Method for increasing photosynthetic capacity of plants using NADPH or NADH water-forming oxidase

Abstract

The present invention relates to polynucleotides comprising a plant cell promoter element operably linked to a nucleotide sequence encoding a water-forming oxidase. The invention also relates to methods of increasing plant growth and yield.

Description

Technical Field

[0001] This invention relates to polynucleotides comprising plant cell promoter elements operatively linked to a nucleotide sequence encoding a water-forming oxidase. The invention also relates to methods for increasing plant growth and yield. Background Technology

[0002] Photosynthesis

[0003] A growing population and climate change are placing enormous pressure on agricultural production, necessitating a significant increase in crop yields by 2050 to meet anticipated demand. This increased production must be achieved through sustainable methods to avoid exacerbating climate change and accelerating biodiversity loss through agricultural expansion.

[0004] Enhancing photosynthesis is considered one of the most promising ways to increase crop yield. In photosynthesis, ribulose-1,5-bisphosphate carboxylase / oxygenase (rubisco) is an enzyme that converts atmospheric CO2 into sugars necessary for plant growth. The rate at which rubisco catalyzes CO2 fixation determines the maximum rate of biomass accumulation in the plant (and consequently, growth and yield). However, rubisco also catalyzes a competing reaction with O2, which depletes the pool of photosynthetic metabolites and leads to energy and resource consumption in a process called photorespiration. The rate at which rubisco fixes CO2 and produces sugars, or fixes O2 and loses energy, depends on the concentrations of these two gases in the chloroplasts where rubisco resides.

[0005] Under normal field conditions, photorespiration can reduce crop yields by up to 36%, resulting in the loss of millions of tons of crop yield annually. Furthermore, since photorespiration increases with rising temperatures, climate change is expected to exacerbate crop losses by increasing the rate of photorespiration. Therefore, there is great interest in methods to reduce photorespiration to improve plant growth and crop yields, thereby helping to protect plants from the effects of future temperature increases.

[0006] Previous attempts to mitigate yield losses due to photorespiration have focused on introducing photorespiration bypasses (Kebeish et al., 2007 Nature Biotechnology 25(5):593-9; Maier et al., 2012 Frontiers in Plant Science 28;3:38; South et al., 2019 Science 4;363(6422); Shen et al., 2019 Molecular Plant 12: 199-214; Basler et al., 2016 Frontiers in Bioengineering and Biotechnology 4:31; Maurino 2019 Frontiers in Bioengineering and Biotechnology 47:6 1805-1813). Each of these methods attempts to provide a more energy-efficient pathway to recover photorespiratory CO2, either by reducing the ATP and NADPH consumed in converting 2-phosphoglycolic acid to 3-phosphoglycerate, or by releasing photorespiratory CO2 very close to the location of Rubisco. Despite numerous attempts to mitigate the effects of photorespiration by constructing alternative, low-cost pathways, no study has yet been able to reduce photorespiration by altering the relative concentrations of CO2 and O2 in the chloroplasts containing Rubisco. This invention provides a novel and innovative solution to modify the relative abundance of O2 and CO2 around Rubisco in plants to enhance growth and yield.

[0007] pores

[0008] Stomata mediate the exchange of gaseous CO2 and water vapor between plants and their external environment. Specifically, CO2 provides the primary substrate for photosynthesis, and its absorption is the fuel required for plant growth. Conversely, water vapor loss is a component of transpiration, helping to regulate internal water state, temperature, and nutrient uptake. Since CO2 and H2O gases share a diffusion path but in opposite directions, controlling the flow along this path is crucial for balancing the physiological needs of individual plants. Therefore, optimizing stomatal distribution and function is a core component of maximizing plant productivity in terrestrial habitats.

[0009] Given their role at the plant-environment interface, stomata can modulate their diffuse conductance in response to various stimuli, including light (quality and quantity), humidity, temperature, soil water availability, and atmospheric CO2. Utilizing these signals and natural diurnal rhythms, stomata open during the day to promote CO2 fixation and close at night or under adverse conditions to limit excessive water loss. In addition to this dynamic behavior, stomata exhibit significant developmental plasticity in response to different environmental changes. For example, the number and distribution of stomata on the abaxial and adaxial surfaces of leaves can vary between species and even among individuals of the same species, depending on light availability. Stomatal density also exhibits significant plasticity and varies among individuals based on soil water availability, temperature, ambient CO2 concentration, and relative humidity. These environmentally induced density variations are also associated with secondary adjustments to stomatal size (leading to higher stomatal density and smaller stomata, and vice versa), although density always takes precedence over size, becoming the primary anatomical constraint on leaf gas conductance. Therefore, the dynamics and developmental changes of stomata play an important role in plant growth and environmental adaptation.

[0010] The considerable plasticity of stomatal morphology and function, coupled with the importance of stomata in regulating plant growth and environmental interactions, has inspired numerous attempts to modify their properties for crop improvement. These successes can be traced back to the pioneering work of Farquhar and Richards in the 1980s (Farquhar GD. And Richards RA. 1984 Australian Journal of Plant Physiology, 11(6), 539–552), who used stable carbon isotope ratios to screen wheat for improved water use efficiency. Subsequently, research expanded to include a range of forward genetic approaches that manipulate stomatal distribution, as well as the mechanical, transport, and metabolic properties of stomatal guard cells. Among these approaches, altering stomatal density has received the most attention, with many mutants exhibiting increases and decreases in stomatal number across different plant species.

[0011] A common theme in methods for altering stomatal density is the genetic manipulation of stomatal development gene expression levels. This includes fundamental helical-loop-helical transcription factors SPEECHLESS (SPCH) (Gudesblat GE et al., 2012 NatureCell Biology 14, 548–554), MUTE (Pillitteri, LJ. et al., 2007 Nature 445, 501–505), and FAMA (Bergmann DC. et al., 2004 Science 304, 1494–1497), which regulate the initiation, proliferation, and differentiation phases of stomatal lineages, respectively. Engineering has also been successful by targeting other important components of stomatal development, including the epidermal patterning factor family of signal peptides and their receptor components TOOMANY MOUTHS and ERECTA protein kinases, stomatal density and distribution 1 (SDD1) and its corresponding interacting components, as well as many other genes involved in hormone signaling and plant development processes. However, the molecular and biochemical mechanisms linking changes in gene networks to changes in environmental and physiological factors remain poorly understood. Consequently, there have been relatively few successes in controlling plant stomatal density through metabolic engineering.

[0012] Water forms NADH and NADPH oxidases

[0013] Water-forming NADH or NADPH oxidases are enzymes capable of oxidizing NADH or NADPH to NAD+ or NADP+, respectively. They function by reducing molecular oxygen through a four-electron transfer from a reducing equivalent in the presence of a proton, forming water and an electron acceptor (Petschacher B et al., Computational and Structural Biotechnology Journal. 2014 Feb 26;9:e201402005. doi: 10.5936 / csbj.201402005). Different isoforms of water-forming NADH or NADPH oxidases can selectively use NADH or NADPH, or both (Gao H et al., International Journal of Biological Macromolecules 2019. 15;123:629-636. doi: 10.1016 / j.ijbiomac.2018.11.096). Because these enzymes use oxygen as a substrate and produce water as a byproduct, they are frequently used in biotechnology applications for cofactor regeneration.

[0014] To date, a variety of water-forming NADH or NADPH oxidases have been identified in a wide range of organisms, from bacteria (Higuchi M et al., 1993 The Journal of General Microbiology. 139, 2343–2351) to archaea (Ward DE, 2001 FEBS J. 268:5816–5823) and eukaryotes (Brown DM et al., 1996 European journal of biochemistry, 241(1), 155–161). In addition to naturally occurring enzyme variants, water-forming NADH or NADPH oxidases have been the subject of enzyme engineering efforts (Petschacher B et al., 2014 Computational and structural biotechnology journal, 9(14), p.e201402005) to modify their substrate and / or cofactor specificity. Therefore, a variety of water-forming NADH or NADPH oxidases (natural or engineered) exist in a wide range of organisms known in this art. This invention specifically relates to a water-forming oxidase that reduces molecular oxygen to form water by converting NADPH or NADH into NADP or NAD, respectively.

[0015] This invention provides a novel polynucleotide comprising a plant cell promoter element operatively linked to a nucleotide sequence encoding a water-forming oxidase. Plants can be genetically engineered to contain the polynucleotide of this invention, which in turn can promote enhanced growth and biomass production. The polynucleotide of this invention, in particular, can drive enhanced plant growth and biomass production by modulating the relative abundance of O2 and CO2 near the rubisco. The polynucleotide of this invention can also drive enhanced plant growth and biomass production by reducing stomatal density and enhancing plant water use efficiency. Therefore, this invention has the potential to positively impact global crop yields. Summary of the Invention

[0016] The inventors have devised a novel method to enhance plant growth and yield. This is achieved by engineering plants to contain polynucleotides comprising promoter elements operatively linked to nucleotide sequences encoding water-forming oxidases.

[0017] The polynucleotides can advantageously enhance plant growth and yield by reducing photorespiration by altering the relative concentrations of CO2 and O2 near rubisco in chloroplasts. This invention may be particularly advantageous in many applications, including but not limited to industrial biotechnology (where enhanced growth and / or yield will result in enhanced production of proteins, peptides, metabolites, molecules, compounds, etc.), and food, feed, biomass, and biofuel production (where enhanced growth and / or yield will result in enhanced production of food, feed, biomass, or biofuel).

[0018] This invention demonstrates that expression of one or more water-forming NADH or NADPH oxidases in plant cells leads to enhanced plant growth and biomass production. This invention also specifically demonstrates that a variety of different natural or engineered water-forming oxidase (e.g., NADH or NADPH) genes from different species can provide this function. This invention further demonstrates that this function can be provided when these proteins are expressed in their unmodified form, and / or as fusion proteins, and / or as proteins targeting specific subcellular compartments, and / or as proteins anchored to the cell membrane. This invention also demonstrates that proteins in the various constituent forms described above can be expressed in plant cells using promoters that are universally active in every tissue of the plant, and expressed only in photosynthetically active cells using cell- and / or tissue-specific promoters.

[0019] The present invention provides a polynucleotide comprising a plant cell promoter element operatively linked to a nucleotide sequence encoding a water-forming oxidase.

[0020] The present invention also provides a vector comprising the polynucleotides of the present invention.

[0021] The present invention further provides compositions for transforming plant cells, the compositions comprising the polynucleotides of the present invention and / or the carriers of the present invention, preferably wherein the compositions comprise microparticles complexed with the polynucleotides and / or the carriers.

