacagcagtt

[0419] SEQ ID NO:33 NtNTP2-S mut tgA reverse primer

[0420] cggagcttttcttgtatcggtacc

[0421] SEQ ID NO:34 NtNTP2-S mut tga probe

[0422] agctgagcttacggtgt

[0423] SEQ ID NO:35 NtNTP2-T wt forward primer

[0424] catagggactttgacagcagtaacc

[0425] SEQ ID NO:36 NtNTP2-T wt reverse primer

[0426] ccgagcttttcttgtatcggtact

[0427] SEQ ID NO:37 NtNTP2-T wt probe

[0428] agctgggcttatgg

[0429] SEQ ID NO:38 NtNTP2-T mut forward primer

[0430] catagggactttgacagcagtaacc

[0431] SEQ ID NO:39 NtNTP2-T mut reverse primer

[0432] ccgagcttttcttgtatcggtact

[0433] SEQ ID NO:40 NtNTP2-T mut probe

[0434] agctgagcttatggtg

[0435] SEQ ID NO:41 Sequence in the 5' position of ntp2-S W211stop SNP mutation

[0436] acggagctg

[0437] SEQ ID NO:42 - Sequence in the 3' position of ntp2-S W211stop SNP mutation

[0438] gcttacggtg

[0439] SEQ ID NO:43 - Sequence in the 5' position of ntp2-S W211stop SNP mutation

[0440] acggagct

[0441] SEQ ID NO:44 - Sequence in the 3' position of ntp2-S W211stop SNP mutation

[0442] ggcttacggtg

[0443] SEQ ID NO:45 DNA sequence of synthetic RNAi loop with 35S CaMV terminator

[0444]

Claims

1. A mutant, non-naturally occurring or transgenic plant or plant part having reduced or inhibited expression or activity of NtNTP2-T, or reduced or inhibited expression or activity of NtNTP2-T and NtNTP2-S, wherein the NtNTP2-T and NtNTP2-S comprise or consist of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 11; or (iii) a polypeptide encoded by the polynucleotide shown in (i) or (ii); or (iv) an NtNTP2-S polypeptide having at least 77% sequence identity to SEQ ID NO:7; or (v) an NtNTP2-T polypeptide having at least 77% sequence identity to SEQ ID NO: 12; Wherein the expression or activity of the NtNTP2-T or the expression or activity of the NtNTP2-T and the NtNTP2-S is reduced or inhibited compared with the control plant.

2. The mutant, non-naturally occurring or transgenic plant or plant part according to claim 1, wherein said plant or said plant part: (i) do not have reduced nitrate levels compared to control plants grown under the same fertilization conditions; and (ii) having increased biomass compared to said control plants grown under the same fertilization conditions; and (iii) have an increased NUE response, expressed as biomass per unit of nitrogen applied, compared to said control plants grown under the same fertilization conditions.

3. The mutant, non-naturally occurring or transgenic plant or plant part according to claim 1 or claim 2, wherein the mutant, non-naturally occurring or transgenic plant or plant part in which the expression or activity of NtNTP2-T or the expression or activity of NtNTP2-T and NtNTP2-S is reduced or inhibited comprises: (i) one or more sequence-specific polynucleotides capable of interfering with the transcription of NtNTP2-T or NtNTP2-T and NtNTP2-S; and / or (ii) one or more sequence-specific polypeptides that are capable of interfering with the stability of NtNTP2-T or NtNTP2-T and NtNTP2-S; and / or (iii) one or more sequence-specific polynucleotides that are capable of interfering with the enzymatic activity of NtNTP2-T or NtNTP2-T and NtNTP2-S or the binding activity of NtNTP2-T or NtNTP2-T and NtNTP2-S with respect to substrates or regulatory proteins; and / or (iv) gene-edited NtNTP2-T or NtNTP2-T and NtNTP2-S; and / or (v) at least one genetic alteration in the NtNTP2-T polynucleotide sequence, or at least one genetic alteration in the NtNTP2-T polynucleotide and the NtNTP2-S polynucleotide sequence, or at least one genetic alteration in the NtNTP2-T polypeptide sequence, or at least one genetic alteration in the NtNTP2-T polypeptide sequence and the NtNTP2-S polypeptide sequence, suitably, at least one genetic alteration that results in the encoded polypeptide terminating or completing translation earlier than the control plant.

4. A mutant, non-naturally occurring or transgenic plant or plant part according to claim 3(v), wherein the at least one genetic alteration is at least one mutation, suitably wherein the at least one genetic alteration comprises at least one nonsense mutation in the NtNTP2-T polynucleotide or the NtNTP2-T polypeptide or at least one nonsense mutation in the NtNTP2-T polynucleotide or the NtNTP2-T polypeptide and at least one nonsense mutation in the NtNTP2-S polynucleotide or the NtNTP2-S polypeptide.

