Enhancing productivity in C3 plants

Abstract

Vascular sheath tissue-specific expression of Phytochrome B or its variants in C3 plants increases photosynthesis rate and / or introduces a carbon refixation mechanism. The heritable genetic material of C3 plant cells is modified so that one copy of Phytochrome B, or an active variant, or a functional fragment thereof, is expressed specifically in vascular sheath cells. Whole plants are regenerated from these genetically modified plant cells. Alternatively, Crispr modification of the native phytochrome locus in plant cells is used to insert vascular sheath-specific regulatory elements, such as promoter or enhancer elements, so that Phytochrome B is expressed in vascular sheath cells of the regenerated whole plants. The genetically modified whole plants have yield-increasing related traits, such as increased seed yield as a result of enhanced photosynthesis and / or the introduction of a carbon refixation mechanism.

Description

[Technical Field]

[0001] The present invention generally relates to the field of plant molecular biology and to methods for tissue-specific expression of certain genes that enhance yield-related characteristics of plants by increasing photosynthesis. The present invention relates to expression constructs useful in the methods of the present invention. The present invention also relates to genetically modified plants that have increased yield-related characteristics as a result of enhanced photosynthesis. The present invention further relates to parts of such modified plants, such as plant cells, plant parts, plant organs, fruits, seeds, embryos, embryonic traits, and processed plant products.

[0002] Inclusion by reference Each patent, publication, and non-patent document cited herein is incorporated herein by reference in its entirety, as if each were incorporated individually by reference. [Background technology]

[0003] Phytochrome B (PHYB) is a red / far-red light receptor involved in regulating numerous plant processes, such as germination, de-etinolysis, photokinetic plant development (photomorphogenesis), flowering, response to shade, and chloroplast biosynthesis. PHYB also regulates temperature responses by interacting with the promoters of major target genes in a temperature-dependent manner and subsequently repressing their expression. PHYB can function as a thermal timer, integrating temperature information for the processes of the day / night cycle.

[0004] PHYB exists in two interconvertible forms: Pr (inactive in the dark) and Pfr (active in light). Active Pfr PHYB accumulates in the nucleus after exposure to red light and functions to initiate numerous regulatory cascades that control the aforementioned plant processes. A constitutively active variant of PHYB exists known as YHB. This variant contains a single Y-to-H amino acid change at 276 sites in the Arabidopsis variety of the PHYB gene. YHB performs the same regulatory function as active PHYB but does not require light to be activated. Throughout this application, the term “active variant” in relation to PHYB means all constitutively active variants of PHYB, as well as YHB. Due to its regulatory role in multiple plant processes, all previous manipulations of PHYB or YHB have resulted in developmental abnormalities that make manipulation of the timing or location of this gene expression unsuitable for crop improvement. Recurring abnormalities resulting from manipulation of PHYB or YHB expression include dwarfism, delayed flowering, thick leaves, smaller tubers (in potatoes), decreased water use efficiency, and increased drought sensitivity. Furthermore, it has been demonstrated that when the photosynthetic rate is normalized to nitrogen investment, the photosynthetic rate does not increase in plants overexpressing PHYB or YHB.

[0005] Most of the PHYB regulatory processes found in the Arabidopsis genus, such as germination, de-yellowing, photo-mediated plant development (photomorphogenesis), flowering, response to shade, and chloroplast biosynthesis, are also regulated by PHYB in other plant species. Furthermore, many plants have genes encoding multiple orthologs of PHYB. The genomes of flowering plants also contain genes encoding other phytochromes, such as Phytochrome A (PHYA), whose gene products have antagonistic correlations with PHYB and often promote antagonistic effects, for example, in the response to shade. Plants that overexpress PHYA also have detrimental effects on plant productivity.

[0006] The following is a list of examples where overexpression of PHYB or YHB (or other related phytochrome genes) resulted in detrimental effects on plant productivity: Wagner et al., (1991) "Overexpression of Phytochrome B induces a short hypocotyl phenotype in transgenic Arabidopsis," Plant Cell. 3(12): 1275~1288. This paper describes how systemic overexpression of natural PHYB in Arabidopsis plants or rice PHYB in Arabidopsis plants alters photomorphogenesis, resulting in shortened hypocotyls and shorter plants.

[0007] Thiele et al., (1999) "Heterologous Expression of Arabidopsis Phytochrome B in Transgenic Potato Influences Photosynthetic Performance and Tuber Development," Plant Physiology. 120:73~81. This paper describes the overexpression of PHYB in potatoes. It was found to cause various negative changes in the plants. These included delayed flowering, increased branching, a greater number of smaller and thicker leaves due to larger mesophyll cells, and a slowdown in chlorophyll degradation. When the fixation rate was normalized to the unit of chlorophyll, there was no difference in carbon dioxide fixation between plants overexpressing PHYB and wild-type plants. Modified plants had negative effects, such as smaller tubers and delayed tuber formation, resulting in lower yields of modified plants than unmodified control plants under the same growth conditions.

[0008] Rao et al., (2011) "Overexpression of the phytochrome B gene from Arabidopsis thaliana increases plant growth and yield of cotton (Gossypium hirsutum)" J. Zheijiang Univ.Sci.B.12:326~334. This paper describes how overexpression of PHYB in cotton resulted in faster growth, but also caused numerous negative effects, such as a quadrupling of transpiration rates (i.e., making the plant more drought-sensitive and less water-use efficient), dwarfism, fleshy leaves, and consequently more branching, as well as a decrease in apical dominance.

[0009] Halliday et al., (1997) "Expression of heterologous phytochromes A, B or C in transgenic tobacco plants alters vegetative development and flowering time," The Plant Journal 12:1079~1090. This paper describes the overexpression of PHYB in tobacco, which results in negative effects such as delayed flowering and dwarfism.

[0010] Husaineid et al., (2007) "Overexpression of homologous phytochrome genes in tomato: exploring the limits in photoperception," J. Exp.Bot.58:615~626. This paper describes tomato lines that overexpress PHYA, PHYB1, or PHYB2 under the control of a constitutive dual-35S(CaMV) promoter. This results in the negative effects of dwarfism and increased anthocyanin production.

[0011] Holefors et al., (2000) "The Arabidopsis phytochrome B gene influences growth of the apple rootstock M26," Plant Cell Reports 19:1049~1056. This paper describes the overexpression of the M26 gene in the apple rootstock M26 (Malus domestica). This results in negative effects such as reduced stem length and decreased dry mass of shoots, roots, and plants.

[0012] Distefano et al., (2013) "Ectopic expression of Arabidopsis Phytochrome B in Troya citrange affects photosynthesis and plant morphology." Scientia Horticulturae 159:1~7. This paper explains how overexpression of PHYB in citrus fruits increases photosynthetic gene expression and leaf chlorophyll content, as well as stomatal density, alters branching angles, and decreases photosynthetic rate.

[0013] Zheng et al., (2001) "Modification of Plant Architecture in Chrysanthemum by Ectopic Expression of the Tobacco Phytochrome B1 Gene," J. Am.Hort.Soc.Sci.126(1):19~26. This paper describes the ectopic expression of the tobacco PHYB1 gene in Chrysanthemum under the control of the CaMV 35S promoter. The resulting plants showed negative effects, such as shorter height due to larger branching angles than wild-type plants. The effect of PHYB1 expression was comparable to that of commercially available growth inhibitors, and therefore the authors suggest that the application of PHYB1 overexpression could be an alternative to the application of exogenous growth inhibitors.

[0014] Yang et al., (2013) "Deficiency of Phytochrome B alleviates chilling-induced photoinhibition in rice," Am.J.Bot. 100(9):1860~1870. This paper describes how mutant rice plants with reduced PHYB expression experienced less photoinhibition during and after chilling stress than wild-type plants, and also exhibited significantly higher optical system II efficiency and chlorophyll content than wild-type control plants. Thus, this paper demonstrated that reducing PHYB expression leads to increased photosynthesis. These findings suggest that crop improvement should follow a strategy of reducing PHYB expression rather than increasing it.

[0015] Su and Lagarias (2007) "Light-Independent Phytochrome Signaling Mediated by Dominant GAF Domain Tyrosine Mutants of Arabidopsis Phytochromes in Transgenic Plants. Phytochrome B-Y276H (YHB)" The Plant Cell, Vol 19:2124~2139. This paper describes a variant form of the Arabidopsis thaliana PHYB protein known as YHB, in which tyrosine (Y) at position 276 is converted to histidine (H). The Y276H mutant is profluorescent and photoinsensitive. When YHB is expressed in plants, various altered photosignaling activities are found associated with this mutation, resulting in small, stunted plants.

[0016] U.S. Patent No. 8,735,555 discloses a mutant phytochrome that alters the photomorphogenic characteristics of plants when introduced into the genus Arabidopsis. The Y276H mutant of PHYB is described as being photostable in plants and consequently resulting in altered photomorphogenesis compared to the same species or varieties lacking the mutant. Transgenic plants expressing the mutant Y276H Arabidopsis phytochrome showed reduced shade avoidance and altered photomorphogenesis resulting in dwarfism compared to the same species of plants lacking the mutant phytochrome.

[0017] Hu et al., (2019) "Regulation of monocot and dicot plant development with constitutively active alleles of phytochrome B." Plant Direct, 4:1~19. This paper describes experiments in which either Arabidopsis YHB or rice YHB is overexpressed in Arabidopsis, rice, tobacco, tomato, and Alpinia species. In all cases, a series of developmental changes were consistently induced, resulting in altered plant structure and reduced plant height. Furthermore, both shoot branching and seed yield were negatively affected by YHB overexpression in all of these species.

[0018] U.S. Patent Application Publication 2004 / 0268443 (Wu et al.) describes increasing the accumulation of heterologous PHYAs in plants, such as Basmati rice, to alter plant structure and, as a result, minimize or overcome the plant's shade-avoidance response. More specifically, the elite indica rice Pusa Basmati-1 ("PBNT") was transformed with Arabidopsis PHYAs under the control of a light-regulated tissue-specific rice RbcS promoter, resulting in numerous independent transformation lines. Results from the fifth-generation (generation "T4") homozygous transformation line showed high levels of PHYA accumulation and altered plant structure in the leaves of light-grown plants compared to unmodified plants.

[0019] U.S. Patent Application Publication 2005 / 0120412 discloses long-day plants modified to overexpress PHYA or PHYB proteins in at least a subset of plant cells such that their flowering shoots, flowers, blossoms, seeds, or fruits develop in substantially shorter days than the corresponding flowering shoots, flowering pots, blossoms, seeds, or fruits in similar unmodified long-day plants. An expression cassette containing a phytochrome coding sequence under the control of a functional promoter is provided. In particular, the cauliflower mosaic virus (CaMV) 35S promoter is used.

[0020] Chinese Patent Application Publication No. 106854240 (BIOTECHNOLOGY RES CENTER SHANDONG ACAD OF AGRICULTURAL SCIENCES) discloses the nucleotide and amino acid sequences of the peanut phytochrome AhphyB. Phytochrome AhphyB is proposed to modulate and control the high-dose response to shade. AhphyB in peanuts is expressed in the Arabidopsis genus, and the effect of light conditions on hypocotyl elongation is tested. Upregulation of phyB expression is proposed to control peanut pod development and to enable the growth of high-yielding peanut varieties in a maize-peanut intercropping mode.

[0021] International Publication No. 2005093054(A1) (KANSAI TECH LICENSING ORG) discloses how the N-terminal region of phytochrome molecules possesses nuclear signal transduction capabilities. The N-terminal fragment of phytochrome fused with domains involved in quantification and nuclear localization exhibits more than 100 times higher photosensitivity than the full-length phytochrome molecule. This artificial phytochrome molecule is used to modify plants, such as rice, to enhance photosensitivity, resulting in increased pigmentation, extended flowering period, ovary enlargement, or stem enlargement.

[0022] International Publication No. 99 / 31242 (A1) (KWS) relates to plants that overexpress phytochrome B by introducing the phytochrome B gene into plants or activating the phytochrome B gene of plants. The chimeric Arabidopsis thaliana phyB gene was transformed into potato plants by Agrobacterium tumefaciens-mediated gene transfer. Transgenic plants expressing phytochrome B derived from Arabidopsis thaliana exhibit dwarfism, reduced apical dominance, and dark green leaves. Various phenotypic changes were thought to be associated with an increase in photosynthetic output. In the transformed plants, an increase in the number and yield of tubers was found. Transformation of potato with phytochrome B derived from Solanum tuberosum can also improve the characteristics of the plant, but it can improve fewer characteristics than the gene derived from Arabidopsis thaliana.

[0023] US Patent Application Publication No. 2007295252 (Dasgupta) discloses nucleic acid molecules identified from Zea mays, such as promoters, leaders, and enhancers, and combinations of such regulatory elements in chimeric molecules. The identified regulatory elements are derived from the fructose 1-6 bisphosphate aldolase (FDA), pyruvate orthophosphate dikinase (PPDK), or ribulose bisphosphate carboxylase activase (RCA) genes. The regulatory element molecules preferably regulate the transcription of genes in leaf tissue. The regulatory element includes such regulatory elements in the form of promoters, enhancers, leaders, and combinations of such regulatory elements in chimeric or hybrid expression elements. Transgenic maize plants and seeds containing the DNA construct, which includes a promoter and a regulatory element operably linked to a heterologous DNA molecule, are described, and by these, the transgenic plants exhibit an agriculturally desirable phenotype.

[0024] Chinese Patent Application Publication No. 108913717 (UNIV HENAN) discloses a Crispr-Cas9-based rice phytochrome PHYB gene editing vector. The vector is used to mutate the rice phytochrome PHYB gene without mutating other genes in the plant. Four mutant phyB mutants are produced in rice and then screened for agronomically useful traits. The gene editing vector simplifies the workload of producing phyB mutants and makes the process of producing mutants more controllable.

[0025] Ganesan et al. (2017) "Development of transgenic crops based on photo-biotechnology" Plant Cell Environ. 40: 2469~2486 is a review article that generally discusses the regulation of photoreceptors. Various attempts related to the regulation of PHYB are mentioned (listed above), but all have resulted in results that are undesirable for plant growth and development and have a negative impact on plant productivity.

[0026] In summary, despite many attempts to manipulate PHYB, YHB, and PHYA expression in plants, none of the aforementioned patent disclosures have been successful in improving photosynthesis, growth, or yield. Instead, they have had a negative impact on plant development, plant structure, and water use efficiency. Phytochromes have a major regulatory role in all plants, and all previous manipulations of these genes have resulted in developmental abnormalities that make the manipulation of the timing or location of the expression of this gene inappropriate for crop improvement.

[0027] Leegood, RC (2008) "Roles of the bundle sheath cells in leaves of C3 plants" J. Exp. Bot. vol 59 pp. 1663-1673 is a review article describing the structure and function of bundle sheath cells surrounding the veins of leaves in many C3 plants. It is clear that the cells of the bundle sheath and its extensions have several metabolic roles, for example, in carbohydrate synthesis and storage, nitrogen and sulfur uptake, metabolism and transport, and antioxidant metabolism, but it is clear that much more needs to be known about their activity in the leaves of C3 plants. [Prior art documents] [Patent Documents]

[0028] [Patent Document 1] U.S. Patent No. 8,735,555 [Patent Document 2] U.S. Patent Application Publication No. 2004 / 0268443 [Patent Document 3] U.S. Patent Application Publication No. 2005 / 0120412 [Patent Document 4] Chinese Patent Application Publication No. 106854240 [Patent Document 5] International Publication No. 2005093054(A1) [Patent Document 6] International Publication No. 99 / 31242(A1) [Patent Document 7] U.S. Patent Application Publication No. 2007295252 [Patent Document 8] Chinese Patent Application Publication No. 108913717 [Non-patent literature]

[0029] [Non-Patent Document 1] Wagner et al. (1991) “Overexpression of Phytochrome B induces a short hypocotyl phenotype in transgenic Arabidopsis” Plant Cell. 3(12): 1275~1288 [Non-Patent Document 2] Thiele et al. (1999) “Heterologous Expression of Arabidopsis Phytochrome B in Transgenic Potato Influences Photosynthetic Performance and Tuber Development” Plant Physiology.120:73~81 [Non-Patent Document 3] Rao et al., (2011) “Overexpression of the phytochrome B gene from Arabidopsis thaliana increases plant growth and yield of cotton (Gossypium hirsutum)” J. Zheijang Univ.Sci.B.12:326~334 [Non-Patent Document 4] Halliday et al. (1997) “Expression of heterologous phytochromes A, B or C in transgenic tobacco plants alters vegetative development and flowering time” The Plant Journal 12:1079~1090 [Non-Patent Document 5] Husaineid et al. (2007) “Overexpression of homologous phytochrome genes in tomato: exploring the limits in photoperception” J. Exp.Bot.58:615~626 [Non-Patent Document 6] Holefors et al. (2000) “The Arabidopsis phytochrome B gene influences growth of the apple rootstock M26” Plant Cell Reports 19:1049~1056 [Non-Patent Document 7] Distefano et al. (2013) “Ectopic expression of Arabidopsis Phytochrome B in Troya citrange affects photosynthesis and plant morphology.” Scientia Horticulturae 159 :1~7 [Non-Patent Document 8] Zheng et al., (2001) “Modification of Plant Architecture in Chrysanthemum by Ectopic Expression of the Tobacco Phytochrome B1 Gene” J. Am.Hort.Soc.Sci.126(1):19~26 [Non-Patent Document 9] Yang et al., (2013) “Deficiency of Phytochrome B alleviates chilling-induced photoinhibition in rice” Am.J.Bot.100(9):1860~1870 [Non-Patent Document 10] Su and Lagarias (2007) “Light-Independent Phytochrome Signaling Mediated by Dominant GAF Domain Tyrosine Mutants of Arabidopsis Phytochromes in Transgenic Plants. Phytochrome B-Y276H (YHB)” The Plant Cell, Vol 19:2124~2139 [Non-Patent Document 11] Hu et al., (2019) “Regulation of monocot and dicot plant development with constitutively active alleles of phytochrome B.” Plant Direct, 4:1~19 [Non-Patent Document 12] Ganesan et al. (2017) “Development of transgenic crops based on photo-biotechnology” Plant Cell Environ.40: 2469~2486 [Non-Patent Document 13] Leegood, RC (2008) “Roles of the bundle sheath cells in leaves of C3 plants” J. Exp. Bot. vol 59 pp. 1663-1673 [Non-Patent Document 14] Bashirullah A, Cooperstock R, Lipshitz H (2001) Spatial and temporal control of RNA stability. PNAS 98: 7025~7028 [Non-Patent Document 15] Certo MT, Gwiazda KS, Kuhar R, Sather B, Curinga G et al. (2012) Coupling endonucleases with DNA end-processing enzymes to drive gene disruption. Nature methods 9:973~975 [Non-Patent Document 16] Christou, P. (1997) Rice transformation: bombardment. Plant Mol Biol. 35 (1-2):197~203) [Non-Patent Document 17] Gasiunas, G., Barrangou, R., Horvath, P., Siksnys, V. (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. PNAS 109(39):E2579~86 [Non-Patent Document 18] Kantor, A. et al. (2020) Int. J. Mol. Sci. 21: 6240 [Non-Patent Document 19] Khatodia, S. et al. (2016) Front. Plant Sci. vol 7 506 pages [Non-Patent Document 20] Patel et al. 2006. J Biol Chem 281(35):25485~91 [Non-Patent Document 21] Patel et al. 2004. Plant Physiology 136(3): 3550~3561 [Non-Patent Document 22] A. Fahn, Plant Anatomy Pergamon Press 1995 [Non-Patent Document 23] Legris et al., (2019) “Molecular mechanisms underlying phytochrome-controlled morphogenesis in plants.” Nat. Comms. 10:5219 [Non-Patent Document 24] Wang et al., (2017) “Transcriptional control of photosynthetic capacity: conservation and divergence from Arabidopsis to rice.” New Phytol., 216: 32–45 [Non-Patent Document 25] Emms and Kelly. Genome Biology 2019. 20: 238 [Non-Patent Document 26] Hwang et al. (2014) International Journal of Photoenergy [Non-Patent Document 27] Wu et al., (2011) PLoS ONE 6(11) [Non-Patent Document 28] Takano et al., (2009) PNAS. 106(34): 14705~14710 [Non-Patent Document 29] Potter et al. (2018) Nucleic Acids Research 46:W200~W204 [Non-Patent Document 30] Oka et al. (2004) “Functional Analysis of a 450-Amino Acid N-Terminal Fragment of Phytochrome B in Arabidopsis” Plant Cell. 16(8): 2104~2116 [Non-Patent Document 31] Engelmann et al. (2008) Plant Physiology 146(4):1773~1785 [Non-Patent Document 32] Zeng et al. New Phytol. 2017 May;214(3):1338~1354 [Non-Patent Document 33] Adwy et al. The Plant Journal 2015 November;84(6) [Non-Patent Document 34] Adwy et al. Plant Gene 2019 June;18 [Non-Patent Document 35] Kirschner et al. (2018) Journal of Experimental Botany 69(20): 4897~4906 [Non-Patent Document 36] Knerova et al. biorxiv https: / / doi.org / 10.1101 / 380188 [Non-Patent Document 37] Lundquist Molecular Plant. 2014 Jan;7(1):14~29

Non-licensed Document 38

Non-licensed Document 39

Non-licensed Document 40

Non-licensed Document 41

Non-licensed Document 42

Non-licensed Document 43

Non-licensed Document 44

Non-licensed Document 45

Non-licensed Document 46

Non-licensed Document 47

Non-licensed Document 48

[0030] The inventors have discovered that when a gene of interest (GOI), specifically PHYB, is predominantly expressed in plant vascular bundle sheath cells compared to other plant cells or tissues, this results in a variety of overall beneficial properties without any harmful properties for plant growth, development, and productivity.