[0022] The present invention also provides cells comprising the polynucleotides of the present invention or the vectors of the present invention.

[0023] The present invention also provides a plant or a portion thereof, comprising: (i) the cell of the present invention; or (ii) a cell containing a polynucleotide, said polynucleotide comprising a promoter element operatively linked to a nucleotide sequence encoding a water-forming oxidase.

[0024] The present invention also provides a method for increasing the photosynthetic capacity of plants, preferably wherein the plant is a C3 or C4 plant, the method comprising modifying the heritable genetic material of the plant such that a water-forming oxidase is expressed in at least one mesophyll cell of the plant, thereby increasing the photosynthetic capacity of the plant.

[0025] The present invention also provides a method for:

[0026] (i) Reduce the stomatal density of plants; and / or

[0027] (ii) Reduce the stomatal conductance of plants; and / or

[0028] (iii) Increase the water retention capacity of plants.

[0029] Preferably, the plant is a C3 or C4 plant, and the method includes modifying the heritable genetic material of the plant to express a water-forming oxidase in at least one cell of the plant leaf epidermis. Preferably, the cell is a stomatal cell, and more preferably, the stomatal cell is a stomatal progenitor cell.

[0030] The present invention also provides plant parts, plant tissues, plant organs, plant cells, plant protoplasts, embryos, callus cultures, pollen grains or seeds derived from the plants of the present invention or from plants produced by the methods of the present invention, optionally wherein the plant parts contain a nucleic acid sequence corresponding to a water-forming oxidase as defined herein.

[0031] This invention also provides the use of water-forming oxidase for:

[0032] (i) Reduce the stomatal density of plants; and / or

[0033] (ii) Reduce the stomatal conductance of plants; and / or

[0034] (iii) Increase the water retention capacity of plants.

[0035] Preferably, the use comprises cultivating plants containing nucleotide sequences corresponding to water-forming oxidases as defined according to the present invention. Attached Figure Description

[0036] Figure 1A simplified schematic diagram of the gene constructs used to express the water-forming oxidase gene in this invention is shown. A) Expression cassette for expressing the C-terminus of the water-forming oxidase gene fused to GFP under the control of the CaMV 35S promoter. B) Same as A), but with an N-terminal fusion protein (NTFP). C) Expression cassette for expressing the water-forming oxidase gene under the control of the chlorophyll a / b binding protein 3 (CAB3) promoter. D) Same as C), but with the addition of an NTFP. All expression cassettes include a caustic acid synthase terminator (NOSt).

[0037] Figure 2 Images of plant cells (protoplasts) expressing a water-forming oxidase gene fused with GFP at their C-terminus are displayed. The top row shows a free GFP control. The second row shows an image of a protoplast expressing LbNOX water-forming oxidase, where the enzyme undergoes in-frame translational fusion with GFP at its C-terminus. The third row shows an image of a protoplast expressing SmNOX water-forming oxidase, which targets the intermembrane space of chloroplasts by translating and fusing the SmNOX gene with outer membrane protein 9 at its N-terminus and with GFP at its C-terminus. The fourth row shows an image of a protoplast expressing SmNOX water-forming oxidase, which targets the chloroplast stroma by translating and fusing the SmNOX gene with the oxygen-evolving protein 16 chloroplast target peptide sequence at its N-terminus and with GFP at its C-terminus. The fifth row shows an image of a protoplast expressing SmNOX water-forming oxidase, which targets the chloroplast stroma by translating and fusing the SmNOX gene with the rubisco small subunit protein sequence at its N-terminus and with GFP at its C-terminus.

[0038] Figure 3 The effects of water-forming oxidase gene expression on plant growth and yield are shown. A) Growth rate of the water-forming oxidase-expressing plant line compared to the unmodified control (WT). B) Visible rosette leaf area on day 20 of the water-forming oxidase-expressing plant line compared to the unmodified control (WT). C) Inflorescence height on day 32 of the water-forming oxidase-expressing plant line compared to the unmodified control (WT). D) Flowering time of the water-forming oxidase-expressing plant line compared to the unmodified control (WT). The letters above the box plots indicate significant differences between groups (p < 0.05), which were determined by one-way ANOVA and Tukey's post-hoc multiple comparison test.

[0039] Figure 4The effects of water-forming oxidase gene expression on stomatal density, stomatal conductance, and photosynthetic water use efficiency are shown. A) Stomatal density (in mm⁻²) on the abaxial surface of mature rosette leaves of water-forming oxidase-expressing plants compared to unmodified control plants (WT). B) Light-saturated stomatal conductance (gs sat, mol m⁻² s⁻¹) of mature rosette leaves of water-forming oxidase-expressing plants compared to unmodified control plants (WT). C) Light-saturated intrinsic water use efficiency (iWUEsat, μmol mol⁻¹) of mature rosette leaves of water-forming oxidase-expressing plants compared to unmodified control plants (WT). Differences between transgenic plants and WT were assessed by post-hoc Fisher LSD analysis followed by two-way ANOVA, where the letter above each box represents a statistically significant difference in the mean (p ≤ 0.05).

[0040] Brief description of the sequence

[0041] SEQ ID NO:1 – Nucleotide sequence of the Arabidopsis thaliana CAB3 promoter

[0042]

[0043] Nucleotide sequence of SEQ ID NO:2–CaMV 35S promoter

[0044] GTCAACATGGTGGAGCACGACACTCTGGTCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGATCAAAGGGCTATTGAGACTTTTCAACAAAGGATAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCT ATCATTCAAGATCTCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGAGGTTCCAACCACGTCTACAAAGCAAGTGGATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATAAGGAAGTTTCATTTCATTTGGAGAGGACACGC

[0045] SEQ ID NO:3 - Nucleotide sequence of the Agrobacterium tumefaciens nos terminator including the 3'UTR.

[0046] GTCAAGCAGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATG ACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGGTGTCATCTATGTTACTAGATCGA

[0047] SEQ ID NO:4 - Codon-optimized nucleotide sequence of monomer-enhanced GFP

[0048] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGTACTCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAATGA

[0049] SEQ ID NO:5 - Amino acid sequence of monomer-enhanced GFP

[0050] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0051] SEQ ID NO:6 - Codon-optimized nucleotide sequence of the Arabidopsis rubisco small subunit

[0052] ATGGCTTCTTCTATGCTGTCCAGCGCTACTATGGTGGCATCTCCCGCACAGGCTACCATGGTCGCTCCCTTCAATGGCTTGAAATCTTCCGCTGCCTTCCCCGCAACACGCAAGGCGAACAATGATATCACTAGCATTACCTCTAACGGAGGTCGTGTCAATTGTATGCAGGTGTGGCCTCCCATCGGTAAAAAGAAGTTCGAAACCCTGAGCTACCTGCCTGACCTGACTGACTCTGAGCTGGCAAAGGAGGTTGACTATCTGATCCGTAACAAGTGGATTCCCTGCGTTGAGTTTGAGCTCGAGCATGGGTTCGTTTACCGTGAGCACGGAAACTCTCCCGGATATTACGACGGTCGTTACTGGACCATGTGGAAACTGCCACTTTTCGGTTGCACTGATTCTGCACAGGTCCTTAAGGAAGTTGAGGAATGCAAAAAGGAGTATCCAAACGCATTCATCCGTATCATTGGATTTGACAACACTCGCCAGGTGCAATGCATTTCATTCATCGCCTACAAGCCCCCTTCGTTTACCGGTTAG

[0053] SEQ ID NO:7 - Amino acid sequence of the Arabidopsis rubisco small subunit

[0054] MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNCMQVWPPIGKKKFETLSYLPDLTDSELAKEVDYLIRNKWIPCVEFELEHGFVYREHGNSPGYYDGRYWTMWKLPLFGCTDSAQVLKEVEECKKEYPNAFIRIIGFDNTRQVQCISFIAYKPPSFTG

[0055] SEQ ID NO:8 - Codon-optimized nucleotide sequence of Arabidopsis thaliana oxygen evolution protein 16 transport peptide

[0056] ATGGCTTCTATGGGCGGACTGCACGGGGCCTCCCCCGCAGTCTTGGAGGGAAGCCTGAAGATCAACGGTTCTTCACGTCTGAATGGATCTGGACGCGTGGCTGTTGCACAGCGCTCTCGTCTGGTTGTGC GTGCTCAGCAGTCGGAGGAAACCTCTCGTCGTAGCGTCATTGGTCTTGTCGCAGCGGGTCTGGCAGGTGGTTCTTTCGTTCAAGCTGTGCTGGCTGACGCAATCAGCATTAAAGTTGGACCCCCTCCCGCC

[0057] SEQ ID NO:9 - Amino acid sequence of Arabidopsis thaliana oxygen evolution protein 16 transport peptide

[0058] MASMGGLHGASPAVLEGSLKINGSSRLNGSGRVAVAQRSRLVVRAQQSEETSRRSVIGLVAAGLAGGSFVQAVLADAISIKVGPPPA

[0059] SEQ ID NO:10 - Codon-optimized nucleotide sequence of Arabidopsis thaliana oxygen evolution protein 23 transport peptide

[0060] ATGGCCTACAGCGCTTGTTTCCTGCACCAGAGCGCTCTGGCTTCTTCTGCTGCACGTTCTTCCTCCTCATCTTCTTCCCAGCGTCACGTGTCTCTTTCTAAGCCCGTCCAGATTATCTGCAAAGCACAACAGAGCCATGAGGACGATAACTCTGCAGTTTCTCGTCGCCTGGCATTGACCCTGCTGGTTGGAGCGGCAGCTGTCGGTTCAAAGGTTAGCCCTGCAGACGCC

[0061] SEQ ID NO:11 - Amino acid sequence of Arabidopsis thaliana oxygen evolution protein 23 transport peptide

[0062] MAYSACFLHQSALASSAARSSSSSSSQRHVSLSKPVQIICKAQQSHEDDNSAVSRRLALTLLVGAAAVGSKVSPADA

[0063] SEQ ID NO:12 - Codon-optimized nucleotide sequence of Arabidopsis thaliana envelope protein 9

[0064] ATGGGTAACGAAACCAAGACTAATGGGGGTCCTGCTAGCATGGCTGGAGGCGGAGGTTTCCGTGCAAAAATGGAGCATTACGTTTACTCTGGTGAAAAGAAGCACGTCCTTGTCGGTATCGGAATCGTTA CCATTATTTTTGGAGTGCCCTGGTATCTGATGACTCAGGGATCTAAACACCAGAGCCACCAAGACTATATGGATAAGGCCGACAAGGCACGCAAGGCTCGTCTGTCCTCTTCATCTTCTGCAAACAAATAG