5. A mutant, non-naturally occurring or transgenic plant or plant part according to claim 4, comprising a single nucleotide polymorphism in NtNTP2-S at nucleotide position 632 or 633 or 632 and 633 of SEQ ID NO:3, suitably wherein the single nucleotide polymorphism is a 'g' to 'a' mutation at nucleotide position 632 or 633 of SEQ ID NO:3, or a 'g' to 'a' mutation at nucleotide positions 632 and 633 of SEQ ID NO:

3.

6. The mutant, non-naturally occurring or transgenic plant or plant part of claim 5, wherein the mutant NtNTP2-S polynucleotide sequence comprises, consists of or consists essentially of SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO:

6.

7. A mutant, non-naturally occurring or transgenic plant or plant part according to claim 4, comprising a single nucleotide polymorphism in NtNTP2-T at nucleotide position 636 of SEQ ID NO: 11, suitably wherein the single nucleotide polymorphism is a 'g' to 'a' mutation at nucleotide position 635 or 636 of SEQ ID NO: 11, or a 'g' to 'a' mutation at nucleotide positions 635 and 636 of SEQ ID NO:

11.

8. The mutant, non-naturally occurring or transgenic plant or plant part of claim 7, wherein the mutant NtNTP2-T polynucleotide sequence comprises, consists of or consists essentially of SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO:

15.

9. The mutant, non-naturally occurring or transgenic plant or plant part of claim 4, wherein the mutant NtNTP2-T polypeptide or the mutant NtNTP2-T polypeptide and the mutant NtNTP2-S polypeptide each have at least one nonsense mutation at position W212 or positions W212 and W211, respectively.

10. The mutant, non-naturally occurring or transgenic plant or plant part of claim 9, wherein the mutant NtNTP2-T polypeptide or the mutant NtNTP2-T polypeptide and the mutant NtNTP2-S polypeptide comprise, consist of or consist essentially of SEQ ID NO: 16 or SEQ ID NO: 8 and SEQ ID NO: 16, respectively; optionally, wherein the mutant NtNTP2-T polypeptide or the mutant NtNTP2-T polypeptide and the mutant NtNTP2-S polypeptide are truncated.

11. A mutant, non-naturally occurring or transgenic plant or plant part according to any one of the preceding claims, wherein the plant part is selected from: (i) green leaves or parts thereof; or (ii) dried leaves or parts thereof, suitably, wherein the dried leaves or parts thereof are air-dried, suitably, sun-dried or fire-dried; or (iii) dried leaves or parts thereof, suitably, wherein the dried leaves are air-dried, more suitably, the leaves are sun-dried or fire-roasted, flue-roasted.

12. A method of preparing a plant or plant part, comprising: (a) providing a plant comprising: (i) a NtNTP2-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 11; or (iii) a polypeptide encoded by the polynucleotide shown in (i) or (ii); or (iv) an NtNTP2-S polypeptide having at least 77% sequence identity to SEQ ID NO:7; or (v) an NtNTP2-T polypeptide having at least 77% sequence identity to SEQ ID NO: 12; (b) reducing the expression or activity of the NtNTP2-T or the combination of NtNTP2-T and NtNTP2-S in the plant; and (c) obtaining plants or plant parts which: (i) do not have reduced nitrate levels compared to control plants grown under the same fertilization conditions; and (ii) have increased biomass compared to control plants grown under the same fertilization conditions; and (iii) have an increased NUE response compared to control plants grown under the same fertilization conditions.

13. A mutant, non-naturally occurring or transgenic plant or part thereof obtained or obtainable by the method of claim 12; or A mutant, non-naturally occurring or transgenic plant or part thereof, wherein nitrate levels are not significantly different compared to control plants grown under the same fertilization conditions, wherein biomass yield is greater compared to control plants grown under the same fertilization conditions, and wherein NUE of the plant is greater compared to control plants grown under the same fertilization conditions.

14. A tobacco product or smoking article comprising a mutant, non-naturally occurring or transgenic plant or plant part according to any one of claims 1 to 11, or comprising a plant or plant part according to claim 12.

15. A method for improving agronomic traits of a plant, the method comprising reducing or inhibiting the expression or activity of NtNTP2-T or NtNTP2-T and NtNTP2-S, wherein the NtNTP2-T and NtNTP2-S comprise or consist of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 11; or (iii) a polypeptide encoded by the polynucleotide shown in (i) or (ii); or (iv) an NtNTP2-S polypeptide having at least 77% sequence identity to SEQ ID NO:7; or (v) an NtNTP2-T polypeptide having at least 77% sequence identity to SEQ ID NO: 12; Wherein the expression or activity of the NtNTP2-T or the expression or activity of the NtNTP2-T and the NtNTP2-S is reduced or inhibited compared with the control plant.

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