[0031] Accordingly, the present invention provides a method for enhancing the photosynthetic capacity of a C3 plant, comprising the step of modifying the heritable genetic material of the plant such that a GOI is expressed in one or more vascular sheath cells of the plant, wherein the GOI is expressed in the vascular sheath cells of the plant under the control of an active gene expression regulatory element.

[0032] As will be readily apparent to those skilled in the art, the method of the present invention is for providing a C3 plant having a modified genetic structure compared to a normal plant or a wild-type plant, or any plant not subjected to the method of the present invention. Currently, many methods exist for modifying the genome of a plant, and various terms are used to describe them. Each of these terms will be familiar to the experienced reader and encompasses "genetically modified," "genetically engineered," or "gene edited," and is often used interchangeably. All refer to a plant having its genome sequence modified compared to an unmodified control plant. This modification could be brought about by the insertion of one or more polynucleotides of the present invention into the genome of the target plant by any transformation, transfection, transduction, or genome engineering technique. This modification can also be brought about by nuclear-mediated genome editing, prime editing, and / or base editing.

[0033] In embodiments of the method of the present invention as defined herein, preferably, first the genetic material of plant cells is modified, and then a genetically modified whole plant is regenerated from the genetically modified cells. The regeneration of plants from cells or plant tissues is familiar to those skilled in the art from established literature.

[0034] In a preferred method, the gene expression regulatory element is active in particular at least some of the vascular sheath cells of the plant, thereby causing the GOI under the control of the regulatory element to be expressed in particular at least some of the vascular sheath cells of the genetically modified whole plant. The term “specific,” as used herein, may also mean “exclusive” or “strongly preferred.”

[0035] Additionally or alternatively, the GOI is Phytochrome B, or an active variant thereof, or a functional fragment, as further defined below in this specification.

[0036] The process of altering heritable genetic material may include the process of inserting polynucleotides into the heritable genetic material of a plant cell.

[0037] In some methods, the process of modifying heritable genetic material may include introducing a gene repair oligonucleotide (GRON)-mediated mutation into a target DNA sequence in the heritable genetic material of a plant cell. In further methods, plant cells may be exposed to a DNA cutter and GRON. The DNA cutter may include a meganuclease, a transcription activator-like effector nuclease (TALEN), a Zn finger, an antibiotic, or a Cas protein.

[0038] The process of modifying heritable genetic material may include the use of zinc finger nucleases (ZNFs) and / or transcription activator-like effector nucleases (TALENs) for site-specific homologous recombination of heritable genetic material in plant cells. Accordingly, the present invention provides a method for modifying the genetic material of a plant such that it performs C3 photosynthesis in at least a portion thereof, wherein the modification step is the step of the modified plant expressing an active mutant or functional fragment thereof, such as PHYB or YHB, in at least some; and possibly all, of the vascular sheath cells of the plant. This expression in vascular sheath cells is added to the normal expression pattern of at least one copy of the plant's PHYB gene. As understood, at least one copy of PHYB and its associated regulatory elements are preferably left unchanged so that the growth and development of the modified plant are substantially different compared to an unmodified plant of the same genotype.

[0039] The method according to the present invention may utilize classic, well-known techniques of gene modification involving transformation, thereby enabling the incorporation of one or more additional copies, active variants, or functional fragments thereof of a native or exogenous PHYB gene into the plant genome, along with the necessary vascular sheath cell expression regulatory elements. Such incorporation is preferably stable and heritable to enable the introduction of modifications into a specific lineage of crop plants; advantageously, for the purpose of crop improvement or breeding programs. Furthermore, as already mentioned above, a CRISPR-Cas gene modification method may be used, thereby selecting a guide RNA (gRNA) to target the action of a CRISPR-associated protein (Cas) on a desired genomic locus, resulting in a homologous recombination (HR) event, i.e., the insertion-deletion of a desired polynucleotide into the plant genome.

[0040] In some embodiments, the methods of the present invention may simply involve introducing vascular sheath expression regulatory elements, such as promoter sequences or DNA regulatory elements, to an upstream position of existing innate PHYB coding gene sequences in the genome by various gene editing approaches. In operating such embodiments of the present invention, a guided approach is convenient, which involves using a CRISPR-related protein (Cas) that can be directed, for example, by gRNA or any other genome editing nuclease (ZFN, TALEN, and other Cas proteins), to cleave a specific genomic region and introduce the necessary polynucleotides as a repair DNA template by homologous recombination.

[0041] According to the aforementioned method of the present invention involving CRISPR-Cas, one or more polynucleotides used to transform plant material may comprise a polynucleotide encoding a Cas protein and optionally further a guide RNA (gRNA), in which case the gRNA directs the Cas protein to the locus of at least one copy of the endogenous PHYB gene in the plant cell genome, thereby inserting a regulatory element to induce the expression of the endogenous copy of PHYB, particularly in at least some of the vascular sheath cells of the regenerated plant.

[0042] In some embodiments, gRNA is synthesized as a single-stranded guide RNA (sgRNA) or as a CRISPR-RNA (crRNA):trans-activated CRISPR RNA (tracrRNA) double helix. In some embodiments, multiple gRNAs, crRNAs, or tracrRNAs may be used simultaneously to target multiple genomic regions, for example. In some embodiments, different types of CRISPR-Cas systems and orthogonal Cas proteins may be used simultaneously.

[0043] As used herein, the terms “Cas,” “Cas protein,” “CRISPR-Cas protein,” “Cas nuclease,” “Cas moiety,” or “Cas domain” mean CRISPR-related proteins, including any equivalent or functional fragment thereof, any Cas homolog, ortholog, or paralog from any organism, and any naturally occurring or engineered variants or variants of Cas. CRISPR-Cas proteins may be, for example, Cas9, Cas12a, or Cas12b. CRISPR endonucleases can be produced using Escherichia coli (E. coli) expression systems. One such mechanism is, for example, encoding the Cas gene into E. coli driven by the T7 promoter. CRISPR-Cas proteins may include Cas12c (or C2C3), Cas12d (or CasY), Cas12e (or CasX), Cas13a (or C2C2), Cas13b (or C2C6), Cas13(c), or C2C7, Cas13d (or Casrx), or their functional fragments.

[0044] As used herein, the terms “Cas9,” “Cas9 nuclease,” “Cas9 moiety,” “Cas9 domain,” or “Csn1” mean CRISPR-associated protein 9 or a functional fragment thereof, and include any naturally occurring Cas9, any naturally occurring Cas9 equivalent or functional fragment thereof from any organism, any Cas9 homolog, ortholog, or paralog from any organism, and any naturally occurring or engineered variant or variant of Cas9. More broadly, Cas9 is a type of “RNA programmable nuclease” or “RNA-guided nuclease,” and more broadly, a type of “nucleic acid programmable DNA-binding protein (napDNAbp).” The term Cas9 is not particularly restrictive and may be referred to as “Cas9 or equivalent.” Exemplary Cas9 proteins are described in detail herein and / or in the Art thereof and incorporated herein by reference. This disclosure is not limited to specific Cas9s used in the advanced base editor of the present invention.

[0045] As used herein, the terms “Cas12a,” “Cas12a nuclease,” “Cas12a moiety,” or “Cas12a domain” are used interchangeably with Cpfl. The term “Cas12a” may also include CRISPR-related protein 12a or its functional fragments, and encompasses any naturally occurring Cas12a from any organism, any naturally occurring Cas12a equivalent or functional fragment thereof, any Cas homolog, ortholog, or paralog from any organism, and any naturally occurring or engineered variant or variant of Cas12a. This extends to orthologs of Cas12a, and polynucleotide sequences encoding such orthologs or systems, and vectors or vector systems containing them, and delivery systems containing them. In a broader sense, Cas12a is a type of "RNA-programmable nuclease" or "RNA-guided nuclease," and in an even broader sense, a type of "nucleic acid-programmable DNA-binding protein (napDNAbp)." The term Cas12a is not particularly restrictive and may also be referred to as "Cas12 or equivalent." Exemplary Cas12a proteins are described in detail herein and / or in the Art, and are incorporated herein by reference.

[0046] As used herein, the terms “Cas12b,” “Cas12b nuclease,” “Cas12b moiety,” or “Cas12b domain” are used interchangeably with C2c1 or Cpf2. The term “Cas12b” may also include CRISPR-related protein 12b or its functional fragments, and encompasses any naturally occurring Cas12b from any organism, any naturally occurring Cas12b equivalent or functional fragment thereof, any Cas homolog, ortholog, or paralog from any organism, and any naturally occurring or engineered variant or variant of Cas12b. This extends to orthologs of Cas12b, and polynucleotide sequences encoding such orthologs or systems, as well as vectors or vector systems containing them, and delivery systems containing them. In a broader sense, Cas12b is a type of "RNA programmable nuclease" or "RNA-guided nuclease," and in an even broader sense, a type of "nucleic acid programmable DNA-binding protein (napDNAbp)." The term Cas12b is not particularly restrictive and may be referred to as "Cas12b or equivalent." Exemplary Cas12b proteins are described in detail herein and / or in the art and incorporated herein by reference.

[0047] As described above, the method according to the present invention may also utilize emerging genetic modification technologies. For example, the technology may involve introducing a gene repair oligonucleotide (GRON)-mediated mutation into a target deoxyribonucleic acid (DNA) sequence in a plant cell, as described in detail in U.S. Patent No. 9,957,515. The technology may involve combining the GRON-mediated mutation with other DNA editing or recombination technologies, such as, but not limited to, Zn finger nucleases, transcription activator-like effector nucleases (TALENs), or Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), into the target DNA sequence in the plant cell. The technology may also include exposing the plant cell to a DNA cutter (a portion that acts to break strands) and GRON. Non-limiting examples of DNA cutters that may be used include meganucleases, TALENs, antibiotics, Zn fingers, and CRISPR or CRISPR / Cas systems.

[0048] The techniques may involve introducing purified nuclease proteins into plant cells without requiring the insertion of exogenous genetic material. These techniques may involve the techniques described in European Patent No. 3008186. In particular, the techniques may involve providing plant cells containing the exogenous gene to be modified; providing a Cas9 endonuclease protein targeted to the gene; and transfecting plant cells with the Cas9 endonuclease protein using bioristic or protoplast transformation such that the Cas9 endonuclease introduces one or more double-strand DNA breaks (DSBs) into the genome, in order to produce plant cells having detectable targeted genomic modifications without the presence of any exogenous Cas9 genetic material in the plant genome, as disclosed in European Patent No. 3008186. Transfection can be carried out by the delivery of sequence-specific nucleases to isolated plant protoplasts. For example, transfection can be carried out by the delivery of sequence-specific nucleases to isolated plant protoplasts using polyethylene glycol (PEG)-mediated transfection, electroporation, bioristic-mediated transfection, sonication-mediated transfection, or liposome-mediated transfection.

[0049] RNA templates may also be used. For example, another aspect of the present invention relates to a conjugate of a CRISPR-Cas protein-guide RNA complex, in which case the guide RNA is a conjugate of crRNA, dual guide RNAs, sgRNA, or 1gRNA with one or more single-stranded DNA (ssDNA) as a donor template for gene editing. Thus, according to the aforementioned method of the present invention with CRISPR-Cas, one or more polynucleotides used to transform plant material may comprise a polynucleotide encoding the CRISPR-Cas protein and optionally further at least one guide RNA (gRNA), in which case the gRNA directs the CRISPR-Cas protein to the locus of at least one copy of endogenous Phytochrome B in the plant cell genome, thereby inserting a regulatory element to cause expression of the Phytochrome B copy, particularly in at least some of the vascular sheath cells of the regenerated plant. The at least one copy to be inserted into the plant cell genome may be inserted using a viral vector-based system. In relation to genetic engineering, all references to the insertion of regulatory elements may mean any donor, donor sequence, or donor polynucleotide inserted into the plant cell genome using, for example, the system described above. The donor (donor sequence, or donor polynucleotide) may mean a polynucleotide, RNA, DNA, or genomic insertion.

[0050] The sequence-specific nuclease delivered may be in the form of a purified nuclease protein or an mRNA molecule that can be translated into a protein after transfection. The nuclease protein can be prepared by several means known to those skilled in the art, using available protein expression vectors, such as, but not limited to, pQE or pET. Suitable vectors allow for the expression and subsequent purification of the nuclease protein in various cell types (E. coli, insects, mammals). Synthesis of the nuclease in mRNA format can also be carried out by various means known to those skilled in the art, such as using a T7 vector (pSF-T7) that enables the production of capped RNA for transfection into cells. The mRNA can be modified with optimal 5' untranslated region (UTR) and 3' untranslated region. UTRs have been shown to play a crucial role in posttranslational regulation of gene expression by regulating localization, stability, and translational efficiency (Bashirullah A, Cooperstock R, Lipshitz H (2001) Spatial and temporal control of RNA stability. PNAS 98: 7025~7028). As mentioned above, mRNA delivery is desirable due to its non-transgenic nature; however, mRNA is a very fragile molecule and is easily degraded during the plant transformation process. The use of UTRs in plant mRNA transformation enables increased stability and localization of the mRNA molecule, which in turn gives increased transformation efficiency for non-transgenic genome modifications.

[0051] In some embodiments, CRISPR reagents may be delivered using Agrobacterium-mediated or particle-impact-mediated transformation with DNA having a CRISPR expression cassette. For example, in some embodiments, mRNA encoding the Cas protein may be co-delivered to plants together with gRNA by particle impact. In other embodiments, the Cas protein and gRNA can be pre-assembled to form ribonucleoprotein (RNP) and introduced into plants by donor templates. Delivery of RNP to plants can be achieved by various methods, including, for example, polyethylene glycol (PEG)-mediated cell transfection, particle impact, electroporation, and lipofection. The term “donor template” means an transgene cassette or gene editing sequence flanked by homologous regions for recombination by a host locus and replacement of mutated DNA with the correct sequence by homologous gene repair (HDR) / single-stranded DNA repair (SSDR). As used herein, a donor template may also be called a “donor polynucleotide.” The donor polynucleotide may be ssDNA or dsDNA or plasmid / vector, and may be chemically conjugated to guide RNA or Cas protein by a covalent linker. The donor template may be chemically synthesized and may possess the chemical function for conjugation / ligation. The conjugating donor template may also be prepared by in vitro gene synthesis in the presence of DNA polymerase, with chemical functional groups, such as amines and alkynes, enzymatically incorporated into its 5' or 3' end for chemical conjugation / ligation from a nucleoside triphot phosphate analogue.

[0052] Purified nucleases are delivered to plant cells by various means. The delivered sequence-specific nuclease may be in the form of a purified nuclease protein or an mRNA molecule that can be translated into a protein after transfection. Nuclease proteins can be prepared by several means known to those skilled in the art, using available protein expression vectors, such as, but not limited to, pQE or pET. Suitable vectors allow for the expression and subsequent purification of nuclease proteins in various cell types (E. coli, insects, mammals). Synthesis of nucleases in mRNA form can also be carried out by various means known to those skilled in the art, such as the use of a T7 vector (pSF-T7) that enables the production of capped RNA for transfection into cells. mRNA can be modified with optimal 5' untranslated region (UTR) and 3' untranslated region. UTRs have been shown to play a crucial role in posttranslational regulation of gene expression by regulating localization, stability, and translational efficiency (Bashirullah A, Cooperstock R, Lipshitz H (2001) Spatial and temporal control of RNA stability. PNAS 98: 7025-7028). As mentioned above, mRNA delivery is desirable due to its non-transgenic nature; however, mRNA is a very fragile molecule and is easily degraded during the plant transformation process. The use of UTRs in plant mRNA transformation enables increased stability and localization of the mRNA molecule, which in turn gives increased transformation efficiency for non-transgenic genome modifications.

[0053] Furthermore, bioristic particle delivery systems can be used to transform plant tissues. Standard PEG and / or electroporation methods can be used for protoplast transformation. After transformation, plant tissues / cells are cultured to allow cell division, differentiation, and regeneration. DNA derived from individual events can be isolated and screened for mutations. Any type of sequence-specific nuclease can be used to carry out the method provided herein, insofar as it has similar capabilities to TAL effector nucleases. Therefore, it must be possible to induce double-strand DNA breaks at one or more target loci, resulting in one or more targeted mutations at those loci (mutations resulting from erroneous repair of the breaks by NHEJ or other mechanisms) (Certo MT, Gwiazda KS, Kuhar R, Sather B, Curina G et al. (2012) Coupling endonucleases with DNA end-processing enzymes to drive gene disruption. Nature methods 9:973~975. Christou, P. (1997) Rice transformation: bombardment. Plant Mol Biol. 35 (1-2):197~203). Examples of such sequence-specific nucleases, but not limited to these, include ZFNs, homing endonucleases such as I-SceI and I-CreI, restriction endonucleases, and other homing endonucleases or TALEN®.In certain embodiments, the endonuclease used includes CRISPR-related Cas proteins, such as Cas9 (Gasiunas, G., Barrangou, R., Horvath, P., Siksnys, V. (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. PNAS 109(39):E2579~86).