[0065] SEQ ID NO:13 - Amino acid sequence of Arabidopsis thaliana envelope protein 9

[0066] MGNETKTNGGPASMAGGGGFRAKMEHYVYSGEKKHVLVGIGIVTIIFGVPWYLMTQGSKHQSHQDYMDKADKARKARLSSSSSSANK

[0067] SEQ ID NO: 14 - Codon-optimized nucleotide sequence of Lactobacillus brevis NAD(P)H oxidase

[0068]

[0069] SEQ ID NO: 15 - Amino acid sequence of Lactobacillus brevis NAD(P)H oxidase

[0070] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTNHAQTTESYDKLVMTSG SWPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAGYIGAELAEAYSTTGHDVTLIDAMARVMPKYFDADFTDVIEQDYRDHGVQLALGETVESFTDSATGLTIK TDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVST GLTLAAAKQQGLNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0071] SEQ ID NO: 16 - Codon-optimized nucleotide sequence of Lactobacillus brevis NAD(P)H oxidase, containing mutations 159A / 177A / 178R / 179S / 184R

[0072]

[0073] SEQ ID NO: 17 - Amino acid sequence of Lactobacillus brevis NAD(P)H oxidase, containing mutations 159A / 177A / 178R / 179S / 184R

[0074] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTNHAQTTESYDKLVMTSG SWPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAGYIAAELAEAYSTTGHDVTLIARSARVMRKYFDADFTDVIEQDYRDHGVQLALGETVESFTDSATGLTIK TDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVST GLTLAAAKQQGLNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0075] SEQ ID NO: 18 - Codon-optimized nucleotide sequence of NAD(P)H oxidase from Streptococcus mutans, containing mutations 192A / 193R / 194H / 199R

[0076]

[0077] SEQ ID NO: 19 - The amino acid sequence of Streptococcus mutans NAD(P)H oxidase, containing mutations 192A / 193R / 194H / 199R.

[0078] MSKIVIVGANHAGTAAINTVLDNYGSENEVVVFDQNSNISFLGCGMALWIGKQISGPQGLFYADKESLEAKGAKIYMESPVTAIDYDAKRVTALVNGQEHVESYEKLILATGST PILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVINKLQDKTQNLNRIAVVGAGYIGVELAEAFKRLGKEVILIARHDTCLRGYYDQDLSEMMRQNLEDHGIELAFGETVK AIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYDNAINDTNYIALASNALRSGIVAGHNAAGHKLESLGVQGSNGI SIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDFQKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTIERLALLDYFFLPHFNQPYNYMIKAALKAK

[0079] SEQ ID NO:20 - Codon-optimized nucleotide sequence of the Arabidopsis rubisco small subunit fused with Streptococcus mutans NAD(P)H oxidase, containing mutations 192A / 193R / 194H / 199R

[0080]

[0081] SEQ ID NO:21 - The amino acid sequence of the Arabidopsis rubisco small subunit fused with Streptococcus mutans NAD(P)H oxidase, containing mutations 192A / 193R / 194H / 199R

[0082] MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNCMQVWPPIGKKKFETLSYLPDLTDSELAKEVDYLIRNKWIPCVEFELEHGFVYREHGNSPGYYDGRYWTMWKLPLFGCTDSAQVLKEVEECKKEYPNAFIRIIGFD NTRQVQCISFIAYKPPSFTGGSGGGMSKIVIVGANHAGTAAINTVLDNYGSENEVVVFDQNSNISFLGCGMALWIGKQISGPQGLFYADKESLEAKGAKIYMESPVTAIDYDAKRVTALVNGQEHVESYEKLILATGSTPILPPIKGAAIKEGSRDFEATL KNLQFVKLYQNAEDVINKLQDKTQNLNRIAVVGAGYIGVELAEAFKRLGKEVILIARHDTCLRGYYDQDLSEMMRQNLEDHGIELAFGETVKAIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCA TVYDNAINDTNYIALASNALRSGIVAGHNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDFQKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTIERLALLDYFFLPHFNQPYNYMIKAALKAK

[0083] SEQ ID NO:22 - Codon-optimized nucleotide sequence of the Arabidopsis thaliana oxygen evolution protein 16 transport peptide fused with Streptococcus mutans NAD(P)H oxidase, containing mutations 192A / 193R / 194H / 199R

[0084]

[0085] SEQ ID NO:23 - The amino acid sequence of the Arabidopsis thaliana oxygen evolution protein 16 transport peptide fused with Streptococcus mutans NAD(P)H oxidase, containing mutations 192A / 193R / 194H / 199R

[0086] MASMGGLHGASPAVLEGSLKINGSSRLNGSGRVAVAQRSRLVVRAQQSEETSRRSVIGLVAAGLAGGSFVQAVLADAISIKVGPPPAGGMSKIVIVGANHAGTAAINTVLDNYGSENEVVVFDQNSNISFLGCGMA LWIGKQISGPQGLFYADKESLEAKGAKIYMESPVTAIDYDAKRVTALVNGQEHVESYEKLILATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVINKLQDKTQNLNRIAVVGAGYIGVELAEAFKRL GKEVILIARHDTCLRGYYDQDLSEMMRQNLEDHGIELAFGETVKAIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYDNAINDTNYIALASNALRSGI VAGHNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDFQKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTIERLALLDYFFLPHFNQPYNYMIKAALKAK

[0087] SEQ ID NO:24 - Codon-optimized nucleotide sequence of the Arabidopsis thaliana oxygen evolution protein 23 transport peptide fused with Streptococcus mutans NAD(P)H oxidase, containing mutations 192A / 193R / 194H / 199R

[0088]

[0089] SEQ ID NO:25 - The amino acid sequence of the Arabidopsis thaliana oxygen evolution protein 23 transport peptide fused with Streptococcus mutans NAD(P)H oxidase, containing mutations 192A / 193R / 194H / 199R

[0090] MAYSACFLHQSALASSAARSSSSSSSQRHVSLSKPVQIICKAQQSHEDDNSAVSRRLALTLLVGAAAVGSKVSPADAGGMSKIVIVGANHAGTAAINTVLDNYGSENEVVVFDQNSNISFLGCGMALWIGKQIS GPQGLFYADKESLEAKGAKIYMESPVTAIDYDAKRVTALVNGQEHVESYEKLILATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVINKLQDKTQNLNRIAVVGAGYIGVELAEAFKRLGKEVI LIARHDTCLRGYYDQDLSEMMRQNLEDHGIELAFGETVKAIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYDNAINDTNYIALASNALRSGIVAG HNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDFQKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTIERLALLDYFFLPHFNQPYNYMIKAALKAK

[0091] SEQ ID NO:26 - Codon-optimized nucleotide sequence of Arabidopsis thaliana envelope protein 9 fused with Streptococcus mutans NAD(P)H oxidase, containing mutations 192A / 193R / 194H / 199R

[0092]

[0093] SEQ ID NO:27 - The amino acid sequence of Arabidopsis thaliana envelope protein 9 fused with Streptococcus mutans NAD(P)H oxidase, containing mutations 192A / 193R / 194H / 199R

[0094] MGNETKTNGGPASMAGGGGFRAKMEHYVYSGEKKHVLVGIGIVTIIFGVPWYLMTQGSKHQSHQDYMDKADKARKARLSSSSANKGGMSKIVIVGANHAGTAAINTVLDNYGSENEVVVFDQNSNISFLGCGMAL WIGKQISGPQGLFYADKESLEAKGAKIYMESPVTAIDYDAKRVTALVNGQEHVESYEKLILATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVINKLQDKTQNLNRIAVVGAGYIGVELAEAFKRL GKEVILIARHDTCLRGYYDQDLSEMMRQNLEDHGIELAFGETVKAIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYDNAINDTNYIALASNALRSGI VAGHNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDFQKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTIERLALLDYFFLPHFNQPYNYMIKAALKAK

[0095] SEQ ID NO:28 - Codon-optimized nucleotide sequence of the Arabidopsis thaliana oxygen evolution protein 16 transport peptide fused with Lactobacillus brevis NAD(P)H oxidase, containing mutations 159A / 177A / 178R / 179S / 184R

[0096]

[0097] SEQ ID NO:29 - The amino acid sequence of the Arabidopsis thaliana oxygen evolution protein 16 transport peptide fused with Lactobacillus brevis NAD(P)H oxidase, containing mutations 159A / 177A / 178R / 179S / 184R

[0098] MASMGGLHGASPAVLEGSLKINGSSRLNGSGRVAVAQRSRLVVRAQQSEETSRRSVIGLVAAGLAGGSFVQAVLADAISIKVGPPPAGGMKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIA LYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTNHAQTTESYDKLVMTSGSWPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAGYIAAELAEAYSTTGHDVTLIARSA RVMRKYFDADFTDVIEQDYRDHGVQLALGETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPT VKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGLNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0099] SEQ ID NO:30 - Codon-optimized nucleotide sequence of the Arabidopsis thaliana oxygen evolution protein 23 transport peptide fused with Lactobacillus brevis NAD(P)H oxidase, containing mutations 159A / 177A / 178R / 179S / 184R

[0100]

[0101] SEQ ID NO:31 - The amino acid sequence of the Arabidopsis thaliana oxygen evolution protein 23 transport peptide fused with Lactobacillus brevis NAD(P)H oxidase, containing mutations 159A / 177A / 178R / 179S / 184R

[0102] MAYSACFLHQSALASSAARSSSSSSSQRHVSLSKPVQIICKAQQSHEDDNSAVSRRLALTLLVGAAAVGSKVSPADAGGMKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVA DPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTNHAQTTESYDKLVMTSGSWPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAGYIAAELAEAYSTTGHDVTLIARSARVMRK YFDADFTDVIEQDYRDHGVQLALGETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVK YMGTQSSSGLALYDRTIVSTGLTLAAAKQQGLNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNA

[0103] SEQ ID NO:32 - Codon-optimized nucleotide sequence of Streptococcus mutans NAD(P)H oxidase, containing mutations 192A / 193R / 194H / 199R, fused with monomerically enhanced GFP.

[0104]

[0105] SEQ ID NO:33 - The amino acid sequence of Streptococcus mutans NAD(P)H oxidase, containing mutations 192A / 193R / 194H / 199R, fused with monomerically enhanced GFP.