[0054] Furthermore, the present invention may also include at least one polynucleotide comprising a 5' to 3' active expression regulatory element, a nucleotide sequence encoding a PHYB, an active variant, or a functional fragment thereof, and a terminator, so that an exogenous PHYB, an active variant, or a functional fragment thereof under the control of a vascular sheath regulatory element is inserted into a desired locus in the plant genome, particularly in plant vascular sheath cells, as well as a further polynucleotide encoding a genome editing nuclease, and optionally the same or further polynucleotides encoding a gRNA or crRNA that directs the genome editing nuclease protein to a desired locus in the plant genome.

[0055] In some embodiments of the present invention, there may also be at least one polynucleotide comprising an active regulatory element, particularly in plant vascular sheath cells, and a nucleotide sequence encoding the PHYB, active variant, or functional fragment, which is inserted into the plant genome from 5' to 3'.

[0056] In some embodiments, methods that do not involve the induction of double-strand DNA breaks may be used to incorporate a desired DNA sequence. For example, prime editing is such a method that can be used to override native nucleotide sequences. As is well known to those skilled in the art, prime editing uses a DNA nickase enzyme conjugated with an engineered reverse transcriptase to target a specific genomic region and override it with an arbitrary DNA sequence (see, for example, Kantor, A. et al., (2020) Int. J. Mol. Sci. 21: 6240, which provides a review of CRISPR-Cas9 DNA base editing and prime editing). A prime editor uses an engineered reverse transcriptase fused with a nickase, such as Cas9 nickase, and prime editing guide RNA (pegRNA). The pegRNA contains a sequence complementary to the target site that directs the nickase to its target sequence, as well as an additional sequence that spells the desired sequence change. Prime editors can extend the scope of DNA editing to include, but not all, transposition or transposition mutations, as well as small insertion and deletion mutations. Examples of nickases that can be used in prime editing include, but are not limited to, Cas9 nickase or Cas12 nickase. For example, Cas9 D10A nickase or Cas9 H840A nickase may be used. Furthermore, to broaden the number of clearly recognized bases for target cleavage, Cas9n, which uses a paired nickase system with two different gRNAs, can be used, which can improve specificity and help mitigate off-target phenomena (see, for example, Khatodia, S. et al. (2016) Front. Plant Sci. vol 7 p. 506, which is another review article providing information on CRIPSR / Cas genome editing tools).

[0057] Prime editing may be used to override the expression regulatory element of an endogenous native gene sequence, such as one copy of the native PHYB, so that the resulting modified plant expresses PHYB, particularly in at least some vascular sheath cells. Alternatively, prime editing may be used to further modify a native or exogenous sequence introduced into the plant's genetic material, for example, by modifying the coding sequence of PHYB, so that it becomes an active variant such as YHB.

[0058] In some embodiments, the method may utilize a Cas endonuclease, in which case the Cas endonuclease may include a modified form of the Cas polynucleotide. The modified form of the Cas polypeptide may include amino acid changes (e.g., deletion, insertion, or substitution) that reduce the naturally occurring nuclease activity of the Cas protein. In some cases, the modified form of the Cas polypeptide may have substantially no nuclease activity and be referred to as catalytically "inactivated Cas" or "deactivated Cas (dCas)." Inactivated Cas / deactivated Cas includes, for example, deactivated Lapis Cas endonuclease (Lapis dCas). For example, in some embodiments, nuclease-deactivated Cas9 (dCas9) is used to carry out such insertions. RNA or DNA can be modified by using a dCas protein conjugated with a base editing enzyme (cytidine or adenine deaminase). In some embodiments, direct effector fusion design may be used, and the regulation (CRISPRi) or activation (CRISPRa) of targeted genes may be achieved by genetically fusing effector proteins—or their active domains—to dCas9 and expressing them as a single recombinant protein. For example, transcription activator domains (VP64, p65) or transcription repressor domains (KRB, SID) may be fused to dCas9, in particular, to increase or decrease target gene expression. In some embodiments, the effector domain is recruited by a functional scaffold incorporated into the sgRNA-dCas9 complex either by fusion to dCas9 or as an RNA aptamer in scaffold RNA (scRNA). In other embodiments, spatiotemporal control of effector activity is achieved by controlled recruitment of the effector to the reconstitution of split-dCas9 directly fused to the effector via the sgRNA-dCas9 complex or a photo-inducible or chemi-inducible heterodimerization partner.

[0059] In other embodiments, the methods of the present invention may include the possibility of base editing, which allows for the modification of individual nucleotides. Base editing may be performed using DNA base editors, two classes of which have been reported: cytosine base editors and adenine base editors. DNA base editors comprise two key components: Cas enzymes for programmable DNA binding and single-strand DNA modifying enzymes for targeted nucleotide modification. When cytosine base editors are used, cytosine deamination yields uracil, whose base pairs like thymidine in DNA. Fusion with a uracil DNA glycosylase inhibitor (UGI) can inhibit the activity of uracil N-glycosylase (UNG) and increase the editing efficiency of cytosine base editing in cells. In the case of adenine base editors, adenosine deamination yields inosine, which has the same base pairing preference as guanosine in DNA. In summary, cytosine and adenine base editing can install all four base-transition mutations (C→T, T→C, A→G, and G→A). Therefore, for example, by using the site-directed action of the cytosine deaminase enzyme, the conversion of targeted cytosine bases to uracil can be catalyzed, which is then read as thymine by the native polymerase. Thus, if desired, there are multiple options available for introducing vascular sheath expression regulatory elements to act on native phytochrome sequences and for converting native PHYBs to YHB sequences. The present invention also provides isolated DNA polynucleotides comprising a nucleotide sequence encoding a PHYB, an active variant, or a functional fragment thereof, and a terminator, with the expression regulatory element active in vascular sheath cells, particularly C3 plant cells, from 5' to 3'.

[0060] In embodiments of the present invention, the promoter may be a vascular bundle sheath cell-specific promoter of a plant, or a mestome sheath cell-specific promoter, or a promoter that is particularly active in both vascular bundle sheath cells and mestome sheath cells.

[0061] In some embodiments, the isolated DNA polynucleotide may further comprise a nucleotide sequence encoding a transcription factor and a nucleotide sequence encoding a second promoter (not the vascular sheath promoter described above) recognized by the transcription factor, in which case the nucleotide sequence of the second promoter lies upstream of the nucleotide sequence encoding PHYB, an active variant, or a functional fragment thereof, and the vascular sheath-specific promoter drives the expression of the transcription factor.

[0062] DNA polynucleotides may be synthesized in whole or in part; or, as may be, cloned in whole or in part. Promoters active in vascular sheath cells, in particular, of C3 plants may also be active in other cells of the vascular bundle, whether vascular bundle sheath cells or mestom sheath cells (or both), non-limiting examples of which include phloem and / or xylem cells. The term "vascular bundle," as used in this application, means all cells of the vascular bundle, including vascular sheath cells. Promoters active in vascular sheath cells may also be active in other non-vascular cell types, non-limiting examples of which include root cells, epidermal cells, or stomatal cells, such as guard cells. Promoters active in vascular sheath cells may also be active in extensions of the vascular sheath, such as extensions of the vascular bundle sheath and paraveinal mesophyll.

[0063] In particular, promoters active in C3 vascular sheath cells are also within the scope of the present invention; that is, these promoters are active in C3 vascular sheath cells but not in any other leaf tissue or leaf cell, but may be active in any of several possible plant cell or tissue types other than those found in leaves.

[0064] Termination sequences are well known to those skilled in the art, and any suitable terminator can be used, for example, if the terminator is Nos ter As shown in the embodiment of the present invention, it may be selected and used.

[0065] Preferably, in any embodiment of the present invention as defined herein, the promoter is a vascular sheath promoter (e.g., a vascular bundle sheath cell promoter, or a mestome sheath cell promoter, or a promoter expressed in both vascular bundle sheath cells and mestome sheath cells). This may be a synthetic promoter comprising various selected elements. For example, such a synthetic promoter may include a vascular sheath cell-specific transcription factor binding element upstream of the promoter element. There may be two or more transcription factor binding elements, which may be the same or different. Multiple such transcription factor binding elements may help to enhance the activity and / or specificity of the promoter in vascular sheath cells.

[0066] For example, the promoters included in the synthetic vascular sheath promoter mentioned above may be selected from the minimal ZmUbi1 promoter, the NOS core promoter, the CHSA core promoter, or the minimal 35S promoter. Other minimal and / or core promoters well known to those skilled in the art may also be used. Preferred promoters have the nucleotide sequence of SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 13, or a sequence with at least 80% identity to them.

[0067] In other embodiments, the vascular sheath-specific promoter may be derived from a gene that is preferentially or specifically expressed in the vascular bundle sheath or mestom sheath (or both), and such a promoter is a naturally occurring promoter. The gene may be expressed in other cell types and vascular sheath cells, but preferably not or only very slightly in mesophyll cells. The gene may be expressed in guard cells, vascular sheath extensions, epidermal cells, guard cells, or other vascular tissues, e.g., xylem and / or phloem; or in other locations in plants other than leaf tissue, e.g., flowers, fruits, roots, stems, etc. Preferably, such a naturally occurring vascular sheath promoter may be related to a gene that is expressed particularly in plant vascular bundle sheath cells or mestom sheath cells or both, e.g., only in vascular bundle sheath cells, but not in any other plant tissue or cell type.

[0068] Vascular sheath-specific promoters may originate from, for example, one of the following genes: Arabidopsis thaliana MYB76, Flaveria trinervia GLDP, Arabidopsis thaliana SULTR2;2, Arabidopsis thaliana SCR, Arabidopsis thaliana SCL23, Urochloa panicoides, PCK1, Zoysia japonica PCK, and Hordeum vulgare PHT1;1 (including homologs of these genes). Promoters are specified by referencing the plant species, but naturally, the same or similar promoters may be found and used in plant species different from the original plant species.

[0069] In some embodiments, the vascular sheath promoter can be obtained from a non-plant organism, such as the rice tungro bacilliform virus (RTBV) promoter.

[0070] The vascular sheath promoter in question may be obtained from forward screening of mutant populations to identify promoters that drive gene expression in vascular structures.

[0071] In some embodiments, preferential expression of the vascular sheath can be achieved by using UTR sequences that, when fused to a target coding sequence for a PHYB, active variant, or functional fragment thereof, give protein-specific expression to cells, even if transcript expression is driven by a constitutive promoter. Examples of such vascular sheath-specific UTR elements include UTR sequences from the Rubisco small subunit derived from either Flaveria bidentis (Patel et al. 2006. J Biol Chem 281(35):25485~91) or Amaranthus hypochondriacus (Patel et al. 2004. Plant Physiology 136(3):3550~3561) (both giving phagocytic and preferential vascular bundle sheath cell expression).

[0072] The PHYB or amino acid sequence variants that can be encoded in the DNA polynucleotide of the present invention may correspond to any of the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12. In addition to the aforementioned reference sequences, any of the coding sequences of sequences identified by accession numbers listed in Table 1 may be used as reference sequences instead. With respect to the reference sequence variants for the PHYB, these may include sequences with at least 65% identity to it; preferably at least 70% identity to it; more preferably at least 80% identity to it.

[0073] In an example of the present invention, the PHYB variant YHB SEQ ID NO: 4 is used, which is encoded by the nucleotide sequence of SEQ ID NO: 1. In a further example of the present invention, the PHYB variant YHB SEQ ID NO: 12 is used, which is encoded by the nucleotide sequence of SEQ ID NO: 11.

[0074] Therefore, in the polynucleotide of the present invention, the nucleotide sequence encoding PHYB is a sequence that is at least 65% identical to any of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 8, or SEQ ID NOs: 11, or any of the above sequences; preferably a sequence that is at least 70% identical to any of the above sequences; more preferably a sequence that is at least 70% identical to any of the above sequences.

[0075] In certain embodiments of the present invention, a functional fragment or variant thereof of PHYB is used. Such a functional fragment has wild-type phytochrome signaling activity but lacks photosensitivity. In other words, the PHYB variant is a sub-full-length amino acid sequence and is photosensitive as a result of the absence of a photosensing domain or amino acids essential for photosensing function. Preferably, the phytochrome fragment referred to herein consists only of the PAS and GAF ​​domains.

[0076] The present invention comprises a DNA polynucleotide, in which case the PHYB protein molecule, active variant, or functional fragment thereof encoded thereby is a photosensitive sequence variant; in other words, there is one or more amino acid substitutions, deletions, or insertions, resulting in photosensitiveness of the protein while retaining normal PHYB signaling activity. The number of consecutive amino acid changes in such a variant may be any number of amino acids selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids. The number of amino acid changes (which may have some consecutive letters but not all) can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

[0077] In some embodiments, the present invention may include a plasmid comprising the DNA polynucleotides described herein above, a replication origin and a T-DNA right-bound repeat sequence of a Ti or Ri plasmid, and at least one bacterial selection marker. Often, the plasmid also includes a left-bound repeat sequence of a Ti or Ri plasmid.

[0078] The plasmid according to the present invention may further comprise one or more other elements selected from: enhancers, plant-selectable markers, multicloning sites, or recombinant sites.

[0079] The present invention also provides Ti or Ri plasmids containing DNA polynucleotides as defined herein. The structure, modification, propagation, and generation of vectors incorporating such plasmids are well known to those skilled in the art.

[0080] In some embodiments, the present invention may include compositional transformation of plant cells using a biolistic method. The composition thus comprises microparticles coated with DNA polynucleotides or plasmids as defined herein. These microparticles may be made of metal or synthetic materials. For example, the microparticles may contain tungsten or gold.

[0081] The present invention also provides a bacterium comprising a plasmid, i.e., a shuttle vector, as defined herein, and in some embodiments of the present invention, the bacterium is Escherichia coli.

[0082] When Ti or Ri plasmids are used to transform plant material, they may contain suitable bacteria, such as those of the Agrobacterium genus; preferably Agrobacterium tumefaciens, etc.

[0083] The present invention includes any plant or plant material obtained from or available by any of the methods of the present invention as defined herein, i.e., cells, tissues, organs, parts, seeds, or fruits.

[0084] The products of the present invention include a plant that performs C3 photosynthesis in at least a portion thereof, wherein the plant contains a DNA polynucleotide as defined herein that is stably incorporated into its genome, and expresses PHYB, or an active variant, or a functional fragment thereof as defined herein, in at least some of its vascular sheath cells (i.e., bundle sheath cells and / or mestome sheath cells). As already described, this DNA polynucleotide can be introduced into the plant genome by either incorporating a full-length promoter and PHYB, an active variant, or a functional fragment by a genetic modification method, or by gene editing the expression regulatory region of the natural PHYB genome to alter its expression domains. Both approaches result in the same outcome, namely, heritable expression of PHYB in vascular sheath cells. The PHYB gene, active variant, or functional fragment thereof can be expressed in substantially all bundle sheath cells and / or mestome sheath cells.

[0085] The present invention further includes a plant that performs C3 photosynthesis in at least a portion thereof, wherein the plant has at least one copy of the PHYB gene, an active variant, or a functional fragment thereof as defined herein, and the plant is genetically modified compared to an equivalent unmodified plant, such that the expression regulatory element of at least one copy of the PHYB gene, active variant, or functional fragment thereof is modified to result in expression in at least some of the vascular bundle sheath cells and / or mestome sheath cells of the plant. In such a plant, the expression regulatory element is preferably a promoter that is active particularly in C3 plant vascular bundle sheath cells, as defined herein.

[0086] The coding sequence of at least one PHYB gene may be identical to that of the plant's native PHYB gene. Therefore, at least one native copy of the PHYB gene is modified to be expressed in at least some of the plant's vascular sheath cells. As a result, in species with two or more copies of the PHYB gene, at least one native PHYB gene remains under unmodified, naturally occurring expression control.

[0087] In certain embodiments of the modified plant, at least one PHYB gene is different from other PHYB genes in the plant.

[0088] The plants according to the present invention may be monocotyledons (monocot) or eudicotyledons (eudicot, dicot); preferably crop plants, such as fruits, vegetables, grains, oilseed crops, legumes, biofuel crops, and fiber crops, which are commonly used for food, animal feed, biofuels, or biomass production; and further, horticultural plants.

[0089] In preferred plants, the DNA polynucleotides as defined herein are stable and genetically integrated into their genomes.

[0090] In some embodiments, the PHYB gene expressed in at least some of the plant vascular sheath cells of the present invention has a functional fragment defined by encoding any of the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or any of the sequence accessions listed in Table 1, or an active variant, or an amino acid sequence with at least 65% identity to any of the above sequences; preferably a sequence with at least 70% identity to any of the above sequences; more preferably a sequence with at least 70% identity to any of the above sequences. In some embodiments, the PHYB gene has any of the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or any of the sequence accessions listed in Table 1. In other embodiments, the PHYB gene encodes any of the amino acids having at least 65% identity to any of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or any of the sequence accessions listed in Table 1. In other embodiments, the PHYB gene encodes either an amino acid sequence having at least 70% identity with SEQ ID NOs. 4, 5, 6, 9, and 12, or a sequence accession listed in Table 1. In other embodiments, the PHYB gene encodes either an amino acid sequence having at least 80% identity with SEQ ID NOs. 4, 5, 6, 9, and 12, or a sequence accession listed in Table 1. In other embodiments, the PHYB gene encodes either an amino acid sequence having at least 90% identity with SEQ ID NOs. 4, 5, 6, 9, and 12, or a sequence accession listed in Table 1. In the plant of the present invention expressing functional fragments of PHYB, these functional fragments are as defined above in this specification.

[0091] The PHYB gene, active variant, or functional fragment thereof may be a photosensitive sequence variant resulting from one or more mutations, for example, involving substitutions, insertions, or deletions of amino acid residues. The PHYB gene, active variant, or functional fragment thereof may also be modified by substitutions, insertions, or deletions of nucleic acid residues. In some embodiments, for example, as described below, the PHYB sequence is the sequence of the active variant YHB, encoding the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 12, or a sequence with at least 65% identity to them.

[0092] The plants according to the present invention may have chloroplasts present in vascular sheath cells, such as vascular bundle sheath cells and / or mestome sheath cells, which may be larger than those in equivalent cells of unmodified control plants grown for the same period and under the same conditions.

[0093] Plants according to the present invention may have a higher photosynthetic rate than unmodified control plants grown under the same conditions.

[0094] The plants according to the present invention may have higher water use efficiency than unmodified control plants grown under the same conditions.

[0095] Plants according to the present invention may have enhanced photosynthetic efficiency compared to control plants grown under the same conditions.

[0096] The plants according to the present invention may have enhanced photosynthesis, resulting in one or more of the following characteristics compared to control plants grown under the same conditions: increased growth rate, shortened time to flowering, earlier maturation, increased seed yield, increased biomass, increased plant height, and increased canopy area.

[0097] The present invention also provides plant parts, plant tissues, plant organs, plant cells, plant protoplasts, embryos, callus cultures, pollen grains, or seeds derived from or obtained from any of the types of plants described herein.