[0106] MSKIVIVGANHAGTAAINTVLDNYGSENEVVVFDQNSNISFLGCGMALWIGKQISGPQGLFYADKESLEAKGAKIYMESPVTAIDYDAKRVTALVNGQEHVESYEKLILATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVINKLQDKTQNLNRIAVVGAGYIG VELAEAFKRLGKEVILIARHDTCLRGYYDQDLSEMMRQNLEDHGIELAFGETVKAIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYDNAINDTNYIALASNALRSGIVAGHNAAGHKLESLGVQGSNGISIFGLNM VSTGLTQEKAKRFGYNPEVTAFTDFQKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTIERLALLDYFFLPHFNQPYNYMIKAALKAKGGSAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTT LTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0107] SEQ ID NO:34 - A codon-optimized nucleotide sequence of the Arabidopsis rubisco small subunit fused with Streptococcus mutans NAD(P)H oxidase, containing mutants 192A / 193R / 194H / 199R, and fused with monomerically enhanced GFP.

[0108]

[0109] SEQ ID NO:35 - Amino acid sequence of the Arabidopsis rubisco small subunit fused with Streptococcus mutans NAD(P)H oxidase, containing mutants 192A / 193R / 194H / 199R, and fused with monomerically enhanced GFP.

[0110] MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNCMQVWPPIGKKKFETLSYLPDLTDSELAKEVDYLIRNKWIPCVEFELEHGFVYREHGNSPGYYDGRYWTMWKLPLFGCTDSAQVLKEVEECKKEYPNAFIRIIGFDNRQVQCISFIAYKPPSFTGGSGGGMSKIVIVGANGHAGTAAINTVLDNYGSENEVVVFDQNSNISFLGCGMALWIGKQISGPQGLFYADKESLEAKGAKIYMESPVTAIDYDAKRVTALVNGQEHVESYEKKLILATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVINKLQDKTQNLNRIAVVGAGYIGVELAEAFKRLGKEVILIARHDTCLRGYYDQDLSEMMRRQNLEDHGIELAFGETVKAIEGDGKVERIVTDKASHDVDMVILAVGF RPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYDNAINDTNYIALASNALRSGIVAGHNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDFQKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTIERLALLDYFFLPHFNQPYNYMICAALKAKGGSAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0111] SEQ ID NO:36 - Codon-optimized nucleotide sequence of Arabidopsis thaliana oxygen evolution protein 16 transport peptide fused with Streptococcus mutans NAD(P)H oxidase, containing mutants 192A / 193R / 194H / 199R, and fused with monomer-enhanced GFP.

[0112]

[0113] SEQ ID NO:37 - Amino acid sequence of Arabidopsis thaliana oxygen evolution protein 16 transport peptide fused with Streptococcus mutans NAD(P)H oxidase, containing mutants 192A / 193R / 194H / 199R, and fused with monomerically enhanced GFP.

[0114] MASMGGLHGASPAVLEGSLKINGSSRLNGSGRVAVAQRSRLVVRAQQSEETSRRSVIGLVAAGLAGGSFVQAVLADAISIKVGPPPAGGMSKIVIVGANHAGTAAINTVLDNYGSENEVVVFDQNSNISFLGCGMALWIGKQISGPQGLFYADKESLEAKGAKIYMESPVTAIDYDAKRVTALVNGQEHVESYEKLI LATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVINKLQDKTQNLNRIAVVGAGYIGVELAEAFKRLGKEVILIARHDTCLRGYYDQDLSEMMRQNLEDHGIELAFGETVKAIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYDNAIND TNYIALASNALRSGIVAGHNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDFQKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTIERLALLDYFFLPHFNQPYNYMIKAALKAKGGSAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGK LTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGH KLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0115] SEQ ID NO:38 - Codon-optimized nucleotide sequence of Arabidopsis thaliana oxygen evolution protein 23 transport peptide fused with Streptococcus mutans NAD(P)H oxidase, containing mutants 192A / 193R / 194H / 199R, and fused with monomer-enhanced GFP.

[0116]

[0117] SEQ ID NO:39 - The amino acid sequence of an Arabidopsis thaliana oxygen evolution protein 23 transport peptide fused with Streptococcus mutans NAD(P)H oxidase, containing mutants 192A / 193R / 194H / 199R, and fused with monomerically enhanced GFP.

[0118] MAYSACFLHQSALASSAARSSSSSSSQRHVSLSKPVQIICKAQQSHEDDNSAVSRRLALTLLVGAAAVGSKVSPADAGGMSKIVIVGANHAGTAAINTVLDNYGSENEVVVFDQNSNISFLGCGMALWIGKQISGPQGLFYADKESLEAKGAKIYMESPVTAIDYDAKRVTALVNGQEHVESYEKLILATGSTP ILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVINKLQDKTQNLNRIAVVGAGYIGVELAEAFKRLGKEVILIARHDTCLRGYYDQDLSEMMRQNLEDHGIELAFGETVKAIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYDNAINDTNYIA LASNALRSGIVAGHNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDFQKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTIERLALLDYFFLPHFNQPYNYMIKAALKAKGGSAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0119] SEQ ID NO:40 - Codon-optimized nucleotide sequence of Arabidopsis thaliana envelope protein 9, fused with Streptococcus mutans NAD(P)H oxidase, containing mutants 192A / 193R / 194H / 199R, and fused with monomerically enhanced GFP.

[0120]

[0121] SEQ ID NO:41 - The amino acid sequence of Arabidopsis thaliana envelope protein 9 fused with Streptococcus mutans NAD(P)H oxidase, containing mutants 192A / 193R / 194H / 199R, and fused with monomerically enhanced GFP.

[0122] MGNETKTNGGPASMAGGGGFRAKMEHYVYSGEKKHVLVGIGIVTIIFGVPWYLMTQGSKHQSHQDYMDKADKARKARLSSSSSSANKGGMSKIVIVGANHAGTAAINTVLDNYGSENEVVVFDQNSNISFLGCGMALWIGKQISGPQGLFYADKESLEAKGAKIYMESPVTAIDYDAKRVTALVNGQEHVESYEKLI LATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVINKLQDKTQNLNRIAVVGAGYIGVELAEAFKRLGKEVILIARHDTCLRGYYDQDLSEMMRQNLEDHGIELAFGETVKAIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYDNAIND TNYIALASNALRSGIVAGHNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDFQKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTIERLALLDYFFLPHFNQPYNYMIKAALKAKGGSAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGK LTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGH KLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0123] SEQ ID NO:42 - Codon-optimized nucleotide sequence of Lactobacillus brevis NAD(P)H oxidase fused with monomer-enhanced GFP

[0124]

[0125] SEQ ID NO:43 - Amino acid sequence of Lactobacillus brevis NAD(P)H oxidase fused with monomer-enhanced GFP.

[0126] MKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTNHAQTTESYDKLVMTSGSWPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAGYIGAELAEAYSTTGHDV TLIDAMARVMPKYFDADFTDVIEQDYRDHGVQLALGETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQ QGLNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNAGGSAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT YGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0127] SEQ ID NO:44 - Codon-optimized nucleotide sequence of Arabidopsis thaliana oxygen evolution protein 16 transport peptide fused with Lactobacillus brevis NAD(P)H oxidase, containing mutants 159A / 177A / 178R / 179S / 184R, and fused with monomer-enhanced GFP.

[0128]

[0129] SEQ ID NO:45 - Amino acid sequence of Arabidopsis thaliana oxygen evolution protein 16 transport peptide fused with Lactobacillus brevis NAD(P)H oxidase, containing mutants 159A / 177A / 178R / 179S / 184R, and fused with monomerically enhanced GFP.

[0130] MASMGGLHGASPAVLEGSLKINGSSRLNGSGRVAVAQRSRLVVRAQQSEETSRRSVIGLVAAGLAGGSFVQAVLADAISIKVGPPPAGGMKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTNHAQTTESYDK LVMTSGSWPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAGYIAAELAAEAYSTTGHDVTLIARSARVMRKYFDADFTDVIEQDYRDHGVQLALGETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAV RQGILVGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGLNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNAGGSAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKL TLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0131] SEQ ID NO:46 - Codon-optimized nucleotide sequence of Arabidopsis thaliana oxygen evolution protein 23 transport peptide fused with Lactobacillus brevis NAD(P)H oxidase, containing mutants 159A / 177A / 178R / 179S / 184R, and fused with monomer-enhanced GFP.

[0132]

[0133] SEQ ID NO:47 - Amino acid sequence of Arabidopsis thaliana oxygen evolution protein 23 transport peptide fused with Lactobacillus brevis NAD(P)H oxidase, containing mutants 159A / 177A / 178R / 179S / 184R, and fused with monomerically enhanced GFP.

[0134] MAYSACFLHQSALASSAARSSSSSSSQRHVSLSKPVQIICKAQQSHEDDNSAVSRRLALTLLVGAAAVGSKVSPADAGGMKVTVVGCTHAGTFAIKQILAEHPDAEVTVYERNDVISFLSCGIALYLGGKVADPQGLFYSSPEELQKLGANVQMNHNVLAIDPDQKTVTVEDLTNHAQTTESYDKLVMTSGS WPIVPKIPGIDSDRVKLCKNWAHAQALIEDAKEAKRITVIGAGYIAAAELAEAYSTTGHDVTLIARSARVMRKYFDADFTDVIEQDYRDHGVQLALGETVESFTDSATGLTIKTDKNSYETDLAILCIGFRPNTDLLKGKVDMAPNGAIITDDYMRSSNPDIFAAGDSAAVHYNPTHQNAYIPLATNAVRQGIL VGKNLVKPTVKYMGTQSSSGLALYDRTIVSTGLTLAAAKQQGLNAEQVIVEDNYRPEFMPSTEPVLMSLVFDPDTHRILGGALMSKYDVSQSANTLSVCIQNENTIDDLAMVDMLFQPNFDRPFNYLNILAQAAQAKVAQSVNAGGSAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK Detailed Implementation

[0135] The invention will be described with reference to specific embodiments and certain accompanying drawings, but this disclosure is not limited thereto, but is defined solely by the claims. No reference numerals in the claims should be construed as limiting the scope. It should be understood, of course, that not all aspects or advantages can be achieved according to any particular embodiment. Therefore, for example, those skilled in the art will recognize that the disclosed embodiments may be implemented or performed in a manner that achieves or optimizes one or more advantages taught herein, without necessarily achieving other aspects or advantages taught or suggested herein.

[0136] When read in conjunction with the accompanying drawings, the organization and operation of this disclosure, as well as its features and advantages, can be best understood by referring to the detailed embodiments described below. Aspects and advantages of the invention will become apparent from the embodiments described below. Throughout the specification, reference to “an embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one disclosed embodiment. Therefore, the phrases “in one embodiment” or “in an embodiment” appearing throughout this specification do not necessarily refer to the same embodiment, but may refer to the same embodiment. Similarly, it should be understood that in the description of exemplary disclosed embodiments, various features are sometimes combined in a single embodiment, drawing, or description thereof for the purpose of simplifying the disclosure and aiding understanding of one or more various aspects of the invention. However, this method of disclosure should not be construed as reflecting an intention that the claimed invention requires more features than expressly recited in each claim. Rather, as reflected in the following claims, the inventive aspect lies in a portion of the features of a single foregoing disclosed embodiment.

[0137] It should be understood that the “implementations” of this disclosure may be specifically combined together unless the context otherwise indicates. A specific combination of all disclosed embodiments (unless the context otherwise implies) constitutes a further disclosed embodiment of the claimed invention.

[0138] Furthermore, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise. Thus, for example, a reference to “polynucleotide” includes two or more polynucleotides, a reference to “protein” includes two or more proteins, and so on.

[0139] When the term “about” is used herein to refer to a measurable value such as quantity or duration, it means a variation of ±20% or ±10% of a specific value, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, because such a variation is suitable for implementing the disclosed method.

[0140] As used herein, “polynucleotide,” “nucleotide sequence,” “DNA sequence,” or “nucleic acid molecule” refers to a polymeric form of nucleotides of any length, including ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Therefore, the term includes double-stranded and single-stranded DNA as well as RNA. The term “polynucleotide” as used herein can refer to a single-stranded or double-stranded covalently linked nucleotide sequence, wherein the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. Polynucleotides can consist of deoxyribonucleotide bases or ribonucleotide bases. Polynucleotides can be synthesized in vitro or isolated from natural sources. Polynucleotides can also include modified DNA or RNA, such as methylated DNA or RNA, or RNA that has undergone post-translational modifications, such as 5'-capping with 7-methylguanosine, 3'-processing (e.g., cleavage and polyadenylation), and splicing. Polynucleotides can also include synthetic nucleic acids (XNAs), such as hexetol nucleic acids (HNAs), cyclohexene nucleic acids (CeNAs), threonine nucleic acids (TNAs), glycerol nucleic acids (GNAs), locked nucleic acids (LNAs), and peptide nucleic acids (PNAs).

[0141] In the context of this invention, the term "amino acid" is used in its broadest sense and means an organic compound containing amine (NH2) and carboxyl (COOH) functional groups, as well as a side chain (e.g., an R group) specific to each amino acid. In some embodiments, the amino acid refers to a naturally occurring L α-amino acid or residue. Common single-letter and three-letter abbreviations for naturally occurring amino acids are used herein: A=Ala; C=Cys; D=Asp; E=Glu; F=Phe; G=Gly; H=His; I=Ile; K=Lys; L=Leu; M=Met; N=Asn; P=Pro; Q=Gln; R=Arg; S=Ser; T=Thr; V=Val; W=Trp; and Y=Tyr (Lehninger, AL, (1975) Biochemistry, 2d ed., pp. 71-92, WorthPublishers, New York). The general term “amino acid” also includes D-amino acids, retro-inverso amino acids, and chemically modified amino acids (such as amino acid analogs), naturally occurring amino acids that are not typically incorporated into proteins (such as ortholeucine), and chemically synthesized compounds that possess amino acid properties known in the art (such as β-amino acids). For example, analogs or mimics of phenylalanine or proline that restrict the conformation of peptide compounds to that of natural Phe or Pro are included within the definition of amino acids. Such analogs and mimics are referred to herein as “functional equivalents” of the corresponding amino acids. Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., NY 1983, provide other examples of amino acids, which are incorporated herein by reference.

[0142] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acid residues and their variants and synthetic analogs. Therefore, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic, non-naturally occurring amino acids (e.g., chemical analogs of corresponding naturally occurring amino acids), as well as to naturally occurring amino acid polymers. Polypeptides may also undergo maturation or post-translational modification processes, including but not limited to: glycosylation, proteolytic cleavage, lipolysis, signal peptide cleavage, propeptide cleavage, phosphorylation, etc. Peptides can be prepared using recombinant technologies, such as by expressing recombinant or synthetic polynucleotides. Recombinant peptides are generally substantially free of mediators, for example, mediators comprising less than about 20% of the volume of the protein article, more preferably less than about 10%, and most preferably less than about 5%.

[0143] The term "protein" is used to describe folded polypeptides that have secondary or tertiary structures. Proteins can consist of a single polypeptide or can comprise multiple polypeptides that assemble to form a multimer. The multimer can be a homooligomer or a heterooligomer. Proteins can be naturally occurring or wild-type proteins, or modified or non-natural proteins. Proteins can differ from wild-type proteins, for example, by the addition, substitution, or deletion of one or more amino acids.

[0144] Protein “variants” include peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions relative to the unmodified or wild-type protein in question, and have similar biological and functional activities to the unmodified protein from which said variants are derived. The term “amino acid identity” as used herein refers to the degree of similarity of sequences when compared on an amino acid-by-amino acid basis within a comparison window. Therefore, the “sequence identity percentage” is calculated by comparing two best-aligned sequences within a comparison window, determining the number of positions in both sequences where the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage.

[0145] For all aspects and embodiments of the invention, the "variant" has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity with the corresponding wild-type protein. Sequence identity can also correspond to fragments or portions of full-length polynucleotides or polypeptides. Thus, a sequence may have only 50% overall sequence identity with a full-length reference sequence, but the sequence of a specific region, domain, or subunit may have 80%, 90%, or up to 99% sequence identity with the reference sequence.

[0146] The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. Wild-type genes are the most frequently observed genes in a population and are therefore arbitrarily engineered to be in their "normal" or "wild-type" form. Conversely, the terms "modified," "mutant," or "variant" refer to a gene or gene product that, compared to a wild-type gene or gene product, exhibits modifications in its sequence (e.g., substitution, truncation, or insertion), post-translational modifications, and / or functional characteristics (e.g., altered traits).

[0147] Methods for introducing or replacing naturally occurring amino acids are well known in the art. For example, methionine (M) is replaced with arginine (R) by replacing the methionine codon (ATG) with the arginine codon (CGT) at the relevant position of the polynucleotide encoding the mutant monomer. Methods for introducing or replacing non-naturally occurring amino acids are also well known in the art. For example, non-naturally occurring amino acids can be introduced by incorporating synthetic aminoacyl-tRNA into an IVTT system used to express the mutant monomer. Alternatively, they can be introduced by expressing a auxotrophic mutant monomer of a specific amino acid in *E. coli* in the presence of a synthetic (i.e., non-naturally occurring) analog of the specific amino acid. They can also be generated by naked linking if the mutant monomer is generated using partial peptide synthesis. Conservative substitution replaces an amino acid with another amino acid having a similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acid they replace. Alternatively, conservative substitution may introduce another aromatic or aliphatic amino acid to replace a previously present aromatic or aliphatic amino acid. The conserved amino acid variations are well known in the art and can be selected based on the characteristics of the 20 major amino acids defined in Table 1 below. When amino acids have similar polarity, this can also be determined by referring to the hydrophilicity table of amino acid side chains in Table 2.

[0148] Table 1 - Chemical properties of amino acids

[0149]

[0150] Table 2 - Hydrophilicity Measurement

[0151]

[0152] Unless otherwise stated, the nucleic acid sequences in this article are written from left to right in a 5' to 3' direction.

[0153] As used herein, "recombinant cell" will be understood to refer to a cell that has been introduced with recombinant nucleic acids (such as recombinant DNA or recombinant RNA). "Recombinant nucleic acid" is a nucleic acid sequence containing a combination of nucleic acid molecules that do not exist in nature. The recombinant nucleic acid referred to herein may be a synthetic recombinant nucleic acid.

[0154] As used herein, water-forming NADH or NADPH oxidases will be understood as enzymes capable of reducing molecular oxygen to form water by oxidizing NADH or NADPH. Any such enzyme may also produce other products, such as H2O2. Many such proteins are known to those skilled in the art and have been identified in bacteria (Higuchi M et al., 1993 The Journal of General Microbiology. 139, 2343–2351. doi: 10.1099 / 00221287-139-10-2343), archaea (Ward DE, FEBS J. 2001 268:5816–5823. doi: 10.1046 / j.0014-2956.2001.02526.x.), and eukaryotes (Brown DM et al., 1996 European journal of biochemistry, 241(1), pp.155-161). It can be expected that the function of any water-forming oxidase will be identical, since it is the enzyme's activity, not sequence identity, that provides utility in this invention. Those skilled in the art will be able to use sequence search methods such as BLAST (Altschul et al., J. Mol Biol 1990 215(3):403-410), hmmer (Eddy S. Bioinformatics 1998 14(9):755-763) or orthologous genomic reasoning methods such as OrthoFinder (Emms DM et al., 2019 Genome Biology 20:238) or OrthoMCL (Li et al., 2003 Genome Research 13(9): 2178-2189) to identify sequences associated with known water-forming oxidases, and then determine their function as water-forming NADH or NADPH oxidases by enzyme properties. Those skilled in the art will understand that other water-forming NADH or NADPH oxidase genes not listed in this invention will provide the same biochemical function and therefore will provide the same effect if expressed in plant cells in the same manner as the examples provided herein.

[0155] As used herein, NOX is used as an abbreviation for water-forming NADH or NADPH oxidase. For example, SmNOX refers to a bifunctional water-forming NADPH and NADH oxidase from Streptococcus mutans [SEQ ID NO: 18]. LbNOX refers to a water-forming NAD(P)H oxidase from Lactobacillus brevis [SEQ ID NO: 14].

[0156] As used herein, the terms “overexpress, overexpressed, and overexpression” in the context of expressing a given biological entity (e.g., nucleic acid, protein, peptide, etc.) in recombinant cells mean: (i) that the entity is expressed at a higher level in recombinant cells than the same entity is expressed at the corresponding wild-type cells; or (ii) that the entity is expressed at a detectable level in recombinant cells when the corresponding wild-type cells express the same entity at an undetectable level or not at all.

[0157] As used herein, the term "corresponding wild-type" in the context of modified cells, organisms, nucleic acid sequences, proteins, peptides, etc., refers to the native form of that entity. For example, in the case where recombinant cells are engineered to contain a vector comprising a foreign nucleic acid sequence, the "corresponding wild-type" cell would be the cell that existed in its native form before being engineered to contain said vector. As a further non-limiting example, the "corresponding wild-type" of a codon-optimized nucleic acid or amino acid sequence would be the sequence that existed in its native form before codon optimization.