[0098] The present invention also includes any processed plant product obtained from any plant described herein, wherein the processed product comprises (i) a PHYB gene or active variant or functional fragment thereof bound to an active gene expression regulatory element in at least some of the vascular sheath cells of the plant, or (ii) a detectable nucleic acid sequence encoding at least a portion of the polynucleotide of the present invention. Such detection may be performed using techniques well known in the art, such as PCR, qPCR, or the application of any DNA or RNA sequencing technique of a suitably prepared sample of the processed plant material.

[0099] In summary, the inventors have made novel modifications to C3 plants to enhance their photosynthetic capacity. When using the term "C3" plant, the term also includes plants that perform C3 photosynthesis in any part of the plant at any point in its life cycle (non-limiting examples include leaf sheath tissue, villi, or photosynthetically active parts of the roots, stems, and seeds).

[0100] Previous attempts to increase plant productivity by increasing phytochrome signaling have either reduced photosynthesis and yield, or achieved increased photosynthesis, but only in proportion to chlorophyll investment (which requires more nitrogen investment), resulting in decreased water use and / or yield. These applications of this gene have also repeatedly resulted in undesirable side effects in crops, such as dwarfism, canopy reconstruction, delayed flowering, smaller tubers, and thicker leaves.

[0101] The overall effect of this C3 plant modification is to enhance photosynthesis, plant growth, and yield without any adverse effects on plant morphology, development, or other agricultural characteristics. The present invention is broadly applicable to all C3 plants and can produce an increase of 30% or more in photosynthetic rate, growth rate, and seed yield without any disruption to normal plant development.

[0102] The present invention as a whole achieves enhanced photosynthesis, growth, and yield without any observable negative or harmful anatomical, physiological, biochemical, or developmental effects on the modified plant.

[0103] Embodiments of the present invention will be described in detail below with reference to the examples and accompanying drawings. [Brief explanation of the drawing]

[0104] [Figure 1] This diagram illustrates a simplified PHYB signaling cascade, including a non-limiting example of genes affected by PHYB activity at both the transcript and / or protein levels. PHYB activity releases several genes from repression, which then promote the development of photosynthetic capacity. Full gene names: PHYB=Phytochrome B, PIF=Phytochrome Interacting Factor, COP1=Constitutive Photomorphogenic 1, GLK=Golden-2 Like Transcription factor, CGA1=Cytokinin Responsive GATA Factor 1, GNC=GATA, Nitrate-inducible, Carbon Metabolism-involved, HY5=Elongated Hypocotyl 5, HYH=HY5-Homolog. [Figure 2] This figure shows a phylogenetic tree for Phytochrome B, including non-restrictive examples of flowering plant members of the Phytochrome B gene family. The phylogenetic tree has roots at the base of the flowering plants. Representative species include monocots (rice (Oryza sativa)) and two major dicotyledonous branches: roses (Arabidopsis thaliana and soybean (Glycine max)) and chrysanthemums (tomato (Solanum lycopersicum)). In all three representative dicotyledonous species, independent duplication of PHYB resulted in the presence of two homologs of PHYB in each genome. [Figure 3]This figure shows a schematic diagram of a gene vector used by the Agrobacterium-mediated floral dip method to express the YHB protein in the vascular bundles of *Atractylodes thaliana*. [Figure 4] This figure shows the magnitude of YHB expression in modified plants and unmodified control plants compared to the control gene elF-4E1. The control bars correspond to wild-type plants, and the bars labeled "C12" correspond to Arabidopsis plants containing the gene vector for YHB vascular bundle sheath expression. Error bars indicate the 95% confidence interval of the mean. [Figure 5] This figure shows the results of leaf thickness measurements for transgenic Arabidopsis plants containing gene vectors for vascular bundle sheath expression in YHB (bar labeled "C12") and control Arabidopsis plants (bar labeled "control"). 95% confidence intervals are shown. "ns" indicates that no significant difference was found between the C12 plants and the control plants using a t-test. Comparisons are shown between the control wild-type plant and one mutant, but all three mutants examined were phenotypic identical. [Figure 6] This figure shows the photosynthetic capacity measured by the morphology of the A / Ci curve in transgenic Arabidopsis plants (〇) containing a gene vector for YHB vascular bundle sheath expression and control plants (△) (CO2 assimilation rate: CO2 assimilation rate, Intercellular carbon dioxide concentration: Intercellular carbon dioxide concentration). [Figure 7] This figure shows the results of stomatal conductance measurements for transgenic Arabidopsis plants containing a gene vector for YHB vascular bundle sheath expression (〇) and control Arabidopsis plants (△). [Figure 8]This figure shows whether water use efficiency is enhanced when photosynthesis is operating at maximum capacity in transgenic Arabidopsis plants containing the gene vector for YHB vascular bundle sheath expression (labeled "C12") and control Arabidopsis plants (labeled "control"). 95% confidence intervals are shown. Asterisks indicate statistically significant differences at p<0.05 using a t-test. Comparisons are shown between the control wild-type plant and one mutant, although all three mutants examined were phenotypically identical. [Figure 9] This figure shows that vascular bundle sheath cells (BSCs) of transgenic Arabidopsis plants containing the YHB gene vector for vascular bundle sheath expression (bar labeled "C12") have more chlorophyll than mesophyll cells (MSCs) compared to control Arabidopsis plants (bar labeled "control"). 95% confidence intervals are shown. Asterisks indicate statistically significant chains with p<0.05 using a t-test, while "ns" indicates no significant difference between the compared values. The comparison is shown between the control wild-type plant and one mutant, but all three mutants examined were phenotypic identical. [Figure 10] This figure compares chloroplasts in vascular bundle sheath cells between a control plant and a transgenic Arabidopsis plant containing the YHB gene vector for vascular bundle sheath expression. (A) is a representative image of vascular bundle sheath cell chloroplasts in a control plant. (B) is a representative image of vascular bundle sheath cell chloroplasts in a transgenic Arabidopsis plant containing the YHB gene vector for vascular bundle sheath expression. (C) is a representative image of vascular bundle sheath cell chloroplasts and mesophyll cell chloroplasts in a control plant. (D) is a representative image of vascular bundle sheath cell chloroplasts and mesophyll cell chloroplasts in a transgenic Arabidopsis plant containing the YHB gene vector for vascular bundle sheath expression. BSC = vascular bundle sheath cell, MSC = mesophyll cell, scale bar = 2 micrometers. [Figure 11] This figure shows the results of stable carbon isotope (13C isotope) measurements from leaf material. These data are consistent with the increased refixation of respirationd carbon dioxide in transgenic Arabidopsis plants containing the YHB gene vector for vascular bundle sheath expression (labeled "C12") compared to control Arabidopsis plants (labeled "control"). 95% confidence intervals are shown. Asterisks indicate statistically significant chains with p<0.05 using t-tests. Comparisons are shown between control wild-type plants and one mutant, although all three mutants examined were phenotypically identical. [Figure 12] This figure shows the results of vegetative growth rate measurements between 2 and 3 weeks after germination in transgenic Arabidopsis plants containing the gene vector for YHB vascular bundle sheath expression (bars labeled "C12") and control Arabidopsis plants (bars labeled "control"). 95% confidence intervals are shown. Asterisks indicate statistically significant chains with p<0.05 using a t-test, while "ns" indicates no significant difference between the compared values. Comparisons are shown between the control wild-type plant and one mutant, but all three mutants examined were phenotypically consistent (increase in leaf rosette area). [Figure 13] This figure shows the results of bolt height measurements (higher bolts appearing 35 days after germination) in transgenic Arabidopsis plants containing the YHB gene vector for vascular bundle sheath expression ("C12" labeled rod) compared to control Arabidopsis plants ("control" rod). 95% confidence intervals are shown. Asterisks indicate statistically significant chains with p<0.05 using a t-test. Comparisons are shown between the control wild-type plant and one mutant, although all three mutants examined were phenotypically identical. [Figure 14]This is a photograph of trays containing transgenic and wild-type Arabidopsis plants ("C12") and control Arabidopsis plants ("wild-type") that have undergone normal photomorphogenesis and contain the gene vector for YHB vascular bundle sheath expression. [Figure 15] This figure shows the results of measuring the time to bolting in transgenic Arabidopsis plants containing the gene vector for YHB vascular bundle sheath expression (bar labeled "C12") and control Arabidopsis plants (bar labeled "control"). 95% confidence intervals are shown. Asterisks indicate statistically significant chains with p<0.05 using a t-test. Comparisons are shown between the control wild-type plant and one mutant, although all three mutants examined were phenotypically identical. [Figure 16] These are photographs showing silique formation and terrestrial biomass at 8 weeks in transgenic Arabidopsis plants (labeled C12) containing a gene vector for YHB vascular bundle sheath expression and in control Arabidopsis plants (labeled wild type). Comparisons are shown between the control wild-type plant and one mutant, but all three mutants examined were phenotypic identical. [Figure 17] These are photographs of dried seeds collected from Arabidopsis plants (right tube) and control plants (left tube) containing the gene vector for YHB vascular bundle sheath expression. The comparison is shown between the control wild-type plant and one mutant, but all three mutants examined were phenotypic identical. [Figure 18]This figure shows the measurement of dry seed biomass production in transgenic Arabidopsis plants containing the gene vector for YHB vascular bundle sheath expression (bar labeled "C12") and control Arabidopsis plants (bar labeled "control"). Measurements were taken at two different time points: "early" (seeds dried 6.5 weeks after germination) and "late" (seeds dried 8 weeks after germination). 95% confidence intervals are shown. Asterisks indicate statistically significant chains with p<0.05 using a t-test. Comparisons are shown between the control wild-type plant and one mutant, but all three mutants investigated were phenotypically identical (seed dry weight per plant). [Figure 19] This is a diagram of a proposed model for a novel, enhanced carbon refixation pathway in C3 plants. [Figure 20] This figure shows the ambient photosynthetic rate measured under ambient growing chamber conditions in transgenic wheat plants (labeled "C12") containing the gene vector for YHB vascular bundle sheath expression, compared to control wheat plants (labeled "control"). 95% confidence intervals are shown. Asterisks indicate statistically significant chains at p<0.05 using t-tests. [Figure 21] This photograph shows the enhanced plant growth in a typical transgenic wheat plant containing the YHB gene vector for vascular bundle sheath expression, compared to a control wheat plant (left). [Figure 22] This figure shows the results of height measurements representing plant growth in transgenic wheat plants (labeled "C12") containing the gene vector for YHB vascular bundle sheath expression, compared to control wheat plants (labeled "control"), after 7 weeks of growth. 95% confidence intervals are shown. Asterisks indicate statistically significant results at p<0.05 using a t-test. [Figure 23]This figure illustrates the conservation of function in five vascular sheath promoters known to function in distantly related plant genera. Evolutionary relationships among 11 plant genera spanning three major plant branching groups (Roses, Asteraceae, and Monocots) are shown by phylogenetics (branch lengths are arbitrary). For each of the five promoters (SULTR2;2, GLDP, PCK, PHT1;1, and RBTV), the arrow indicates the species of origin, and the arrowhead points to the distantly related genus in which consistent vascular sheath expression has been demonstrated. The divergence time indicates how many millions of years have passed since the two species were connected by an arrow sharing a common ancestor. For example, although the genera Flaveria and Arabidopsis diverged approximately 125 million years ago, the Flaveria GLDP promoter drives consistent expression in Arabidopsis. [Figure 24] This figure shows published experiments demonstrating the conservation of function among distantly related plants of PHYB orthologs originating from different species. Phylogenetic and evolutionary distances are shown as in Figure 23. Bold text indicates genera in which natural PHYB expression has been altered, such as overexpression in the genera Arabidopsis and Solanum (tomato) and gene lockout in the gene Oryza (rice). Arrows indicate the origin of the PHYB gene and point to the plant in which this PHYB homolog is overexpressed. For example, the Arabidopsis PHYB was overexpressed in the genera Arabidopsis, Solanum (tomato), and Miscanthus (Japanese pampas grass). Regardless of the origin and recipient species of the PHYB, increased PHYB expression results in a consistent phenotype (darker green leaves, shorter internodes, and delayed flowering). [Figure 25]This diagram shows the conservation of functional domains in the amino acid sequence of PHYB protein selection over >400 million years of land plant evolution. The phylogenetic tree illustrates the evolutionary relationships between Brassica napus, tomato, rice, Selaginella moellendorfii, and Physcomitrella patens. In species with duplicate copies of PHYB, such as Brassica napus and tomato, all copies of PHYB are shown. Characteristic PHYB domains are conserved in all PHYB proteins and consist of the domains (in order from N-terminus to C-terminus): PAS_2, GAF, PHY, PAS, PAS, HisKA, and HATPase_c. Three key events in the evolution of land plants—the emergence of vascular plants (>400 million years ago), flowering plants (>160 million years ago), and the Brassicaceae family (>40 million years ago)—are annotated with an "X". Branching lengths are arbitrary and do not reflect evolutionary distance. [Figure 26] This figure shows the expression (Transcipts Per Million) of three different Brassica napus PHYB genes in the leaves of 16 different cultivars. [Figure 27] This figure shows the alignment of 50 base pairs adjacent to a single nucleotide that needs to be altered to convert the rapeseed PHYB into a structurally active form equivalent to the Audrey thaliana YHB (highlighted). The asterisks below the multiple sequence alignments indicate nucleotides that are conserved in all three full-length rapeseed copies of the PHYB gene. The 14 underlined bases indicate the location of variations between PHYB copies that allow individual copies to be targeted for editing. [Figure 28]This figure illustrates two designs illustrating different approaches to gene editing for PHYB expression in two species: the Solyc05g053410 tomato PHYB gene (top) and the soybean Glyma.09G035500 gene (bottom). PHYB genomic regions are shown, annotated with natural exons, 5' and 3' untranslated regions (UTRs), and inserted promoter and enhancer sequences that would confer vascular expression to these genes. Genomic features are labeled according to their position relative to the start codon (beginning at position 0). [Modes for carrying out the invention]

[0105] Different embodiments of the present invention are described in more detail below. Each embodiment described or defined may be combined with any other embodiment unless it is clearly shown to be contrary to it. In particular, any feature indicated as preferred or advantageous may be combined with any other feature indicated as preferred or advantageous.

[0106] The prior arts of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry, recombinant DNA technology, and bioinformatics used in the application of this invention are all readily known and available to those skilled in the art. Specific techniques are well described in the literature.

[0107] The inventors have created a system comprising a vascular sheath-specific regulator of gene expression along with a chloroplast activation regulator that increases photosynthesis and yield-related characteristics. The inventors have demonstrated that this technique is broadly applicable to C3 plants by showing that it works in both eudicots (e.g., Alocasia thaliana) and monocots (e.g., wheat). The inventors have shown that this technique works regardless of the species origin of the PHYB gene and regardless of the vascular sheath promoter used. An important aspect of the invention is that PHYB, an active variant, or a functional fragment thereof is expressed in vascular sheath cells (which may include other cells in the vascular structure or vascular structure sheath extensions as defined herein) but not in leaf mesophyll cells. Transgenic plants incorporating this system surprisingly and advantageously do not exhibit developmental abnormalities associated with overexpression of YHB or PHYB. Transgenic plants undergo normal photomorphogenesis (without dwarfism, reduced apical dominance, delayed flowering, or decreased water use efficiency), possess the same leaf thickness as control plants, and flower normally. However, these plants have higher photosynthetic rates, grow faster, have enhanced water use efficiency, mature to the flowering stage earlier, produce more fruit structures, and produce significantly more seeds. The effect is dramatic, with yields increasing by more than 30% in greenhouse trials.

[0108] The inventors have achieved something previously impossible: the manipulation of PHYB expression in plants to improve photosynthesis, plant growth, and yield without interfering with plant development. An unexpected finding for the inventors is that the combination of improvements can be achieved by additionally expressing PHYB only in the plant's vascular bundles or its constituent cells, thereby decoupled from the disruptive aspects of PHYB expression.

[0109] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to amino acids in polymerized forms of any length linked together by peptide bonds.

[0110] The terms “modified,” “altered,” and “modified” may be used interchangeably herein. A control plant, as used herein, is an unmodified plant. Therefore, a control plant is not genetically modified to alter the expression of any of the polynucleotides of the present invention as described herein. A control plant may be a wild-type (WT) plant. Even if a plant is transgenic, if it is not transgenic with respect to the polynucleotides of the present invention, it could still function as a control plant. The WT or control does not need to be so specific as to provide a reliable reference that allows for comparison of vascular bundle sheath expression of PHYB against modified plant material.

[0111] The terms “increase,” “improve,” and “enhance” are used interchangeably in this specification.

[0112] The term “specific” is, as used herein, considered equivalent to “exclusive” or “highly preferred.”

[0113] Vascular sheath and vascular sheath cells In C3 plants (most crops), the cells surrounding the leaf veins (i.e., the vascular sheath) are known as vascular bundle sheath cells. In dicotyledonous plants, the vascular bundle sheath consists of a single layer of cells encircling the leaf veins, whereas in monocotyledonous plants, the vascular bundle sheath can consist of a single layer of cells or two concentric layers of cells (A. Fahn, Plant Anatomy Pergamon Press 1995). When two layers of cells are present, the outer layer of cells is generally called the vascular bundle sheath, and the inner layer of cells is generally called the mestom sheath (A. Fahn, Plant Anatomy Pergamon Press 1995). When two layers are present, both layers together constitute the vascular bundle sheath (A. Fahn, Plant Anatomy Pergamon Press 1995). Therefore, the term vascular bundle sheath is used to describe either a single layer of vascular bundle sheath cells or a two-layered system including an outer vascular bundle sheath layer and an inner mestom sheath layer. When used throughout this specification, the terms “vascular bundle sheath,” “vascular bundle sheath cell,” “vascular sheath,” or “vascular sheath cell” may be used interchangeably and, unless explicitly indicated in the context, encompass all types of vascular bundle sheath cell layers. Vascular bundle sheath cell layers (i.e., a single vascular bundle sheath layer, or an outer vascular bundle sheath and an inner mestom sheath) may contain chloroplasts. The number of chloroplasts in these vascular bundle sheath layers may be the same as or less than the number in mesophyll cells, and in some cases, vascular bundle sheath cells may lack chloroplasts. Furthermore, when they are present in C3 plants, the size of chloroplasts in vascular bundle sheath cell layers is generally much smaller than that of mesophyll cells (A. Fahn, Plant Anatomy: Pergamon Press (1995)). Vascular bundle sheath cells surround the leaf veins, and therefore they are ideally positioned to ensure a good water supply and to deliver sugars into the leaf veins for distribution to growing plant structures.

[0114] Vascular bundle sheath-specific expression The term "specific," when used in relation to gene expression, describes the biological phenomenon of enhanced gene expression within a limited subset of cell types within a plant. The term "vascular bundle sheath-specific expression" is used synonymously with "vascular bundle sheath-specific expression" to describe the phenomenon where a gene is expressed at substantially higher levels in the vascular bundle sheath than in the surrounding mesophyll cells within the leaf. This does not prevent the gene from being expressed in the leaf or other non-mesophyll cells within the plant; it simply means that the level of expression is higher in the vascular bundle sheath and lower in the mesophyll of the leaf. Genes can also be expressed in other vascular cell types besides vascular bundle sheath cells. These cell types include some or all of the cells of the vascular bundle, such as xylem and / or phloem, as well as related cell types. The gene may be expressed in non-vascular cells, such as guard cells, cells in the extensions of the vascular sheath, cells in the extensions of the vascular bundle sheath, epidermal cells, parabainal mesophyll cells (extensions of the vascular bundle sheath, not mesophyll cells), or in other locations in plants other than leaf tissue, such as flowers, fruit roots, and stems. A key determining factor is that the gene is activated in the vascular bundle sheath but not in the mesophyll.