[0158] As used herein, “C3 photosynthetic plant” will be understood to include any plant whose photosynthesis is all or most of C3 photosynthesis. “C3 photosynthesis” refers to a photosynthetic pathway that uses the Calvin-Benson cycle to fix carbon dioxide from the atmosphere without requiring additional biophysical or biochemical mechanisms to concentrate that CO2. Cell types referred to as “C3” in this article will be understood to originate from “C3 photosynthetic plants”.

[0159] As used herein, “C4 photosynthetic plant” will be understood to include any plant in which all or most of its photosynthesis is C4 photosynthesis. Cell types referred to as “C4” in this article will be understood to originate from “C4 photosynthetic plant”.

[0160] As used herein, “plant part” can refer to a fruit, leaf, root, or plant vascular system (e.g., xylem). Fruits include tissues such as pulp and peel. “Plant part” can also refer to a seed. The term “seed” as used herein can refer to the reproductive unit of a flowering plant, capable of developing into another such plant. The term “plant organ” refers to a plant tissue or group of tissues that constitute the morphologically and functionally distinct parts of a plant. The term “genome” refers to all the genetic material (genes and non-coding sequences) present in every cell of an organism, virus, or organelle; and / or a complete set of chromosomes inherited as a (haploid) unit from one parent. “Offspring” includes any subsequent generations of a plant.

[0161] As used herein, the percentage of "sequence identity" will be understood as a comparison of two sequences that are aligned together to obtain the maximum correlation between the sequences. This may include inserting "gaps" in one or both sequences to improve the alignment. The percentage of sequence identity in length for each compared sequence can then be determined. For example, a nucleotide sequence ("target sequence") having at least 95% "sequence identity" with another nucleotide sequence ("query sequence") means that the target sequence is identical to the query sequence, except that the target sequence may include up to 5 nucleotide changes per 100 nucleotides in the query sequence. In other words, to obtain a nucleotide sequence with at least 95% sequence identity with the query sequence, up to 5% (i.e., 5 out of 100) of the nucleotides in the target sequence may be inserted or replaced with another nucleotide or deleted. The same percentage of identity is used for protein sequences, but involves comparisons of the corresponding amino acids.

[0162] As used herein, a regulatory sequence that is "operably linked" to another sequence refers to a functional relationship between the two sequences such that the regulatory sequence has the ability to influence the expression and / or localization and / or activity of the sequence to which it is linked. For example, a promoter operably linked to a coding sequence will be able to regulate the transcription of that coding sequence. A targeting peptide or N-terminal or C-terminal fusion protein operably linked to a polypeptide will be able to direct the polypeptide to a specific location (e.g., organelle or cell membrane).

[0163] As used herein, a photosynthetically active cell is defined as any cell in a plant capable of using energy obtained from light to fix atmospheric CO2 into sugars. In most plants, the primary photosynthetically active cells are the mesophyll cells of the leaves. Multiple mesophyll cell layers may exist in a leaf, including but not limited to spongy mesophyll cell layers and palisade mesophyll cell layers. The term mesophyll cell, as used herein, includes all types of mesophyll cells and mesophyll cell layers unless the context clearly indicates otherwise. Furthermore, as used herein, the term mesophyll cell promoter or promoter active in photosynthetically active tissue refers to any promoter capable of driving gene expression in mesophyll cells or photosynthetically active cells. Many such promoters are known to those skilled in the art. It can be anticipated that the promoter of any gene expressed in mesophyll cells and / or photosynthetically active cells, when fused with a target gene, will direct the expression of that target gene in mesophyll cells and / or photosynthetically active cells. Many such mesophyll cell promoters have been characterized in the literature, including but not limited to the promoters of the following genes. Rubisco small subunit (pRBCS1A): Mustroph et al., 2009 PNAS 106 (44): 18843-18848. Chlorophyll a / b binding protein (cab) promoter: Mitra et al., 1989 Plant Molecular Biology 12:169-179, 1989. PEPC: DasGupta et al., 2019 The Plant Journal 101 (1):204-216. IQD22: Procko et al., 2022 The Plant Cell 34 (9): 3261–3279. AT1G70958: Procko et al., 2022 The Plant Cell 34 (9): 3261–3279. SQE6: Procko et al., 2022 The Plant Cell 34 (9): 3261–3279. XTH6: Procko et al., 2022 The Plant Cell 34 (9): 3261–3279. PAL1: Procko et al., 2022 The Plant Cell 34 (9): 3261–3279. CORI3: Procko et al., 2022 The Plant Cell 34 (9): 3261–3279. YAB3: Procko et al., 2022 The Plant Cell 34 (9): 3261–3279. LHCB2.4: Procko et al., 2022 The Plant Cell 34 (9): 3261–327.CRR23: Procko et al., 2022 The Plant Cell 34 (9): 3261–3279. SPS4F: Procko et al., 2022 The Plant Cell 34 (9): 3261–3279. ENH1: Procko et al., 2022 The Plant Cell 34 (9): 3261–3279. COR414-TM1: Procko et al., 2022 The Plant Cell 34 (9): 3261–3279.

[0164] Features, integrals, properties, compounds, chemical portions, or groups described in connection with a particular aspect, embodiment, or example of the invention should be understood to be applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith. All features disclosed in this specification (including any appended claims, abstract, and drawings), and / or all steps of any method or process so disclosed, may be combined in any combination, except where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments. The invention includes any single feature or any novel combination of features disclosed in this specification (including any appended claims, abstract, and drawings), or any novel single feature or any novel combination of steps extending to any method or process so disclosed.

[0165] Polynucleotides

[0166] This paper presents a novel method for enhancing plant growth and yield. This is achieved by engineering the plant to contain polynucleotides that include promoter elements operatively linked to a nucleotide sequence encoding a water-forming oxidase.

[0167] The polynucleotides can advantageously enhance plant growth and yield by reducing photorespiration by altering the relative concentrations of CO2 and O2 near rubisco in chloroplasts. This invention may be particularly advantageous in many applications, including but not limited to industrial biotechnology (where enhanced growth and / or yield will result in enhanced production of proteins, peptides, metabolites, molecules, compounds, etc.), and food, feed, biomass, and biofuel production (where enhanced growth and / or yield will result in enhanced production of food, feed, biomass, or biofuel).

[0168] As explained in more detail herein, a polypeptide is provided that can enhance plant growth and yield in plants containing said polypeptide.

[0169] This article provides a polynucleotide comprising a plant cell promoter element operatively linked to a nucleotide sequence encoding a water-forming oxidase.

[0170] promoter element

[0171] A promoter or promoter element is a sequence capable of driving the operatively linked expression of a gene. The promoter element may comprise or be derived from, or consist of, the promoter of any gene expressed in plant cells. The promoter element is preferably capable of driving gene expression in photosynthetic cells, more preferably in mesophyll cells. Transcription factor-activated promoters, such as those described in STAP (Brückner K. et al., 2015 Plant Journal. 82: 707–716) or Lie et al., 2016 Current Opinion in Biotechnology. 37:36–44, may also be used. Alternatively, any multiple target gene sequences may be independently linked to a corresponding such promoter, whether fused, sequentially linked, or otherwise linked. The promoters that activate transcription factors can be operatively linked to at least one transcription factor such as dTALE (see Brückner K. et al., (2015) Plant Journal. 82: 707-716.), or they can be linked to promoters that are active in photosynthetic cells.

[0172] Therefore, the promoter element may comprise or be derived from promoters of genes universally expressed in all plant cells, genes expressed in mesophyll cells, or genes expressed in photosynthetic cells, or may consist of promoters of genes universally expressed in all plant cells, genes expressed in mesophyll cells, or genes expressed in photosynthetic cells. The gene is preferably expressed in C3 plant cells.

[0173] Alternatively, the promoter element may include or be derived from tissue-specific promoters, or may consist of promoters of tissue-specific promoters, such as promoters of genes expressed only in photosynthetic cells or mesophyll cells.

[0174] The plant cell promoter element may be derived from or composed of one of the following gene promoters, but is not limited to: rubisco small subunit (pRBCS1A), chlorophyll a / b binding protein (cab), more preferably CAB3 promoter, PEPC, IQD22, AT1G70958, SQE6, XTH6, PAL1, CORI3, YAB3, LHCB2.4, CRR23, SPS4F, ENH1 or COR414-TM.

[0175] The plant cell promoter element may be derived from or composed of promoters of genes expressed or specifically expressed in plant leaf epidermal cells. Preferably, the cell is a stomatal cell; more preferably, the cell is a stomatal progenitor cell. Those skilled in the art will understand that stomatal progenitor cells are undifferentiated leaf epidermal cells before the initiation or completion of stomatal lineage differentiation. As will be understood by those skilled in the art, stomatal progenitor cells can be identified by the expression of their SPCH, MUTE, and FAMA genes.

[0176] The promoter element can be any known promoter sequence capable of driving the expression of a gene operatively linked thereto. Therefore, the promoter element is not limited by its sequence length. For example, the promoter element may contain at least one or more transcription factor binding sites necessary to induce transcription of the operatively linked nucleotide sequence, or may consist of at least one or more transcription factor binding sites necessary to induce transcription of the operatively linked nucleotide sequence. Those skilled in the art will understand how to modify plant cell promoters that contain, are derived from, or consist of promoters of a given gene, such that the operatively linked nucleotide sequence can be transcribed in a given organism.

[0177] Any promoter element described herein may be generally active or inducible. Those skilled in the art will understand how to derive or modify plant cell promoter elements to make them generally active or inducible.

[0178] Water-forming oxidase

[0179] The sequence encoding water-forming oxidase contained in the polynucleotides described herein can be modified to enhance expression in plants or plant cells. Numerous publicly available online tools enable those skilled in the art to optimize nucleotide or protein sequences used in this invention (see, for example, http: / / genomes.urv.es / OPTIMIZER). Preferably, the sequence is codon-optimized, for example, for expression in plants or specific genera or species of plants. As is known to those skilled in the art, organisms differ in their tendency to use specific codons rather than others to encode the same amino acid. Therefore, codon-optimized sequences can enhance the expression of water-forming oxidase in plants, or in specific genera or species of plants, or in specific cell types.

[0180] The sequences encoding water-forming oxidases can be modified by adding or removing one or more introns. These sequences can be further modified by operatively linking them to other regulatory sequences besides plant cell promoter elements (such as enhancers) to manipulate their transcriptional levels.