[0115] Phytochrome protein for use in the present invention In this specification, "PHYB" (Phytochrome B) is defined as a regulatory photoreceptor. As shown in Figure 1, PHYB activity induces a regulatory cascade by inhibiting the action of transcriptional repressors, such as Phytochrome-Interacting Factor (PIF), and proteins that target other proteins for degradation (e.g., Constitutive Photomorphogenic 1, COP1) (Legris et al., (2019) "Molecular mechanisms underlying phytochrome-controlled morphogenesis in plants." Nat. Comms. 10:5219). In darkness, this layer of repressor proteins inhibits the transcription of photosynthetic proteins by preventing the accumulation of transcription factors that activate the expression of photosynthetic proteins, such as Elongated Hypocotyl 5 (HY5 and its paralog HYH), Golden-2 Like transcription factor (GLK1 and its paralog GLK2), and Cytokinin Responsive GATA Factor 1 (CGA1 and its paralog GNC) (Wang et al., (2017) "Transcriptional control of photosynthetic capacity: conservation and divergence from Arabidopsis to rice." New Phytol., 216: 32~45). The transcription of hundreds of genes, including those involved in the core mechanisms necessary for photosynthesis, can be considered a result of the action of these three groups of transcription factors. Under light, the PHYB protein present in mesophyll cells is activated, releasing these transcription factors from repression. The resulting transcriptional cascade ultimately leads to chloroplast development and activation of photosynthesis.

[0116] Regardless of whether the PHYB protein, active variant, or functional fragment thereof is expressed in the same plant (homologous expression) or in different plants (non-homologous expression), PHYB derived from any plant species can be used in embodiments of the present invention.

[0117] The terms “active variant” and / or “functional fragment,” as used herein in relation to PHYB, mean a variant or fragment of a PHYB gene or a peptide sequence that preserves the signal-activating function of PHYB. Active variants also include variants of a gene of interest that encode a peptide having a sequence modification, for example, at a non-conserved residue, that does not affect the signal-activating function of the resulting protein.

[0118] The present invention also includes functional fragments of PHYB and any variants of the PHYB protein for use in any aspect of the present invention.

[0119] Sequence identity and orthography When the term “variant” is used herein in relation to a given PHYB protein or functional fragment thereof derived from a plant species, it means any PHYB ortholog of a different amino acid sequence derived from another plant species. Such variants may be expressed with percentage identity to any of the reference nucleotide reference sequences disclosed herein (i.e., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 8, or SEQ ID NO: 11). With respect to percentage identity with respect to an amino acid reference sequence, such as Sequence ID No. 4, variants of PHYB may have at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% overall sequence identity with respect to that amino acid reference sequence, in order of increasing priority. The following table provides a non-inclusive list of accession numbers for PHYB orthologs in 50 commercially cultivated plant species. Orthologs of the Arabidopsis PHYB gene were found in the publicly available sequence database NCBI. Two or more PHYB accessions were found for many species, indicating that PHYB has split into two in different plant lineages; many of these paralogs arose as a result of whole-genome duplication.For each species, one representative full-length orthologous amino acid sequence was compared to the Arabidopsis and wheat PHYB orthologs (AT2G18790.1 and Traes_4AS_1F3163292.1, respectively), and the percentage identity for each was quantified using multiple sequence alignments generated by Clustal Omega 2.1 with default parameters (alignments were generated by a hidden Markov model using an mBed-like clustering guide tree and HHalign). The median PHYB percentage identity of these orthologs to the Arabidopsis or wheat PHYB ortholog was approximately 75%. There were several instances where identity of less than 75% was shared between the PHYB ortholog of a given species and both the Arabidopsis and wheat orthologs. For example, the PHYB orthologs of carrot (Daucus carota) and tomato (Solanum lycopersicum) were >70% identical to either the Arabidopsis or wheat PHYB ortholog. Similarly, PHYB orthologs in more distantly related gymnosperm species, such as European spruce (Picea abies) and Sitka spruce (Picea sitchensis), were exactly 66-68% identical at the amino acid level to either the Arabidopsis or wheat PHYB protein. Recently, overlapping PHYB paralogs have fallen within the PHYB similarity range shown in the table, while more distantly related phytochromes have not. For example, multiple sequence alignments of the Arabidopsis species PHYB [SEQ ID NO: 5], PHYD [SEQ ID NO: 9], and PHYA (NCBI accession NP_001322907.1) show that PHYB and paralogous PHYD share 81.98% identity, while PHYA shares exactly 52.35% identity with PHYB and 52.20% identity with PHYD.

[0120] [Table 1A]

[0121] [Table 1B]

[0122] [Table 1C]

[0123] Overall sequence identity can be determined using global alignment algorithms known in the art, such as the Needleman-Wunsch algorithm in the program GAP (GCG Wisconsin Package, Axellis).

[0124] Further examples of suitable PHYB genes can be easily identified by skilled technicians using ortholog finding programs such as OrthoFinder (Emms and Kelly. Genome Biology 2019. 20: 238). The function of such genes can be identified as described herein, and thus skilled technicians can confirm their function when expressed in plants.

[0125] Figure 2 shows the PHYB gene family for four representative plant species spanning three major branching groups of flowering plants (roses, chrysanthemums, and monocots). The phylogenetic tree has a root at the origin of flowering plants, and branch lengths are arbitrary. The phytochrome B gene is duplicated in the lineage that gives rise to the Brassicaceae family, resulting in the paralogous gene pair known as Phytochrome B (AT2G18790) and Phytochrome D (AT4G16250) in Atractylodes thaliana. Similarly, Glycine max (soybean) and Solanum lycopersicum (tomato) have two copies of PHYB, which arose from independent gene duplication events. In these species, these duplications are instead called PHYB1 and PHYB2. Therefore, O. sativa (rice) PHYBs are equally related to both Arabidopsis thaliana (A. thaliana) PHYBs (B and D) and both tomato PHYBs (1 and 2). In species with multiple copies of a PHYB, there is evidence that both copies function redundantly. For example, overexpression of either tomato PHYB1 or PHYB2 in tomato produces the same phenotype (Husaineid et al., (2007) "Overexpression of homologous phytochrome genes in tomato: exploring the limits in photoperception" J. Exp. Bot. 58: 615~626). Thus, as used in this application, the term PHYB encompasses the complete PHYB gene family exemplified by representative members of this gene family shown in Figure 2, as well as all PHYB paralogs such as Phytochrome D.

[0126] In the case of vascular bundle sheath cell-specific promoters, all of these variants and orthologs are included in the present invention. If a reference nucleotide sequence exists for such a promoter, such variants and orthologs include a nucleotide sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% overall sequence identity with respect to the reference promoter sequence.

[0127] The degree of sequence identity of any polynucleotide described in connection with the present invention may be defined in terms of hybridization to any polynucleotide of one of the reference sequences disclosed herein [SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 8, or SEQ ID NO: 11], rather than being expressed as a percentage identity to a reference sequence. Hybridization of such sequences may be carried out under stringent conditions. "Stringent conditions" or "stringent hybridization conditions" are intended to be conditions under which the probe hybridizes to its target sequence to a detectably larger degree than other sequences (e.g., at least twice as large as the background). Stringent conditions are sequence-dependent and will vary in different situations. By controlling the stringency of the hybridization and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homological probing). Alternatively, the stringency conditions can be adjusted to tolerate some mismatches in the sequences so that a lower degree of similarity is detected (non-homological probing). Generally, the probes are less than approximately 1000 nucleotides in length, preferably less than 500 nucleotides.

[0128] Typically, stringent conditions are when the salt concentration is less than approximately 1.5 M Na at pH 7.0 to 8.3. + Ions, typically about 0.01 to 1.0 M Na + The ionic concentration (or other salt) and temperature would be at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., more than 50 nucleotides). The hybridization period is generally less than about 24 hours, usually about 4 to 12 hours. Stringent conditions can also be achieved by adding destabilizers such as formamide.

[0129] PHYB is a highly conserved protein, and its function is highly conserved in all vascular plants. This has been repeatedly demonstrated by increasing the expression of the native PHYB protein, expressing exogenous PHYB protein derived from other plant species, or knocking out the native PHYB gene. Figure 24 shows that PHYB expression can be achieved through genetic engineering: overexpression of either the natural PHYB or YHB in the Arabidopsis genus (Su and Lagarias, (2007) Plant Cell. 19(7): 2124~2139), expression of Arabidopsis PHYB in Solanum (tomato) (Thiele et al., (1999) Plant Physiology. 120: 73~81) and Miscanthus (switchgrass) (Hwang et al., (2014) International Journal of Photoenergy), overexpression of the natural tomato PHYB gene in Solanum (one of two PHYBs in the tomato genome, Husaineid et al., (2007) J. Exp. Bot. 58: 615~626), and glycine (soybean) PHYB in Arabidopsis thaliana (Wu et al., (2011) PLoS ONE). This section summarizes specific examples of modification, such as 6(11)), or by locking out phytochrome in the genus Oryza (rice) (Takano et al., (2009) PNAS. 106(34): 14705~14710). Even though this large amount of experimental evidence includes various species and PHYB proteins (Miscanthus and Arabidopsis genera, which diverged approximately 160 million years ago), characteristic phenotypic effects have been consistently observed in these experiments: namely, consistent changes in chlorophyll (leaf color), dwarfism (internode length), and flowering period are observed in all plant species, regardless of the origin species of the expressed PHYB gene. Therefore, a PHYB gene derived from any plant species can provide PHYB function in any other species when expressed in that plant. Thus, it can be expected that all PHYB proteins induce similar mechanistic functions when expressed in any vascular plant.

[0130] functional fragment PHYB proteins typically contain seven readily recognizable protein domains. These include three Per-Arnt-Sim (PAS) domains (either PF08446 and / or PF00989), a GAF domain (PF01590), a PHY domain (PF00360), a His kinase A phosphoreceptor domain (PF00512), and a GHKL domain (PF02518). Figure 25 illustrates these characteristic PHYB functional domains derived from PHYB proteins found in five different land plant species: rapeseed, tomato, rice, cypress, and physocarpa. Despite 400 million years of evolution (from Physcomitrella to Brassica) and multiple examples of gene duplication (e.g., rapeseed and tomato), all PHYB proteins are the same length and contain the same arrangement of PAS_2, GAF, PHY, PAS, HisKA, and HATase_c( / GHKL) domains. The domains were identified using the EBI HMMR tool (Potter et al., (2018) Nucleic Acids Research 46:W200~W204).

[0131] While these protein domains are highly conserved, excised forms of the PHYB gene can also function to initiate PHYB signaling. For example, Oka et al. (2004) "Functional Analysis of a 450-Amino Acid N-Terminal Fragment of Phytochrome B in Arabidopsis" Plant Cell. 16(8): 2104~2116 showed that a 450-amino acid fragment of PHYB lacking the PHY domain (PF00360), the His kinase A phosphoreceptor domain (PF00512), and the GHKL domain can initiate PHYB signal transduction when targeting the nucleus. Therefore, functional fragments of PHYB can provide PHYB signaling, and such functional fragments are also included in the present invention.

[0132] Vascular sheath (i.e., vascular bundles, vascular bundle sheaths, and / or mestome sheaths) promoter Those skilled in the art are well aware of many vascular bundle, vascular sheath, vascular bundle sheath, or mestom sheath-specific promoters.

[0133] There exist several such promoters isolated from different species that those skilled in the art would expect to function in various plant species; five such examples are shown in Figure 23. The promoter derived from the gene encoding the P-subunit of glycine decarboxylase in Flavelia trinevia, described by Engelmann et al. (2008) Plant Physiology 146(4):1773~1785, drives expression in the vascular bundle sheath cells and vascular bundles of Flavelia videntis, and even in the more distantly related eudicotyledonous Aucuba thaliana. These species last shared a common ancestor about 125 million years ago, and therefore the activity of this promoter is conserved in eudicots (Zeng et al. New Phytol. 2017 May;214(3):1338~1354). Indeed, further studies on the GLDP promoter have revealed that its cross-functionality among species is conferred by a regulatory sequence conserved in the Brassicaceae family, including species of the genera Arabidopsis, Brassica, Capsella, and Moricandia (Adwy et al. The Plant Journal 2015 November;84(6) and Adwy et al. Plant Gene 2019 June;18). Similarly, the promoter for the gene encoding the sulfur transporter SULTR2;2 in Atractylodes thaliana, described by Kirschner et al. (2018) Journal of Experimental Botany 69(20): 4897~4906, drives expression in the vascular bundle sheath and veins of Arabidopsis species, and even in the more distantly related species Flavelia videntis. In further examples, promoters derived from genes expressed in the vascular bundle sheath of C3 plants can also confer vascular bundle sheath-specific expression in these plants. This is exemplified by the promoter derived from the MYB76 gene, which is expressed in the vascular bundles of Arabidopsis species.The promoter derived from this gene is sufficient to drive vascular bundle-specific expression of the reporter gene in the genus Arabidopsis and is found in a highly conserved region of the genome in members of the Brassicaceae family (Knerova et al. biorxiv https: / / doi.org / 10.1101 / 380188), and it shares characteristics with the cross-functional GLDP promoter. There are many other examples of promoters that drive expression in the vascular bundle when fused to a reporter gene. For example, a promoter derived from a gene that gives a reticular phenotype when knocked out provides dominant (or exclusive) expression in vascular or bundle sheath (BS) cells (Lundquist et al. Molecular Plant. 2014 Jan;7(1):14~29). Furthermore, both the SCARECROW (SCR) and SCARECROW-LIKE 23 (SCL23) gene promoters drive the expression of reporter genes, particularly in vascular bundle sheath cells (Cui et al. The Plant Journal. 2104 78(2): 319~327).

[0134] There are also vascular bundle sheath cell promoters described in the literature for monocots. For example, Nomura et al. (2005) Plant Cell Physiology. 46(5):754~61 show that the Shiba PCK promoter functions to drive expression in the rice vascular bundle sheath. Similarly, the River Seedgrass PCK1 promoter targets vascular bundle sheath expression of reporter genes in rice and maize (Suzuki and Burnell. Plant Science. 2003 165(3):603~611). Furthermore, Kloti et al. (1999) Plant Molecular Biology 40(2): 249~266 also describe a rice tungrobacillus virus promoter that functions in vascular bundles and other vascular cells. This promoter functions to drive expression in vascular bundles in both monocots (rice) and dicots (tobacco), despite the fact that these species diverged approximately 160 million years ago. Petruccelli et al. 2001 PNAS 98(13) 7635~7640. Furthermore, Schunmann et al. (2004) Plant Physiol. 136(4): 4205~4214 also demonstrates rice vascular bundle sheath expression using the barley Pht1 promoter (see Figure 3I in the said literature). Since vascular tissue is a universally conserved feature in plant leaf veins, it would be expected by someone of average skill in the art that vascular bundle sheath promoters derived from eudicots, such as those publicly available and known to function in distantly related species, such as Asteraceae and Brassica, would also function in monocots, and vice versa (as in the case of the rice tungrobacillus virus promoter described above, which functions in both monocots and eudicots). Furthermore, there is a great diversity of vascular bundle sheath promoters already known to the average person in this field, and any of these promoters (individually or in combination) would be suitable for driving the expression of PHYB or YHB in vascular bundles or vascular bundle sheath cells in any plant.

[0135] Recombinant constructs Any suitable cloning system may be used. For example, the Golden Gate module system described by Weber, E et al. (2011) PLoS ONE doi.org / 10.1371 / journal.pone.0016765. Alternatively, the gene construct can be newly synthesized entirely or assembled using other molecular biological approaches.

[0136] The PHYB, active variant, or functional fragment sequence of the present invention can be operatively ligated, directly or indirectly, to a vascular sheath promoter used in the present invention for transcription and expression.

[0137] Plant transformation Currently, plant transformation is a common practice in many species. Advantageously, several transformation methods can be used to introduce genes of interest into plants. Several methods described for plant transformation and regeneration from plant tissue or plant cells can be used for transient or stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase the uptake of free DNA, direct injection of DNA into plants, particle gun impact, transformation using viruses or pollen, and microprojection. Methods can be selected from calcium / polyethylene glycol methods for protoplasts, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle impact, and (non-intensive) viral transmission. Transgenic plants, including transgenic crops, can also be produced by Agrobacterium tumefaciens-mediated transformation. Such common methods are also used to introduce genome editing proteins, such as CRISPR Cas nucleases, base-editing nucleases, and other genome editing nucleases. By editing the natural PHYB gene sequence using these genome editing nucleases collectively or separately, vascular sheath promoter sequences, vascular regulatory elements can be introduced, or the natural PHYB sequence can be converted into an active variant or functional fragment.

[0138] Transformation methods are well known in the art. Accordingly, in various aspects of the present invention, the polynucleotides of the present invention are introduced into plants and expressed as transgenes. The nucleic acid sequence is introduced into the plants by a process called transformation. The terms “introduction” or “transformation” are used to encompass all such methods that result in the introduction of exogenous polynucleotides into host plant cells, regardless of the method used for introduction, including “transformation,” “transfection,” and “transduction.” Plant tissues capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, can be transformed by the gene constructs of the present invention, and an entire plant can be regenerated therein. The specific tissues selected will vary depending on the clonal propagation system available for or best suited to the particular species to be transformed. Exemplary tissue targets include leaf discs, pollen, embryos, cotyledons, hypocotyledons, megagametophytes, callus tissue, existing meristems (e.g., apical meristem, axillary buds, and root meristems), and induced meristems (e.g., cotyledonous meristem and hypocotyledonous meristem). The polynucleotide can be introduced into a host cell transiently or stably, and can be maintained in an unintegrated state, for example, as a plasmid. Alternatively, it can be integrated into the host plant genome. The resulting transformed plant cells can then be used to regenerate transformed plants in methods well known in the art.

[0139] To select transformed plants, the plant material obtained in the transformation can, in principle, be subjected to selection criteria that allow the transformed plants to be distinguished from untransformed plants. For example, seeds obtained by the method described above can be planted and, after the initial growth stage, subjected to suitable selection by spraying. A further possibility is to grow the seeds on an agar plate using a suitable selector agent, after disinfection if appropriate, so that only transformed seeds develop into plants. Alternatively, transformed plants are screened for the presence of selectable markers as described above. After DNA transfer and regeneration, plants presumed to be transformed can also be evaluated for the presence, copy number, and / or genomic composition of genes of interest, for example, using Southern blot analysis or whole-genome sequencing. Alternatively or additionally, the expression levels of newly introduced DNA can be monitored using Northern blot analysis and / or Western blot analysis and / or RNA-Seq, all of which are well known in the art.

[0140] The resulting transgenic plants can be propagated by various methods, such as clonal propagation or classical propagation techniques. For example, first-generation (or T1) transgenic plants can be self-pollinated, and homozygous second-generation (or T2) transgenic plants and T2 plants can be further propagated by classical propagation techniques. The resulting transgenic organisms can take various forms. For example, they may be chimeras of transgenic and non-transgenic cells; clonal transgenic bodies (e.g., all cells transformed to contain the expression cassette); or grafts of transgenic or non-transgenic tissue (e.g., in plants, transgenic rootstock grafted onto a non-transgenic scion).

[0141] The modified plants according to the present invention advantageously offer better yield characteristics. Yield characteristics, also known as yield traits, may include one or more of the following non-limiting list of features: yield, biomass, seed yield, seed / grain size, grain starch content, early vigor, greenness index, increased growth rate, increased water use efficiency, and increased resource use efficiency. The term “yield” generally refers to the measurable output of economic value typically associated with a particular crop, area, and period. Yield based on their number, size, and / or mass, or individual plant parts that directly contribute to the actual yield, is the yield per square meter of crop and per growing season, identified by dividing the total output by the square meter planted (including both harvested output and assessed output). The term “yield” of a plant may relate to plant biomass (root and / or shoot biomass), the reproductive organs and / or bulbils of that plant (e.g., seeds or tubers). Accordingly, the present invention allows yield to be measured by evaluating one or more of the following: an increase in seed yield per plant, an increase in seed filling rate, an increase in the number of filled seeds, an increase in harvest index, an increase in viability / germination efficiency, an increase in the number or size of seeds / capsules / pods, an increase in growth or branching, e.g., flowering with more branches, an increase in biomass, an increase in ripening, or an increase in tuber biomass. Preferably, an increase in yield includes an increase in the number of grains / seeds / capsules / pods, an increase in biomass, an increase in growth, an increase in the number of flower pods, an increase in flower branching, or an increase in tubers. Yield can usually be measured relative to control plants.

[0142] Preferably, the plant according to the present invention is a crop. By crop, it means any plant cultivated on a commercial scale for human or animal consumption or use. In a preferred embodiment, the plant is a cereal, an oilseed plant, or a legume.

[0143] Plants according to various aspects of the present invention, including the transgenic plants, methods, and uses described herein, may be monocotyledonous or eudicotyledonous plants.

[0144] Plant or crop species of interest As used herein, the term “plant” encompasses all things that can perform photosynthesis or produce structures that can perform photosynthesis in part or in combination with their constituent parts. Common features that perform or can perform photosynthesis include seeds, fruits, shoots, stems, leaves, roots (including tubers), flowers, tissues, and organs. The term “plant” also encompasses plant cells, suspension cultures, callus tissue, embryos, meristem regions, gametophytes, sporophytes, pollen, and microspores.

[0145] Monocotyledonous plants may be selected from, for example, the Arecaceae, Amaryllidaceae, or Poaceae families. For example, such plants may be cereals such as wheat, rice, barley, oats, rye, millet, or maize, or crops such as garlic, onions, chives, yams, pineapples, or bananas.

[0146] Eudicotyledonous plants are not limited to these, but may be selected from families including Asteraceae, Brassicaceae (e.g., rapeseed), Chenopodiaceae, Cucurbitaceae, Fabaceae (Caesalpinaceae, Mimosoraceae, Papillonaceae, or Fabaceae), Malvaceae, Rosaceae, or Solanaceae. For example, the plant may be selected from buckwheat, lettuce, sunflower, Arabidopsis, broccoli, spinach, canola, watermelon, pumpkin, cabbage, tomato, potato, sweet potato, chili pepper, cucumber, zucchini, eggplant, carrot, olive, cowpea, hops, raspberry, blackberry, blueberry, almond, walnut, tobacco, cotton, cassava, peanut, sesame, rubber, okra, apple, rose, strawberry, alfalfa, legume, soybean, broad bean, pea, lentil, peanut, chickpea, apricot, pear, peach, grape, bell pepper, chili pepper, hemp, camelina, cannabis / marijuana, sugar beet, quinoa, citrus fruits, cocoa, tea, or coffee seeds. In one embodiment, the plant is rapeseed (canola).

[0147] This also includes biofuel and bioenergy crops, such as rapeseed / canola, jute, Jatropha, oil palm, flax, Japanese bean and willow, eucalyptus, poplar, poplar hybrids, etc., or gymnosperms, such as Japanese pine, Norway spruce, or Southern spruce. This also includes crops for silage, grazing, or fodder (grasses, clover, bell beans, alfalfa), fibers (e.g., hemp, cotton, flax), building materials (e.g., pine, oak, rubber), pulp (e.g., poplar), feeder stocks for the chemical industry (e.g., high erucic fatty seeds, rapeseed, flax), and plants for amenity purposes (e.g., turf for golf courses), ornaments for public and private gardens (e.g., snapdragons, morning glories, roses, geraniums, tobacco), and houseplants and cut flowers (African violets, begonias, chrysanthemums, geraniums, coleus, spider plants, dracaena, rubber trees). [Examples]

[0148] (Example 1) Transformation of Arabidopsis thaliana by gene constructs for YHB vascular bundle sheath expression The gene construct was constructed using the Golden Gate cloning system, and the resulting plasmid is shown in Figure 3. LB and RB represent the left and right boundaries of the transfer DNA (T-DNA), respectively. The polynucleotides used by the inventors were the sequences read from LB to RB: the vascular bundle-specific promoter, the PHYB variant coding sequence (YHB in this case), and the plant-friendly terminator sequence. In total, six nucleotide sequence changes were made to the published YHB sequence, none of which altered the corresponding amino acid sequence. These were made to the YHB gene sequence to facilitate the molecular cloning process for constructing the construct. These changes would likely be unnecessary if this process were repeated by synthesizing the construct in a single step, or if an alternative cloning strategy were used. Furthermore, while the construction of this plasmid required the addition of two bacterial marker cassettes, a functionally identical plasmid can be synthesized without requiring a second bacterial selectable marker cassette within the T-DNA region (to the left of RB).

[0149] As described above, before gene synthesis, six nucleotides in the YHB coding sequence [SEQ ID NO: 1] were modified to remove restriction sites, but the amino acid sequence [SEQ ID NO: 4] was left unchanged. A DHS vascular bundle-specific promoter was used. The DHS promoter sequence [SEQ ID NO: 7] was initially cloned from the plasmid described by Knerova et al. (2018) "A single cis-element that controls cell-type specific expression in Arabidopsis" bioRXiv. The two identical vectors contained a herbicide (Basta)-resistant cassette and a cultivated plant-based YHB gene sequence downstream of the vascular bundle promoter. After construction, the vectors were introduced into Agrobacterium tumefasciens (AGL-1 strain) cells by electroporation. Agrobacterium colonies holding the construct were selected on LB plates and cultured on YEB medium.

[0150] Arabidopsis thaliana (Colombian ecotype) plants propagated at the Department of Applied Plant Sciences, University of Oxford, were selected at the time of flowering (approximately 4 weeks old). Several individuals were set aside and propagated to generate wild-type lineages for use as control plants. The remainder were transformed by floral dipping. After dipping, the individuals were grouped into plant batches to distribute seeds to independent transformation events. Seeds were sterilized using ethanol and Triton and stratified in a cold room for 3 days before germination. After germination in soil, T1 plants were screened for transgene insertion by applying Basta herbicide every other day for 1 week. T1 transgenic plants were transferred to larger pots and cultivated to collect T2 seeds. For isolation analysis, T2 seeds were germinated in MS medium using Basta. Single insertion lineages were identified as those showing a 75% viability in selective medium, and they exhibited a single isolated allele. RNA was extracted from these plants to confirm the expression of the YHB transgene in each lineage. Primers were designed and tested to confirm that they specifically amplified YHB but not Phytochrome B from natural Atractylodes thaliana. Three series representing independent transformation events were selected based on segregation and semi-quantitative PCR results, and individual plants from each series were transplanted to soil 12 days after germination. These were grown in a greenhouse under long-day conditions alongside wild-type plants and watered regularly.

[0151] In subsequent embodiments, all phenotypic analyses were performed in all three series unless otherwise specified, and comparisons between the transgenic series derived therefrom (annotated "C12" in the figures) and control plants are shown in subsequent plots. All error bars represent 95% confidence intervals, and t-tests were used to indicate significance ("*") and non-significant ("ns") at p<0.05.

[0152] Figure 4 shows how transfected plants express YHB compared to a housekeeping gene called the eukaryotic cell translation initiation factor elF-4E1.

[0153] Leaf thickness was measured magnetically using a Multispeq V1.0 instrument. In 5.5-week-old plants, nine similar leaves were identified for n=10, and measurements were taken at three locations near the center of the leaf. The median was used for each replicate test. As shown in Figure 5, when measured by t-test (p>0.05), there was no observable difference in leaf thickness between wild-type controls and transgenic Arabidopsis plants containing the gene vector for vascular expression of YHB. Therefore, unlike previous studies in which PHYB expression was manipulated, the present invention described herein does not adversely affect leaf thickness.

[0154] (Example 2) YHB expression in vascular bundle sheath cells enhances the photosynthetic capacity of *Arabidopsis thaliana*. To demonstrate the enhancement of photosynthesis in transgenic plants compared to non-transgenic controls, the plants generated in Example 1 were analyzed by gas exchange measurements using a LICOR 6800 instrument equipped with a multiphase fluorophotometer head. The measurement is the amount of carbon that can be fixed (i.e., their photosynthetic rate) by the control plants and transgenic Arabidopsis plants containing the YHB gene vector for vascular bundle sheath expression, taking into account specific levels of ambient carbon dioxide around the leaves. Arabidopsis plants grown in a greenhouse were clamped to a gas exchange chamber with their leaves clamped, under controlled environmental conditions of 23°C and 65% relative humidity, and a flow rate of 500 μmoles / s. -1 The analysis was performed by setting the fan speed to 10,000 rpm. The same leaves were used for each plant, and all plants were measured between 32 and 35 days of age by testing a mixture of the transgenic series and control plants daily between 10 a.m. and 3 p.m. The plants were 400 μmol / mol -1 CO2 and 1500 μmolm -1 s-1 adapted to light (by a mixture of 90% red and 10% blue), and then the carbon dioxide concentration was gradually reduced from 400 μmol mol -1 to 10 μmol mol -1 and then back to 400 μmol mol -1 and then increased to a maximum of 2000 μmol mol -1 Plants were given 5 minutes to adapt to each new CO2 concentration and then carbon assimilation was measured. The leaf area of the plants was measured to adjust for slight differences in leaf size. The resulting A / Ci curves (Figure 6) demonstrate a significant enhancement of photosynthesis in transgenic plants (n = 8) compared to wild-type controls (n = 12), appearing as a significant increase in maximum photosynthetic capacity and a significant increase in carboxylation efficiency at low carbon dioxide concentrations.

[0155] Transgenic Arabidopsis plants containing the gene vector for vascular expression of YHB were consistently superior to controls until carbon dioxide became too low for either genotype to promote photosynthesis. The initial slope of these curves indicates that these transgenic plants have a higher carboxylation efficiency, and the plateauing phase (for higher values of the carbon dioxide concentrations tested) also demonstrates that the maximum photosynthetic rate of these transgenic plants increased. Considering the ambient carbon dioxide levels (such as those experienced by crops in the field), what the experiment shows is that these transgenic plants fix significantly more carbon dioxide from the ambient air than control plants. Thus, unlike previous studies where the expression of PHYB was manipulated, here the present invention has demonstrated a substantial improvement in the photosynthetic rate at the leaf level.

[0156] (Example 3) Expression of YHB in bundle sheath cells enhances water use efficiency in Arabidopsis Stomatal conductance was measured to demonstrate that transgenic Arabidopsis plants containing a gene vector for YHB vascular expression showed no negative effect on water use efficiency compared to control plants. This is important because previous attempts by others to modulate PHYB / YHB expression (e.g., Rao et al., (2011)) resulted in a significant increase in water consumption. Stomatal conductance was measured at 400 μmol / mol. -1 CO2, 65% relative humidity, 23°C temperature, 500 μmols -1 Measurements were taken at the set flow rate and fan speed of 10,000 rpm. Importantly, compared to the control, transgenic Arabidopsis plants containing the gene vector for YHB vascular bundle sheath expression showed no increase in stomatal conductance (Figure 7).

[0157] Instantaneous water use efficiency (captured carbon per water flux) was calculated by dividing the carbon assimilation rate by stomatal conductance. This demonstrated that while the photosynthetic rate was maximal (as shown in Figure 6), instantaneous water use efficiency was also significantly increased in transgenic Arabidopsis plants containing the gene vector for YHB vascular bundle sheath expression compared to control plants (see Figure 8). Thus, water use efficiency was not impaired by the novel photosynthetic enhancement of the present invention. Moreover, when photosynthesis was operating at maximal rate, transgenic Arabidopsis plants containing the gene vector for YHB vascular bundle expression showed increased water use efficiency compared to control plants. Thus, unlike previous studies that manipulated PHYB expression, the present invention described herein substantially improves the photosynthetic rate at the leaf level while improving water use efficiency.

[0158] (Example 4) YHB expression in vascular bundle sheath cells enhances chloroplast development in vascular bundle sheath cells but not in mesophyll cells in Atractylodes thaliana. In Arabidopsis leaves, mesophyll cells contain fully developed, photosynthetically active chloroplasts, while vascular bundle sheath cells contain smaller, less photosynthetic chloroplasts. To demonstrate that chloroplasts in vascular bundle sheath cells of transgenic Arabidopsis plants containing the YHB gene vector for vascular bundle sheath expression are enhanced compared to control plants, confocal and electron microscopy analyses were performed on the plants. Equivalent leaves (6 leaves) were harvested 25 days after germination from transgenic Arabidopsis plants and control Arabidopsis plants (as developed in Example 1). The lower epidermis was peeled off and the leaves were fixed with formaldehyde. After fixation, parallel skin sections were placed on glass slides and imaged using a confocal microscope. To assign chloroplasts to specific cell types, both chlorophyll and lignin autofluorescence were imaged in cells surrounding the veins. The autofluorescence of lignin and chlorophyll was detected by excitation with 458 nm and 633 nm lasers, and the emission spectra were recorded between 465–599 nm and 650–750 nm, respectively. Z-stacks were imaged around the veins to capture mesophyll cells and bundle sheath cells from a total of five leaves per genotype. For each leaf, five mesophyll cells and five bundle sheath cells were identified from at least two different images, and the chloroplast region plan (i.e., positioned parallel to the Z-plane) of the five largest chloroplasts in each cell was calculated using ImageJ. Thus, the average chloroplast size per genotype was calculated by measuring a total of 125 chloroplasts in 25 cells distributed among five different plants. Furthermore, transmission electron micrographs were obtained by sampling the plants at the same time of day (11 am). The tissues were stained, embedded in resin, and then sectioned using an ultramicrotome diamond knife. Images were captured using a Siemens transmission electron microscope.

[0159] As shown in Figure 9, chloroplasts in vascular bundle sheath cells of transgenic Arabidopsis plants containing the YHB gene vector for vascular bundle sheath expression were significantly larger than chloroplasts in the same cells of control plants. In this cell type, YHB expression induced chloroplast development so that these chloroplasts were the same size as chloroplasts in mesophyll cells. Mesophyll chloroplasts remained unchanged in size between transgenic Arabidopsis plants containing the YHB gene vector for vascular bundle sheath expression and controls.

[0160] Electron microscopy analysis of transgenic Arabidopsis plants containing a gene vector for YHB vascular expression revealed that, in terms of the size and composition of photosynthetic organs, vascular bundle chloroplasts were comparable to mesophyll cell chloroplasts (Figures 10B and 10D), while vascular bundle chloroplasts in control plants were significantly smaller and less photosynthetically efficient compared to mesophyll chloroplasts of the same plants (Figures 10A and 10C). Therefore, the invention of precise YHB expression in vascular bundle sheath cells acts only on vascular bundle sheath chloroplasts, and thus the photosynthetic enhancement described in Example 2 is driven by the activation of photosynthesis in vascular bundle sheath cell chloroplasts.

[0161] (Example 5) YHB expression in vascular bundle sheath cells enhances the refixation of respiration-induced carbon dioxide in Atractylodes thaliana. While photosynthetic cells fix CO2 in sugars, all single plant cells respire, consuming sugars and releasing CO2. Respiration by vein cells releases CO2, which is normally diffused from the vein into the intercellular spaces by surrounding vascular bundle sheath cells and then reabsorbed by the mesophyll or lost from the leaf through stomata. Transgenic plants showed an increased ability to fix carbon, so measurements were performed to determine whether this was due in part to the re-fixation of CO2 that had been respired by the vein and converted back into sugars to promote growth.

[0162] CO2 in the air contains a mixture of carbon-12 and carbon-13 isotopes, but carbon in plant tissues has almost no trace of carbon-13 relative to carbon-12 compared to carbon-12 in the air. This is because the enzyme that fixes carbon from the air, Rubisco in the case of C3 species, discriminates the heavier carbon-13 isotope, resulting in a negative δ13C ratio when measured by dry matter carbon isotope analysis. When a transgenic Arabidopsis plant containing the YHB gene vector for vascular expression (Example 4) refixes respired carbon (i.e., carbon that has already been fixed once before), the carbon ultimately present in the leaves undergoes multiple rounds of Rubisco-mediated fixation, resulting in multiple rounds of discrimination. Therefore, when enhanced refixation of respired CO2 occurs in the transgenic plant containing the YHB gene vector for vascular expression, this trace is expected to be seen in carbon isotope analysis. More specifically, a more negative δ13C ratio is expected to be seen in equivalent tissues derived from control plants.

[0163] At 35 days old (plants developed in Example 1), equivalent leaves (9 leaves) were rapidly frozen in liquid nitrogen and freeze-dried in a freeze-dryer for 4 days. Approximately 1 mg of dried leaf powder was weighed for every 6 samples per genotype (2 genotypes, 1 transgenic lineage and 1 control group were tested), and stable isotope analysis was performed. This demonstrates that transgenic Arabidopsis plants containing the gene vector for vascular expression of YHB have significantly more negative δ13C, indicating that respired CO2 is an important carbon source in these plants (see Figure 11). Therefore, the component of photosynthesis enhancement in these plants is attributable to enhanced refixation of respired CO2. The degree of this enhanced refixation may vary among species depending on the availability of respired / transpired CO2 from vascular sources.

[0164] In normal C3 plants, carbon diffused into the intercellular spaces of leaf cells is fixed to sugars. In photoactivated mesophyll cells, Rubisco fixes this carbon, which is then delivered as sugars into the vascular structure. These sugars promote plant growth throughout the plant through respiration. This releases carbon dioxide, which is returned from the leaf veins and diffused out of the leaf around / through the vascular bundle sheath cells. Figure 19 shows that in the modified C3 plant of the present invention, initial carbon fixation is primarily carried out by mesophyll cells, but vascular bundle sheath cells of the plant of the present invention can also do this. Here, because more active chloroplasts are present in the vascular bundle sheath cells surrounding the leaf veins, the respired carbon dioxide is captured before it can diffuse from the vascular bundle sheath into the intercellular spaces and exit the plant. Thus, this respired CO2 is refixed to sugars, shifting the carbon isotope ratio to a lower level, increasing carbon assimilation efficiency, and further promoting growth per carbon molecule diffused within the leaf. Therefore, the present invention, which drives the precise expression of YHB only in vascular bundle sheath cells, can also yield further advantages in enhanced CO2 refixation.