[0181] Preferably, the water-forming oxidase is NADPH water-forming oxidase, NADH water-forming oxidase, or a bifunctional NAD(P)H water-forming oxidase. The sequence encoding the water-forming oxidase can be derived from any suitable source organism. In particular, the sequence encoding the water-forming oxidase can be derived from any bacterial species, such as Streptococcus mutans and Lactobacillus brevis.

[0182] The water-forming oxidase may be encoded by a nucleotide sequence comprising or consisting of the following sequences: sequences defined according to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46, or variations thereof. The variant may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, or SEQ ID NO: 46. The variant may be a variant with enzymatic activity, i.e., wherein the variant retains the enzymatic function of the water-forming oxidase described herein.

[0183] The water-forming oxidase may comprise or consist of the following sequences: sequences defined according to SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45 or SEQ ID NO: 47, or variations thereof. The variant may comprise or consist of the following amino acid sequences: amino acid sequences having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 47. The variant may be a variant with enzymatic activity, i.e., wherein the variant retains the enzymatic function of the water-forming oxidase described herein.

[0184] More preferably, the water-forming oxidase may be encoded by a sequence comprising or consisting of the following sequences: sequences defined according to SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18, or variants thereof. The variants may comprise or consist of the following sequences: sequences having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18. The water-forming oxidase may comprise or consist of the following sequences: sequences defined according to SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, or variants thereof. The variant comprises or consists of the following sequences: sequences having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19.

[0185] The polynucleotide may preferably further comprise a terminator sequence, preferably located at 3' relative to the sequence encoding water-forming oxidase. Those skilled in the art will understand that any terminator capable of terminating transcription may suitably be included in the polynucleotide provided herein. The terminator sequence may be, for example, the caustic solanine synthase terminator (NOSt) (SEQ ID NO: 3).

[0186] The nucleotide sequence operatively linked to the plant cell promoter element may also encode one or more of the following: i. a protein different from water-forming oxidase; ii. a target peptide; and iii. a transmembrane protein or domain, optionally wherein one or more of i. to iii. are separated from water-forming oxidase by a linker.

[0187] i. to iii. and the linker are preferably co-coded with the water-forming oxidase, thereby fusing in a translational (i.e., genetic) manner. i. to iii. can be encoded into the 5' of the water-forming oxidase, resulting in their fusion into the N-terminus of the water-forming oxidase. i. to iii. can be encoded into the 3' of the water-forming oxidase, resulting in their fusion into the C-terminus of the water-forming oxidase.

[0188] The protein that differs from water-forming oxidase may, for example, be a protein located at a specific subcellular or extracellular location. Therefore, upon expression of the water-forming oxidase encoded by the polynucleotide provided herein, the water-forming oxidase will necessarily be located at a typical subcellular or extracellular location different from that of the protein in i. The protein according to i. may, for example, be a protein encoded by the rubisco small subunit gene (see, for example, the coding sequence of SEQ ID NO: 6), which targets plant cell chloroplasts.

[0189] The protein that is different from water-forming oxidase can be, for example, a protein capable of recognizing the expression of water-forming oxidase, such as a fluorescent protein that can be observed by a fluorescence microscope. According to i, the protein can be, for example, green fluorescent protein (GFP), preferably, wherein the protein is fused to the C-terminus of water-forming oxidase.

[0190] According to ii., the target peptide can be any known peptide sequence that targets a protein to a specific subcellular or extracellular location. The peptide includes known transport peptides, localization sequences, and signal peptides. For example, the peptide sequence can be a chloroplast transport peptide, a peroxisome targeting sequence, a nuclear localization sequence, a mitochondrial transport peptide, an endosome targeting peptide, a signal peptide, or any other cell-targeting peptide. According to ii., the protein can be, for example, a chloroplast transport peptide from the oxygen-evolving protein 16 gene (see, for example, the sequence encoding the chloroplast transport peptide in SEQ ID NO: 8). According to ii., the protein can also be a chloroplast transport peptide from the oxygen-evolving protein 23 gene (see, for example, the sequence encoding the chloroplast transport peptide in SEQ ID NO: 11).

[0191] According to iii., transmembrane proteins or domains can be localized to any membrane within the cell or to the extracellular membrane. Technicians can identify suitable proteins or domains and engineer the coding sequences to ensure that water-forming oxidases are localized to the desired locations within the cell. According to iii., transmembrane proteins can be, for example, proteins encoded by the outer membrane protein 9 gene (see, for example, SEQ ID NO12), which are localized to the chloroplast outer membrane.

[0192] The nucleotide sequence encoding the water-forming oxidase is preferably separated from the sequence encoding any one or more of i. to iii. by a sequence encoding a linker. The linker can be any suitable linker known to those skilled in the art. The linker can be flexible or rigid. Preferred flexible peptide linkers are fragments of 2 to 40, for example 2 to 20, such as 4, 6, 8, 10, or 16 serine and / or glycine amino acids. More preferred flexible linkers include (SG)1, (SG)2, (SG)3, (SG)4, (SG)5, and (SG)8, where S is serine and G is glycine. Even more preferred flexible linkers are polyglycine-serine linkers, such as GGGGSGGGGS. Preferred rigid linkers are fragments of 2 to 30, for example 4, 6, 8, 16, or 24 proline amino acids. Even more preferred rigid linkers include (P)12, where P is proline. Even more preferred rigid linkers are the amino acid sequence AEAAAAKEAAAKEAAAKA.

[0193] carrier

[0194] This document provides vectors containing the polynucleotides of the present invention. Such expression vectors are conventionally constructed in the field of molecular biology and may, for example, include plasmid DNA and suitable initiators, promoters, enhancers, and other elements, such as potentially necessary polyadenylation signals, and are oriented correctly to allow expression of the peptides of the present invention. The vectors may also encode fluorescent proteins other than any fluorescent proteins expressed in the same frame as water-forming oxidases, wherein such other fluorescent proteins are separated from the nucleotide sequence of the polynucleotides of the present invention by an internal ribosome entry site (IRES) or under the control of an additional promoter. The vectors preferably contain an origin of replication and a right-bound repeat of the T-DNA of a Ti or Ri plasmid, optionally also containing a left-bound repeat of the Ti or Ri plasmid, and at least one bacterial selectable marker. The vectors may contain one or more of the following: i. enhancers; ii. plant selectable markers; iii. multiple cloning sites; and iv. recombination sites.

[0195] Suitable vectors for this purpose are well known to those skilled in the art and include, but are not limited to, plasmids, entrapments, vectors, artificial chromosomes, modified viruses, and mobile genetic elements. Other suitable vectors will be apparent to those skilled in the art. As a further example in this regard, we refer to Sambrook et al. (Sambrook, J., Fritsch, ER, & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).

[0196] Composition and transformation

[0197] This document provides compositions for transforming plant cells, the compositions comprising a polynucleotide according to the invention and / or a carrier according to the invention, preferably wherein the compositions comprise microparticles complexed with the polynucleotide and / or carrier, or wherein the polynucleotide and / or carrier are transformed into plant cells using Agrobacterium-mediated transformation.

[0198] Plant transformation is now a routine technique for those skilled in the art. Advantageously, any composition provided herein can be used for any suitable transformation method in order to introduce polynucleotides and / or vectors into suitable ancestor cells. The compositions may contain any known ingredients that make them suitable for the methods of transforming and regenerating plants from plant tissues or plant cells, wherein the compositions 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 bombardment, Agrobacterium-mediated transformation, transformation using viruses or pollen, or microprojection. Methods may include the calcium / polyethylene glycol method for protoplasts, protoplast electroporation, microinjection of plant material, DNA or RNA-coated particle bombardment, infection with (non-integrating) viruses, etc. Methods for coating DNA or RNA onto microparticles are well known to those skilled in the art, for example, as described in Ismagul et al., BMC PlantBiology 2018 18: 135, or Kikkert JR. Cell biology: a laboratory handbook, vol. 4. San Diego: Academic Press; 1998. p. 157–61, or Sanford JC et al., MethodsEnzymol. 1993 217:483–509. Therefore, suitable microparticles complexed with the polynucleotides and / or carriers of the composition are known to those skilled in the art. Methods of the present invention comprising modified heritable genetic material of plants may include transforming plants, preferably wherein said transformation comprises Agrobacterium-mediated transformation. Transgenic plants, including transgenic crops, are preferably produced via Agrobacterium-mediated transformation.

[0199] The methods of the present invention that include modifying the heritable genetic material of a plant may include any suitable transformation methods known in the art, including any transformation process described herein. Preferably, the steps of modifying the heritable genetic material of a plant in any of the methods provided herein do not include inherently biological processes, such as hybridization and selection.

[0200] Transformation methods are well known in the art. Therefore, according to various aspects of the invention, the compositions and / or polynucleotides of the invention can be introduced into plants, such that the polynucleotides and / or vectors are expressed as transgenic structures. Nucleic acids are introduced into plants through transformation. The terms “introduction” or “transformation” as used herein include the transfer of exogenous polynucleotides into host cells, regardless of the method of transfer. Plant tissues capable of subsequent clonal propagation via organogenesis or embryogenesis can be transformed with the gene constructs of the invention, and complete plants can be regenerated from them. The specific tissues selected will vary depending on the clonal propagation system available and best suited to the particular species being transformed. Exemplary tissue targets include leaf discs, pollen, cotyledons, cotyledonary hypocotyls, megagametophytes, callus, existing meristems (e.g., apical meristems, axillary buds, and root meristems), and induced meristems (e.g., cotyledonary meristems and cotyledonary hypocotyl meristems). The polynucleotides and / or vectors can be introduced transiently or stably into host cells and can remain unintegrated, for example, as plasmids. Alternatively, they can be integrated into the host genome. The resulting transformed plant cells can then be used to regenerate transformed plants in a manner known in the art.

[0201] To select for transformed plants, the plant material obtained from transformation is typically placed under selection conditions to distinguish transformed plants from untransformed plants. For example, seeds obtained as described above can be planted and, after the initial growth period, suitable selection can be performed by spraying. Another possibility is to culture seeds on agar plates using a suitable selectant (if appropriate, after sterilization), so that only transformed seeds can grow into plants. Alternatively, transformed plants with selectivity markers can be screened visually using reporter molecules based on color fluorescence, or using molecular techniques to detect the presence of the polynucleotides of the present invention. After DNA transfer and regeneration, the presumed transformed plants can also be evaluated, for example, using Southern blotting or PCR analysis to assess the presence, copy number, and / or genome organization of the target gene. Alternatively or additionally, Northern and / or Western blotting analysis, or monitoring of the expression level of newly introduced DNA by rtPCR or RNA-Seq, all of which are well known in the art.