[0165] (Example 6) YHB expression in vascular bundle sheath cells enhances plant growth in Atractylodes thaliana. Assuming that transgenic Arabidopsis plants containing a gene vector for YHB vascular expression have higher photosynthetic capacity than control plants (Examples 2-5), we identified how this increase in net carbon uptake could promote increased plant growth. Photographs were taken from above the trays of 15 plants (as developed in Example 1) on days 14 and 21 after germination. The images were analyzed in ImageJ to calculate the total rosette area per plant. This showed that the transgenic plants of the present invention grew faster than the control plants in this time window, consistent with the increased photosynthetic rate (Figure 12).

[0166] Bolts are flowering structures in the genus Arabidopsis. When plants have sufficient resources during vegetative growth, they mature into flowering structures and invest those resources in reproductive structures. Bolting time was measured as the number of days from germination until the bolt grew to a height of more than 3 mm. This demonstrated that bolting time was shortened in transgenic Arabidopsis plants containing the YHB gene vector for vascular bundle sheath expression compared to wild-type controls (Figure 15). This is significant because, as previously described in the literature, PHYB / YHB overexpressing plants consistently showed the opposite effect (i.e., delayed time to bolting / flowering). Delayed bolting time is detrimental to crop production because the growing season is extended, plants lose synchronicity with the seasons, and suffer an increased risk of loss. This occurs because photoactivated PHYB / YHB suppresses Flowering Locus T expression in mesophyll cells, thereby inhibiting flowering. In this invention, there is no additional expression of PHYB / YHB in mesophyll cells, and therefore the flowering pathway is not obstructed, thus avoiding this problem. Consequently, the reduction in the time to bolting in the plants of this invention is a novel and advantageous characteristic.

[0167] In addition to measuring the flowering (bolting) time described above, the size of the flowering structure (bolt) was also measured. For each of the n=12 plants, the highest bolt was measured at 12 pm using a ruler. Bolts from transgenic Arabidopsis plants containing the gene vector for YHB vascular bundle expression were higher than those from control plants at 35 days post-germination. Rather than dwarfism, as expected considering previous studies by others on PHYB and YHB overexpression, we found these transgenic plants to be higher at the same time (see Figure 13). Otherwise, the plants underwent normal photomorphogenesis (see photograph of the tray in Figure 14). Thus, unlike previous studies manipulating PHYB expression, the invention described herein substantially improves plant growth without any of the expected adverse developmental effects of PHYB / YHB overexpression.

[0168] Therefore, the present invention, which drives the precise expression of YHB in vascular bundle sheath cells, results in faster growth, earlier flowering, and larger flowering structures. These are all advantageous characteristics for agriculture, as they mean a shorter growing season, a reduced risk of crop loss due to adverse weather or pests / pathogens, and potentially a larger harvest cycle per year, i.e., adding additional value.

[0169] (Example 7) YHB expression in vascular bundle sheath cells enhances yield in *Arabidopsis thaliana*. The plants of this invention exhibited a higher photosynthetic rate, faster growth, earlier flowering, and larger flower structures (Figure 16). We investigated whether these advantageous characteristics resulted in a corresponding increase in yield.

[0170] Figure 17 shows typical seed yields for wild-type plants (left) and transgenic Arabidopsis plants containing the YHB vascular bundle sheath expression gene vector (right) after cessation of watering at week 7.5, harvesting at week 9, and seed sorting and weighing at week 9.5. This represents a >30% increase in yield, which is statistically significant by a t-test statistic < 0.0005. This demonstrates that the amount of seeds produced per plant is significantly higher in the transgenic Arabidopsis plants containing the YHB vascular bundle sheath expression gene vector compared to the control.

[0171] Previous experiments on the overexpression of PHYB / YHB in plants have often reported increased yields, but this can be misleading and may not lead to crop harvests due to multifaceted delays in flowering. For example, Thiele et al. (1999) reported increased yields by overexpressing PHYB in potatoes, producing a smaller but greater number of tubers. However, they also made it clear that this did not occur within the same timeframe as conventional potato harvests; when harvested at the same time as normal potato harvests, the yield of conventional PHYB overexpression was lower than that of controls. In fact, in Hu et al. (2019) mentioned above, YHB (derived from either Arabidopsis or rice) was overexpressed in various different species (Arabidopsis, rice, tobacco, tomato, and Alpinia), and YHB overexpression consistently had a negative impact on seed yield. In stark contrast, our transgenic plants, surprisingly, exhibited significantly higher seed yields than controls, regardless of the stage at which they were harvested, when harvested simultaneously.

[0172] Ultimately, transgenic Arabidopsis plants containing the gene vector for YHB vascular bundle expression filled these siliques to produce significantly more seeds than controls (see Figure 18), and this was consistently enhanced regardless of whether the seeds were harvested early or late. Here, watering was stopped at either 6.5 weeks of age ("early") or 8 weeks of age ("late"). The plants were dried for 1.5 weeks before harvesting the seeds. The dried airborne biomass was collected in paper bags and shaken to release the seeds. Seeds were separated from the plant debris using a fine mesh and poured into plastic tubes for weighing. Thus, the increased photosynthesis was successfully translated into increased yield.

[0173] Therefore, the present invention results in a higher photosynthetic rate, improved water use efficiency, enhanced CO2 refixation, faster growth, earlier flowering, larger flowering structures, and higher yields compared to control plants.

[0174] (Example 8) Transformation of wheat (Triticum aestivum) by gene constructs for YHB vascular bundle sheath expression. To demonstrate the broad general applicability of the present invention and to verify the crop-enhancing ability of bundle-sheath-expressed YHB, a monocotyledon-optimized plasmid was designed and tested in the monocotyledonous crop wheat, specifically the wheat (Triticum aestivum) variety Cadenza. Unlike Example 1, which used a synthetic bundle-sheath promoter, here we used the shiba phosphoenolpyruvate carboxykinase promoter (previously described to provide bundle-sheath-specific gene expression in monocotyledons) (Nomura et al., (2005), Plant Cell Physiol. and Figure 23). This promoter sequence [SEQ ID NO: 10] is obtained from the monocotyledonous shiba rather than the eudicotyledonous Alocasia. This promoter sequence was designed to drive the expression of the endogenous wheat Phytochrome B coding sequence [SEQ ID NO: 11] (Traes_4AS_1F3163292), which was modified to be photoinsensitive by converting the amino acid tyrosine at position 278 to histidine (known as the YHB mutation). When compared using Clustal 2.1, the coding sequence of this wheat gene shared 66.11% identity with the Arabidopsis ortholog used in Example 1, and the amino acid sequence [SEQ ID NO: 12] shared 71.28% identity with the Arabidopsis ortholog.

[0175] The full-length promoter-gene-Nos terminator sequence was newly and completely synthesized. This sequence was incorporated into a binary vector containing an nptII selection cassette and transferred to Agrobacterium species, which were then used to transform wheat callus cultured using standard plant tissue culture and transformation methods. Transformants were screened to confirm successful genome insertion and to identify single-insertion transgenic plants by qPCR. Transformants were planted in pots and cultivated in a cultivation box alongside control plants that had undergone callus regeneration but had not received constructs for YHB vascular bundle sheath expression.

[0176] (Example 9) YHB expression in vascular bundle sheath cells increases the rate of photosynthesis in wheat. Seven weeks after cultivation in the growing box, the photosynthetic rate of the transgenic wheat plants generated in Example 8 was quantified and compared with that of the control plants. As in Example 2, the photosynthetic rate was accurately measured using a LICOR 6800 instrument. The environmental constants were as follows: flow 500 μmols -1 Fan speed 10,000 rpm, leaf temperature 25°C, 65% relative humidity. To measure the ambient photosynthetic rate in the growing box, PAR (photosynthetically active radiation, i.e., the amount of light available for photosynthesis) was set to 350 μmolm -1 s -1 The light intensity was set to (measured at the height of the cultivation box lid), and carbon dioxide was set to 400 μmol / mol. -1 The settings were adjusted as follows: For each plant, a leaf below the flag leaf was selected, and approximately one-third of the leaf from the tip was clamped. After 10 minutes of climate acclimatization (confirmed by observing no changes in assimilation rate, fluorescence, or stomatal conductance after this acclimatization), ambient photosynthesis measurements were recorded. Four control and eight single-insertion wheat plants were screened between 12:00 and 14:00 on the same day. Figure 20 shows the results of this analysis: the photosynthetic rate was, on average, 30% higher in wheat plants containing the gene vector for YHB vascular bundle sheath expression compared to the control, as determined by a t-test at p<0.05.

[0177] (Example 10) YHB expression in vascular bundle sheath cells enhances growth rate in wheat. As demonstrated in Example 6, enhanced Phytochrome B signaling in the vascular bundles of Arabidopsis species is associated with faster growth, as indicated by increased biomass accumulation compared to controls within the same time window, but no change in overall plant structure development. Similarly, wheat plants containing the YHB gene vector for vascular bundle sheath expression showed no changes in development (e.g., dwarfism), and normal flowering was observed. As shown in Figure 21, typical wheat plants containing the YHB gene vector for vascular bundle sheath expression were significantly larger than controls after 7 weeks of growth.

[0178] In fact, plant height (measured as maximum canopy height, which is the distance from the soil surface to the tip of the highest point) was significantly higher in the transgenic plants than in the control (n=4) (by t-test, p<0.05) (Figure 22). At this point, the transgenic plants appeared to have reached full height and had begun flowering, while the control was still only about two-thirds of this maximum height. This approximately 30% faster growth was thought to be mainly due to the 30% increase in photosynthetic rate observed in Example 9. Therefore, despite the large genetic differences and evolutionary distance between the eudicot Arabidopsis thaliana and the monocot Wheat, the present invention consistently enhances photosynthesis, does not hinder development, and increases plant productivity.

[0179] Therefore, those skilled in the art can combine any promoter sequence known to activate vascular bundle expression (either known in the literature or by designing a novel promoter), and can overexpress either the endogenous Phytochrome B gene or the exogenous Phytochrome B gene or the YHB variant or a functional fragment thereof to apply the present invention to any desired crop. Similarly, various callus transformation methods can be used depending on the species of interest (whether flower immersion as in Example 1 or callus transformation as in this example).

[0180] (Example 11) Gene editing of Brassica napus for vascular bundle sheath expression of PHYB and / or YHB As mentioned in Figures 2 and 25, PHYB is duplicated in several agronomically important species, such as rapeseed and soybeans. In fact, most of our crops have recently experienced whole-genome duplication events and contain multiple redundant copies of PHYB. This means that it is possible to convert one copy of PHYB to a vascular-driven YHB without affecting the other copies. This means that introducing YHB by genetic modification would have the same result for the plant (as in Examples 1 and 8), but without requiring the addition of any transgenic material, and therefore, as a result, would instead produce a gene-edited plant. This has the added benefit of ensuring that natural PHYB signaling is not eliminated (otherwise, it would result in developmental abnormalities in the plant).

[0181] Brassica napus provides an example species in which genome editing can be used to achieve vascular bundle sheath expression of YHB using standard genome editing techniques known to those skilled in the art. Figure 26 shows the expression of three PHYB genomes encoded in the Brassica napus genome (BnaA05g22950D, BnaC05g36390D, and BnaC03g39830D, hereafter referred to as BnaA05, BnaC05, and BnaC03, respectively) in the leaves of 16 different cultivars of this crop species (RNA was collected from the second youngest leaf when the plant was in the five true leaf stage, Hong et al., (2019) Nat. Comms. 10:2878). The PHYB homologs BnaA05 and BnaC05 are expressed in the leaves of all cultivars, and both are expressed to the same degree in each cultivar, providing evidence that they function redundantly. An exception to this pattern is the Span cultivar, which does not express BnaC05. However, assuming that Span undergoes normal photosynthetic development, this is further evidence that both PHYBs function in overlapping roles, i.e., that BnaA05 expression compensates for the lack of BnaC05 expression. Therefore, it would be possible to manipulate one variant for the purpose of enhancing photosynthesis without interfering with normal photomorphogenesis.

[0182] Initially, the gene expression domain of the natural PHYB gene will be modified to be expressed in the vascular bundle. This will be achieved by knocking in a short promoter sequence known to those skilled in the art (e.g., Sequence ID No. 7) or any vascular bundle or vascular bundle sheath promoter or vascular bundle sheath enhancer element into the 5' upstream region of the natural PHYB gene (e.g., BnaA05). The vascular bundle sheath promoter described below herein and illustrated in Figure 23 functions over large phylogenetic distances (90 to 160 million years of divergence time). GLDP and SULTR2;2 give constitutive expression patterns to the genera Arabidopsis and Flavelia, which represent deep conservation between the roses and chrysanthemums that diverged approximately 125 million years ago. The genus Flavelia is as closely related to Brassica napus as it is to Arabidopsis thaliana; therefore, promoters that function in both the genera Flavelia and Arabidopsis are expected to function in Arabidopsis as well. The MYB76 regulatory element used in Example 1 is known to be highly conserved between the genera Arabidopsis and Brassica napus, and they are closely related (they diverged only about 20 million years ago). Many promoters are available to those skilled in the art and can be selected from them to target the expression of natural PHYB genes.

[0183] The present invention allows for the editing of the innate PHYB gene by inserting a vascular sheath promoter, resulting in the expected expression of PHYB in the desired tissue. The stably inheritable PHYB sequence is functionally equivalent to the polynucleotides incorporated into the Arabidopsis or wheat genome, as described in Examples 1 and 8. Any region in the 5' upstream area may be a suitable target site for knocking in these promoter sequences. An endonuclease would target a specific site to induce a double-strand DNA break, and a homology arm would direct the promoter polynucleotide, which is incorporated into the DNA by homology-directed repair, to this area. This has already been demonstrated in plants with suitable efficiency. For example, CRISPR-Cpf1 has been used to knock in >3,000 bp DNA fragments into the rice genome with 8% efficiency (Begemann et al., (2017) Sci. Reps. 7:11606). Assuming that the vascular bundle promoter element is much shorter than in this example, and that shorter sequences result in higher knock-in efficiency, this knock-in would be feasible without further steps in the invention. Arabidopsis thaliana can be transformed using Agrobacterium species (as in Example 1) and independent transformation events, and is screened by PCR to find individuals in which the promoter element is successfully incorporated upstream of PHYB. Plants lineage from these individuals will have enhanced PHYB expression in the vascular bundle, which can be tested by gene expression analysis, and will also be expected to exhibit some enhanced chloroplast development, photosynthetic rate, and productivity without developmental abnormalities associated with altered PHYB expression at the whole plant level (e.g., semi-dwarf phenotype resulting from ubiquitous overexpression of PHYB). The phenotype will be expected to be similar to that described herein, from the introduction of PHYB expressed in the vascular bundle using conventional gene modification approaches.

[0184] To further amplify PHYB signaling activity in the vascular structure of Arabidopsis thaliana, a second edit may be necessary to convert vascular bundle-driven PHYBs to YHBs. This can also be delivered by gene editing, but requires only point mutations rather than double-strand DNA breaks. In Arabidopsis PHYBs, the tyrosine-derived residue 276 is converted to histidine, and the "TAT" codon is changed to "CAT" to convert the PHYB to a YHB. In the case of BnaA05, the "TAC" codon codes for an equivalent tyrosine residue, and by changing it to "CAC", the equivalent modification to histidine can be achieved by introducing a single nucleotide change. Figure 27 illustrates the regions of the Arabidopsis thaliana PHYB coding sequence [SEQ ID NOs. 14, 15, and 16] that can make this single base pair change. This editing can be induced by a nickase linked to adenosine deaminase, such as Cas9; the nuclease creates a small window in the single-stranded DNA that directs the deaminase to a specific section of DNA to convert adenine to guanine. This type of editing has been previously demonstrated in Arabidopsis plants and Arabidopsis thaliana protoplasts, with efficiencies of up to 8.8% in the latter species (Beum-Chang Kang et al., (2018) Nat. Plants. 4:427~431). By targeting the reverse strand of the PHYB gene, this system is sufficient to induce adenine-guanine conversion, resulting in a complementary conversion of thymine to cytosine on the forward strand, thereby shifting the codon from "TAC" to "CAC," and consequently shifting from PHYB to YHB. This T-to-C mutation could also be easily achieved by prime editing (Anzalone et al. (2020) Nature Biotechnology, 38:824~844), or by randomly targeted mutagenesis at the correct site using CRISPR-Cas or other genome editing nucleases known to those skilled in the art.

[0185] As shown in Figure 27, despite the high degree of nucleotide sequence conservation of multiple copies of the PHYB gene in the Arabidopsis thaliana genome, each homolog contains several unique variants that can be used to direct targeted base editing only to specific gene variants, i.e., the PHYB gene whose expression domain has been previously edited, thereby ensuring that YHB expression is restricted to the vascular bundle. To find individuals containing this YHB editing, transformed plants are screened by PCR, and it is expected that any increase in photosynthesis and productivity previously induced by the first modification may be further amplified by this second modification.

[0186] Both genome editing methods proposed here have been demonstrated in plants to a high level of efficiency, even in species that are difficult to transform and require methods other than flower immersion, such as callus regeneration or particle impact. Therefore, this Arabidopsis thaliana example provides a general method for introducing YHB expressed in the vascular bundles by genome editing in any species containing two or more copies of PHYB. Moreover, this approach can be employed in any diploid plant, as long as the transformant is maintained as a heterozygous plant containing one unmodified copy in the PHYB allele and one modified copy in the PHYB allele. In summary, a single copy of PHYB is targeted for editing using a nucleotide variant specific to that copy. Firstly, PHYB expression is enhanced in the vascular bundles by knocking in a vascular bundle sheath or a vascular bundle-specific promoter in the 5' upstream region. This same gene is subsequently targeted for a single nucleotide mutation in the CDS (coding sequence); the codon encoding the tyrosine residue that gives the native PHYB the ability to revert from its photoactive form is mutated to histidine. This converts the native PHYB to constitutively active YHB, which further enhances the PHYB signaling cascade in the vascular bundle. Notably, even in species lacking duplicate copies of PHYB, it is possible to initially knock in a full-length PHYB copy, thereby creating a copy that can be further edited. In particular, all of these gene editing proposals achieve the same final result demonstrated in Examples 1 and 8 by the gene editing method: a PHYB homolog expressed in vascular sheath cells.

[0187] Finally, the effect of altering PHYB expression (by knockout or overexpression) is highly conserved among distantly related species (Figure 24), and multiple promoters derived from different distantly related species can drive vascular sheath expression across the width of vascular cells (Figure 23). The exemplary examples provided herein are understood to be illustrative, and thereby, those skilled in the art can deliver this property to any vascular plant species by any of the genetic engineering methods described above.

[0188] (Example 12) Generalized gene editing protocol for activating vascular bundle sheath expression of PHYB or YHB in any plant species In addition to the full promoter knock-in example in Example 11, it is also possible to limit the size of gene editing to a few base pairs by simply introducing a small vascular sheath or vascular bundle motif or enhancer element into the promoter region of the endogenous PHYB gene. Figure 28 provides a comparison between these two approaches, which is demonstrated by designs for tomato (Solanum lycopersicum) and soybean (Glycine max), annotated with respect to genomic models for PHYB orthologs in the species of the former (Solyc05g053410) and the latter (Glyma.09G035500). The glycine decarboxylase P subunit (GLDP) promoter has been characterized in the chrysanthemum Flavelia videntis and the rose Arabidopsis thaliana. A series of deletions revealed that a V-box containing the GLDP1 promoter region is sufficient to drive vascular expression (Adwy et al., (2015) The Plant Journal. 84(6):1231~1238). Therefore, in tomato, vascular expression of PHYB could be introduced by knocking in a GLDP1 V-box containing the promoter [SEQ ID NO: 13] immediately upstream of the first exon of the endogenous PHYB gene identified here, using a method similar to that described in Example 11 (as shown in the image above in Figure 28). That is, those skilled in the art could use such a design to target various genome editing nucleases to a target locus by a DNA repair template encoding a promoter sequence of choice, and thus generate gene-edited plants.

[0189] The MYB76 promoter used in Example 1 is known to drive tissue-specific gene expression through the action of a small, minimal enhancer motif (Dickinson et al., (2020) Nature Plants. 6:1468~1479). Such an enhancer motif sequence could be introduced very close to the transcription start site of the soybean PHYB gene to confer the desired expression pattern. Unlike the tomato design described above, this approach would leave the endogenous core promoter intact, as shown in Figure 28 (image below) due to the presence of the natural 5'UTR. The core promoter can be further characterized by various common techniques, such as, but not limited to, TSS-seq, CAP-seq, and CHIP-seq, to ​​identify the open chromatin region. This additional characterization helps identify the precise location where RNA polymerase binds to initiate transcription, thereby ensuring that the exact genomic location where the vascular bundle enhancer motif is inserted does not interfere with this region (although it is also possible to simply try several locations and confirm success by gene expression analysis in transgenic plants). Therefore, this enhancer element insertion method would enable editing of the native PHYB gene without disrupting the native expression pattern, as well as editing of the PHYB expression profile in species having only one copy of this gene. Considering these advantages, it may be desirable to further reduce known vascular bundle promoters, such as the GLDP1 V-box, to the smallest enhancer sequence that can be introduced by editing as few bases as possible, using the same molecular method already published, for example, when reducing the full-length MYB76 promoter to the smallest enhancer motif sequence that is necessary and sufficient (Dickinson et al., (2020) Nature Plants. 6:1468~1479). In some cases, to further enhance PHYB signaling in vascular bundle cells, subsequent conversion of vascular bundle sheath-expressed PHYB to YHB, as described in Example 11, may be performed.This single nucleotide mutation could be easily achieved by base editing or prime editing, or by randomly targeted mutagenesis at the correct site using CRISPR-Cas or other genome editing nucleases known to those skilled in the art.

[0190] Genetic resources Seeds of Arabidopsis thaliana (Colombian ecotype) were obtained in September 2018 from the greenhouse of the Department of Applied Plant Sciences at Oxford University.

[0191] The Golden Gate cloning components were provided by Sylvestre Marillonnet (Liebnitz Institute of Plant Biochemistry: Weber et al., (2011) PLOS ONE). The DHS vascular bundle promoter was provided by Patrick Dickinson from the Julian Hibberd Laboratory at the University of Cambridge (Knerova et al., (2018) bioRxiv).

[0192] Cadenza wheat plants and wheat transformations were provided by NIAB Crop Transformation Services.

[0193] Nucleotide and amino acid sequences [Sequence ID 1] Cultivated *Alocasia macrorrhizos* PHYB coding sequence containing the YHB mutation. [Sequence ID 2] Cultivated Arabidopsis thaliana PHYB code sequence (Arabidopsis genus_PHYB_AT2G18790.1). [Sequence ID 3] Rice PHYB code sequence (rice_PHYB_LOC_Os03g19590.1). [SEQ ID NO: 4] Amino acid sequence of Arabidopsis thaliana YHB. [SEQ ID NO: 5] Arabidopsis thaliana PHYB amino acid sequence (Arabidopsis genus_PHYB_AT2G18790.1). [SEQ ID NO: 6] Rice PHYB amino acid sequence (rice_PHYB_LOC_Os03g19590.1). [SEQ ID NO: 7] Nucleotide sequence of the MYB76 vascular bundle promoter from the genus Arabidopsis. This is a synthetic promoter containing an oligomerized MYB76 sequence with a minimal enhancer element and a 35S minimal core promoter element. [Sequence ID 8] Phytochrome D nucleotide coding DNA sequence of Arabidopsis thaliana (genus Arabidopsis_PHYD_AT4G16250.1). [SEQ ID NO: 9] Phytochrome D amino acid sequence of Arabidopsis thaliana (genus Arabidopsis_PHYD_AT4G16250.1). [SEQ ID NO: 10] Shiba PCK promoter sequence. [Sequence ID 11] Wheat PHYB coding sequence containing the YHB mutation (derived from Traes_4AS_1F3163292). [SEQ ID NO: 12] Wheat PHYB amino acid sequence containing the YHB mutation (derived from Traes_4AS_1F3163292). [SEQ ID NO: 13] GLDP1 V-box containing promoter DNA lineage. [Sequence ID 14] Extract of the PHYB code sequence of Brassica napus (BnaC03g39830D). [Sequence ID 15] Extract of the PHYB code sequence of Brassica napus (BnaA05g22950D). [Sequence ID 16] Extract of the PHYB code sequence of Brassica napus (BnaC05g36390D).

[0194] Throughout this description and claims, the words “comprise” and “contain” and their variations mean “including but not limited to,” and they are not intended (and do not exclude) other parts, additives, ingredients, integers, or processes. Throughout this description and claims, the singular includes the plural unless the context requires otherwise. In particular, where the indefinite article is used, this specification should be understood to assume the plural as well as the singular unless the context requires otherwise.

[0195] Any feature, integer, property, compound, chemical part, or group described in connection with a particular aspect, embodiment, or example of the present invention should be understood to be applicable to any other aspect, embodiment, or example described herein, provided that they are not incompatible. All of the features disclosed herein (including any appended claims, abstract, and drawings) and / or all of the steps of any method or process so so disclosed may be combined in any combination, except for any combination in which at least some of such features and / or steps are mutually exclusive. The present invention is not limited to any of the details of any prior embodiments. The present invention extends to any novel one or any novel combination of the features disclosed herein (including any appended claims, abstract, and drawings), or any novel one or any novel combination of the steps of any method or process so so disclosed.

[0196] The reader's attention is directed to all documents filed concurrently with or prior to this specification in connection with this application, and which are made available to the public by this specification, and the contents of all such documents are incorporated herein by reference.

Claims

1. C 3 A method for enhancing the photosynthetic capacity of a plant, wherein the gene of interest (GOI) encoding Phytochrome B, or an active variant thereof that retains the signal activation function of PHYB, is C 3 The C is expressed specifically in at least one vascular bundle sheath and / or vascular cell of a plant. 3 The process includes a step of modifying the heritable genetic material of a plant, wherein the GOI is expressed under the control of a gene expression regulatory element, and the gene expression regulatory element is C 3 A method that is specifically active in at least one vascular bundle sheath and / or vascular cell of a plant.

2. The method according to claim 1, wherein the Phytochrome B or the active variant comprises a functional fragment thereof.

3. The method according to claim 1 or claim 2, wherein the GOI is not expressed or is expressed only in very small amounts in mesophyll cells.

4. The step of changing the heritable genetic material is C 3 The method according to any one of claims 1 to 3, comprising the step of inserting at least one polynucleotide into the heritable genetic material of a plant cell.

5. The method according to any one of claims 1 to 4, wherein the step of modifying the heritable genetic material includes the use of a base editor; and may include the use of a prime editor.

6. The process of modifying the heritable genetic material involves gene repair oligonucleotide (GRON)-mediated mutations. 3 The process includes the step of introducing the heritable genetic material into a target DNA sequence in a plant cell; C 3 The method according to any one of claims 1 to 4, which may include the step of exposing the plant cells to a DNA cutter and GRON.

7. The method according to claim 6, wherein the DNA cutter comprises a meganuclease, a transcription activator-like effector nuclease (TALEN), a Zn finger, an antibiotic, or a Cas protein.

8. The step of changing the heritable genetic material is C 3 The method according to any one of claims 1 to 3, comprising the step of using a zinc finger nuclease (ZFN) and / or a transcription activator-like effector nuclease (TALEN) for site-specific homologous recombination of the heritable genetic material of a plant cell.

9. The step of modifying the hereditary genetic material includes introducing a donor template into the hereditary genetic material of the cells of the plant C using a viral vector. 3 The method according to any one of claims 1 to 3, including the step of introducing a donor template into the hereditary genetic material of the cells of the plant C using a viral vector.

10. The method according to claim 9, wherein the viral vector comprises a protein expression vector; and the protein expression vector may comprise a pQE or a pET.

11. One or more polynucleotides comprising a polynucleotide encoding a CRISPR-Cas protein and a donor polynucleotide comprising the sequence of the gene expression regulatory element, and may also include a guide RNA (gRNA); the gRNA encodes the CRISPR-Cas protein, C 3 The method according to any one of claims 1 to 4, wherein the gene expression regulatory element is inserted so as to direct to a locus of at least one copy of the GOI in the genome of a plant cell, thereby causing the expression of the copy of the GOI in at least one vascular bundle sheath and / or vascular cell of a plant regenerated from the cell.

12. The method according to claim 11, wherein the CRISPR-Cas protein and the gRNA are pre-assembled to form a ribonucleoprotein (RNP); and the RNP can be transfected into the cell.

13. The method according to claim 11 or claim 12, wherein the RNP is transfected into the cells using electroporation.

14. The method according to any one of claims 11 to 13, wherein the CRISPR-Cas protein comprises Cas9, Cas12a, or Cas12b.

15. The method according to any one of claims 11 to 13, wherein the polynucleotide encoding the CRISPR-Cas protein is introduced by a plasmid.

16. At least one polynucleotide comprises the expression regulatory element and a nucleotide sequence encoding the GOI, which may include a terminator, and further polynucleotides encoding the CRISPR-Cas protein, and further polynucleotides or additional further polynucleotides encoding the CRISPR-Cas protein 3 The gRNA may encode a gRNA directed to a desired locus in the plant genome, thereby allowing the non-homologous GOI under the control of the vascular bundle sheath and / or vascular regulatory element to be directed to the C 3 The method according to claim 4, which can be inserted into the desired gene locus of the plant cell.

17. The method according to claim 16, wherein the at least one polynucleotide comprises, from 5' to 3', the expression regulatory element and the nucleotide sequence encoding Phytochrome B or an active variant thereof or a functional fragment thereof, and may include the terminator.

18. At least one polynucleotide is C from 5' to 3'. 3 The expression regulatory element is specifically active in at least some vascular bundle sheaths and / or vascular cells of a plant, and comprises a nucleotide sequence encoding Phytochrome B, or an active variant thereof, or a functional fragment thereof, thereby enabling Phytochrome B, or its active variant, or its functional fragment to be C 3 The method according to claim 4, which is inserted into the genome of a plant.

19. From 5' to 3', C 3 An isolated DNA polynucleotide comprising a regulatory element specifically active in at least some vascular bundle sheath cells of a plant, and a nucleotide sequence encoding Phytochrome B or an active variant thereof or a functional fragment thereof, and potentially comprising a terminator.

20. The isolated DNA polynucleotide according to claim 19, wherein the regulatory element comprises a promoter.

21. The isolated DNA polynucleotide according to claim 19 or claim 20, wherein the promoter is a vascular bundle sheath cell-specific promoter and / or a mestome sheath cell-specific promoter.

22. The isolated DNA polynucleotide according to claim 21, wherein the vascular bundle sheath cell-specific promoter and / or the mestome sheath cell-specific promoter is a synthetic promoter; it may include a vascular bundle sheath and / or mestome sheath-specific transcription factor binding element upstream of the promoter; and two or more transcription factor binding elements may be present.

23. The isolated DNA polynucleotide according to claim 22, wherein the vascular bundle sheath-specific promoter or the mestom sheath promoter is selected from the minimum ZmUbi1 promoter, the NOS core promoter, the CHSA core promoter, and the minimum 35S promoter; and the promoter may have the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 10 or a sequence that is at least 90% identical to them.

24. The isolated DNA polynucleotide according to any one of claims 21 to 23, wherein the vascular bundle sheath-specific promoter or mestome sheath promoter is derived from a vascular bundle sheath-specific gene.

25. The isolated DNA polynucleotide according to claim 24, wherein the vascular bundle sheath-specific gene is derived from a plant species selected from the group consisting of Arabidopsis thaliana MYB76, Flavelia trinevia GLDP, Arabidopsis thaliana SULTR2;2, Arabidopsis thaliana SCR, Arabidopsis thaliana SCRL23, Ziziphus japonica PCK, Urocholoa panicoides PCK1, and Barley PHT1;1.

26. The isolated DNA polynucleotide according to any one of claims 19 to 25, wherein the promoter is derived from a non-plant organism.

27. The isolated DNA polynucleotide according to any one of claims 19 to 26, wherein the nucleotide sequence encoding Phytochrome B is any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 8, or a sequence or functional fragment thereof that is at least 90% identical to any of the aforementioned sequences.

28. The isolated DNA polynucleotide according to any one of claims 19 to 27, wherein the functional fragment of Phytochrome B has phytochrome signaling activity but lacks photosensitivity; and the functional fragment may consist of PAS and GAF ​​domains.

29. The isolated DNA polynucleotide according to any one of claims 19 to 28, wherein the Phytochrome B is photosensitive; and YHB and the nucleotide sequence encoding the Phytochrome B may be SEQ ID NO: 1, or a sequence with at least 90% identity thereto, or a functional fragment thereof.

30. A plasmid comprising a DNA polynucleotide according to any one of claims 17 to 29, a replication origin, a right-bound repeat sequence of T-DNA of a Ti or Ri plasmid, and at least one bacterial selectable marker; further comprising a left-bound repeat sequence of a Ti or Ri plasmid.

31. The plasmid according to claim 30, further comprising an element selected from one or more of the following: an enhancer, a plant-selectable marker, a multicloning site, or a recombination site.

32. A Ti or Ri plasmid comprising a DNA polynucleotide according to any one of claims 17 to 29.

33. A composition for transforming plant cells, comprising an isolated DNA polynucleotide according to any one of claims 19 to 29 or a plasmid according to any one of claims 30 to 32; and potentially comprising microparticles coated with the DNA polynucleotide or the plasmid.

34. A bacterium comprising an isolated DNA polynucleotide according to any one of claims 19 to 29 or a plasmid according to any one of claims 30 to 32; which may be Escherichia coli.

35. A bacterium comprising the plasmid described in any one of claims 30 to 32; which may be of the genus Agrobacterium; or which may be Agrobacterium tumephasiensis.

36. At least in part C 3 A photosynthetic plant comprising an isolated DNA polynucleotide according to any one of claims 19 to 29, which is genetically incorporated into its genome.

37. At least in part C 3 A photosynthetic plant that additionally includes at least one additional copy of the Phytochrome B gene or a functional fragment thereof, and is genetically modified compared to a genetically equivalent, unmodified plant, wherein the expression regulatory element of at least one copy of the Phytochrome B gene or a functional fragment thereof in the modified plant results in the specific expression of the additional at least one Phytochrome B gene or a functional fragment thereof in at least some vascular bundle sheath cells and / or mestome sheath cells of the plant, compared to the unmodified plant.

38. The expression regulatory element is C 3 The plant according to claim 37, wherein the promoter is specifically active in at least some vascular bundle sheaths of the plant.

39. The plant according to claim 37 or 38, wherein the coding sequence of the additional at least one Phytochrome B gene is the same as the plant's natural Phytochrome B gene.

40. The plant according to claim 37 or 38, wherein the additional at least one Phytochrome B gene is different from the plant's natural Phytochrome B gene; and the Phytochrome B or active variant or functional fragment thereof may be defined in any one of claims 27 to 30.

41. The plant according to any one of claims 37 to 40, wherein the genetic modification is genetically stable.

42. A plant according to any one of claims 36 to 41, which is a cereal plant, an oilseed crop plant, or a legume.

43. The plant according to any one of claims 37 to 42, wherein the Phytochrome B has the amino acid sequence of any of SEQ ID NOs. 4, 5, 6, or 9, or a sequence that is at least 90% identical to any of the aforementioned sequences, or a functional fragment thereof.

44. The plant according to any one of claims 37 to 43, wherein the functional fragment of Phytochrome B has phytochrome signaling activity but lacks photosensitivity; and the fragment may consist of PAS and GAF ​​domains.

45. The plant according to any one of claims 37 to 44, wherein the Phytochrome B is a photosensitive sequence variant or a functional fragment thereof; it may be YHB having the amino acid sequence of SEQ ID NO: 4, or a sequence that is at least 90% identical to them, or a functional fragment thereof.

46. The plant according to any one of claims 37 to 45, wherein the chloroplasts present in the vascular bundle sheath cells are developmentally enhanced in terms of size or photosynthetic capacity compared to chloroplasts in equivalent vascular bundle sheath cells in an unmodified control plant grown for the same period and under the same conditions.

47. A plant according to any one of claims 36 to 46, wherein photosynthesis is enhanced compared to an unmodified control plant grown under the same conditions.

48. The plant according to any one of claims 36 to 47, wherein the photosynthetic efficiency of the leaves is higher than that of equivalent leaves of an unmodified control plant grown under the same conditions.

49. The plant according to any one of claims 36 to 48, wherein the water use efficiency is higher than that of an unmodified control plant grown under the same conditions.

50. The plant according to any one of claims 36 to 48, wherein, compared to an unmodified control plant grown under the same conditions, the enhanced photosynthesis results in one or more of the following characteristics: enhanced growth rate, shortened time to flowering, earlier maturation, enhanced seed yield, enhanced biomass, increased plant height, and increased canopy area.

51. Plant parts, plant tissues, plant organs, plant cells, plant protoplasts, embryos, callus cultures, pollen grains, or seeds derived from or obtained from the plants described in any one of claims 36 to 50, which are genetically modified compared to genetically equivalent, unmodified plants.

52. Processed plant products obtained from a plant according to any one of claims 36 to 48, or plant parts, plant tissues, plant organs, plant cells, plant protoplasts, embryos, callus cultures, pollen grains, or seeds according to claim 51; which are genetically modified compared to a genetically equivalent unmodified plant; and the processed plant products may comprise (i) Phytochrome B or an active fragment thereof downstream of a gene expression regulatory element that is specifically active in at least some of the vascular bundle sheath cells of a plant, or (ii) a detectable nucleic acid sequence of at least a portion of a polynucleotide according to any one of claims 19 to 29.

53. A method for producing a genetically modified plant, comprising the step of introducing a genome editing protein into plant cells, wherein the genome editing protein causes one or more mutations in the expression regulatory element of a gene encoding a Phytochrome B protein, the one or more mutations induce specific expression of the gene encoding a Phytochrome B protein in vascular bundle sheath cells, mestom sheath cells, or vascular cells of the genetically modified plant, and photosynthesis is enhanced in the genetically modified plant compared to an ungenetically modified plant of the same species.

Images