[0202] The resulting transformed plants can be propagated in various ways, such as through clonal propagation or classical breeding techniques. For example, first-generation (or T1) transformed plants can be self-pollinated to select homozygous second-generation (or T2) transformants, which can then be further propagated using classical breeding techniques. The resulting transformed organisms can take many forms. For example, they can be chimeras of transformed and non-transformed cells; clonal transformants (e.g., all cells are transformed to contain expression cassettes); and grafts of transformed and untransformed tissues (e.g., in a plant, a transformed rhizome grafted onto an untransformed scion).

[0203] The modified plants according to the present invention advantageously provide better yield characteristics. Yield characteristics, also known as yield traits, may include one or more of the following non-limiting listed characteristics: yield, biomass, seed yield, seed / grain size, grain starch content, early vigor, greenness index, Landsat normalized difference vegetation index, increased growth rate, earlier flowering, and enhanced adaptability to environmental changes. The term “yield” generally refers to a measurable product of economic value, usually associated with a specific crop, region, and time period. Various parts of a plant directly affect yield according to their quantity, size, and / or weight. Actual yield is the yield per square meter of a crop per year, determined by dividing the total yield (including harvested yield and assessed yield) by the planted area (square meters). The term “yield” for a plant may refer to the plant biomass (root and / or shoot biomass), reproductive organs, and / or propagules (such as seeds). Therefore, according to the present invention, yield includes one or more of the following, and can be measured by evaluating one or more of the following: increased seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, increased vigor / germination efficiency, increased number or size of seeds / capsules / pods, increased growth, increased number of seeds, increased number of floral organs, increased biomass, or increased grain filling. Preferably, increased yield includes increased number of grains / seeds / capsules / pods, increased biomass, increased growth, and / or increased number of floral organs. Yield is typically measured relative to a control plant.

[0204] cell

[0205] This document provides cells containing polynucleotides according to the invention or vectors according to the invention.

[0206] The cell can be any cell suitable for cloning, such as a bacterial cell, preferably wherein the bacterial species is Escherichia coli, Agrobacterium sp., or Agrobacterium tumefaciens.

[0207] The cells may be plant cells, such as C3 plant mesophyll cells, C3 plant vascular sheath cells, C3 plant bundle sheath cells, C3 plant central bundle sheath cells, C4 photosynthetic plant cells, such as C4 plant vascular sheath cells, C4 plant bundle sheath cells, C4 plant central bundle sheath cells, or C4 plant mesophyll cells; or CAM photosynthetic plant cells, such as CAM plant vascular sheath cells, CAM plant bundle sheath cells, CAM plant central bundle sheath cells, or CAM plant mesophyll cells.

[0208] The cells are preferably plant cells, wherein the plant cells are capable of photosynthesis and / or preferably C3 plant cells.

[0209] The cells may be C3 or C4 plant cells. Preferably, the cells are plant leaf epidermal cells and / or cells capable of photosynthesis. More preferably, the cells are stomatal cells. More preferably, the stomatal cells are stomatal progenitor cells.

[0210] The cells may be photosynthetic cells, which may be photosynthetic bacteria (such as cyanobacteria) or photosynthetic algal cells (such as those found in green algae or streptococci).

[0211] The plant cells can be any plant tissue, such as leaf disc, pollen, plumule, cotyledon, cotyledonary hypoaxial portion, macrogametophyte, callus, existing meristems (e.g., apical meristem, axillary bud and root meristem), and induced meristems (e.g., cotyledonary meristem and cotyledonary hypoaxial meristem). The plant cells are preferably mesophyll cells, stomatal cells, or stomatal progenitor cells. Most preferably, the plant cells are stomatal progenitor cells.

[0212] Cells with modified genetic material according to the methods described herein can be used for bioproduction because they provide enhanced growth and / or biomass. For example, the cells of the present invention can be used for the commercial production of specific metabolites isolated from plant cells, which in turn can serve as building blocks for a wide range of complex chemical substances, including, but not limited to, polymers, solvents, and pharmaceuticals. Other methods of the present invention involve producing transgenic plants as described herein. These transgenic plants will have increased growth rates or biomass compared to their corresponding wild-type plants.

[0213] plant

[0214] This document provides a plant or a portion thereof comprising: i. a cell according to the invention; or ii. a cell comprising a polynucleotide containing a promoter element operatively linked to a nucleotide sequence encoding a water-forming oxidase.

[0215] ii. The promoter element of the cell can be any promoter element capable of driving the expression of an operatively linked gene, and is not limited to plant cell promoter elements. The promoter element may include, be derived from, or be composed of any suitable promoter known to those skilled in the art. For example, the promoter element may be synthetic, or comprise a minimal set of elements, or be derived from a non-plant host (such as the CaMV 35S promoter). The promoter element may also be defined according to the polynucleotide promoter elements provided herein.

[0216] The plants provided herein are preferably transgenic plants. The plants provided herein preferably exhibit enhanced growth compared to unmodified control plants grown under the same conditions. Preferably, the plants are C3 or C4 plants, and preferably, the conditions are water-deficient conditions. Those skilled in the art understand that water-deficient conditions are defined in the context of plant growth. Water-deficient conditions occur in agricultural or natural environments and in any situation where water availability limits photosynthesis or growth. Water-deficient conditions can also include cultivating plants in a water-deficient growth medium. For example, in some experiments, water-deficient conditions can be maintained by providing water to the growth medium (e.g., soil) but subsequently withholding water for an extended period during growth to maintain a suboptimal and deteriorating level of water saturation in the growth medium. For example, water-deficient conditions can be maintained by providing water to the growth medium (e.g., soil) before seed stratification but withholding water for 15 days after stratification is completed to maintain a suboptimal and deteriorating level of water saturation in the growth medium during vegetative growth.

[0217] The plant may be a monocotyledonous plant, such as one selected from the palm family, amaryllidaceae family, or grass family. For example, the plant may be a cereal crop, such as wheat, rice, barley, oats, triticale, rye, or buckwheat, or a non-cereal monocotyledonous crop, such as garlic, onion, leek, yam, oil palm, or banana.

[0218] The plant may be a dicotyledonous plant and may be selected, for example, from the Asteraceae, Brassicaceae (e.g., Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Liliaceae, Mimosaceae, Fabaceae, or Leguminosae), Malvaceae, Rosaceae, or Solanaceae families. For example, the plant may be selected from lettuce, sunflower, broccoli, spinach, watermelon, pumpkin, cabbage, tomato, potato, sweet potato, pepper, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, beans, soybean, broad bean, pea, lentil, peanut, chickpea, apricot, pear, peach, grapevine, bell pepper, chili pepper, citrus, or coffee species.

[0219] The plants may be biofuel or bioenergy crops, such as rapeseed / canola, flaxseed, lupin, and willow, poplar, poplar hybrids, or gymnosperms (such as slash pine). Also included are crops used for silage, grazing or forage (grass, clover, sanfoin, alfalfa), fiber (such as hemp, cotton, flax), building materials (such as pine, oak, teak, rosewood), pulping (such as poplar), and raw materials for the chemical industry (such as high-erucic acid rapeseed, flaxseed, jute, oil palm).

[0220] The plant is most preferably a C3 plant. The C3 plant is preferably a crop plant, particularly one that is commercially available for human or animal consumption. Preferably, the crop is a cereal, oilseed, or legume.

[0221] The C3 plants mentioned in this article can include, for example, soybean (Glycine max), cotton (Gossypium hirsutum), rapeseed / canola rape (B. napus subsp. Napus), potato (Solanum tuberosum), tomato (Solanum lycopersicum), wheat (Triticum aestivum), barley (Hordeum vulgare), oats (Avenasativa), rice (Oryza sativa), rye (Secale cereal), pigeon bean (Cajanus cajan), cowpea (Vigna unguiculata), pea (Pisum sativum), sugar beet (Beta vulgaris), castor oil (castor bean, Ricinus communis), cassava (Manihot esculenta), sweet potato (Ipomoea batatas), yam (Dioscoreaspp.), peanut (Arachis hypogaea), and sunflower (Helianthus). The following are plant species: annuus, flax (Linumspp.), common bean (Phaseolus vulgaris), lima bean (Phaseolus lunatus), mung bean (Phaseolus mung), red bean (Phaseolus angularis), chickpea (Cicer arietinum), buckwheat (Fagopyrum esculentum), tobacco (Nicotiana tabacum), hemp (Cannabis sativa), oil palm (Elaeis guineensis), American sycamore (Acer pseudoplatanus), sweet chestnut (Castanea sativa), poplar (Populus spp.), eucalyptus (Sucalyptus spp.), plantain / banana (Musaacuminate) or rubber (Hevea brasiliensis).

[0222] The plant part provided herein can be any part of a C3 plant. For example, the plant part can be a seed, fruit, branch, stem, leaf, root (including tuber), flower, tissue, or organ.

[0223] The plant is preferably a C4 plant. The C4 plant is preferably a crop plant, particularly one that is commercially available for human or animal consumption or use. C4 plants described herein can be, for example, maize (Zea mays), sorghum (Sorghum bicolor), sugarcane, tamarisk, millet, or other C4 herbaceous plants, grasses, or cereals.

[0224] The plant part provided herein can be any part of a C4 plant. For example, the plant part can be a seed, fruit, branch, stem, leaf, root (including tuber), flower, tissue, or organ.

[0225] Methods and their products

[0226] This article provides a method for increasing plant growth and yield, preferably wherein the plant is a C3 or C4 plant, the method comprising modifying the heritable genetic material of the plant such that a water-forming oxidase is expressed in at least one mesophyll cell of the plant, thereby increasing plant growth and yield.

[0227] This article also provides a method for:

[0228] (i) Reduce the stomatal density of plants; and / or

[0229] (ii) Reduce the stomatal conductance of plants; and / or

[0230] (iii) Increase the water retention capacity of plants,

[0231] Preferably, the plant is a C3 or C4 plant, and the method includes modifying the plant's heritable genetic material to express a water-forming oxidase in at least one cell of the plant's leaf epidermis. Preferably, the cell is a stomatal cell, more preferably, the stomatal cell is a stomatal progenitor cell. Methods for assessing stomatal conductance and increasing water retention are known to those skilled in the art. Preferably, the reduced stomatal conductance and / or reduced stomatal density and / or increased water retention are assessed relative to a reference plant grown under identical conditions, wherein the heritable genetic material of the reference plant is not modified to express a water-forming oxidase in at least one cell of the reference plant's leaf epidermis. Preferably, the cell is a stomatal cell, more preferably, the stomatal cell is a stomatal progenitor cell.

[0232] Stomatal conductance is understood in the art to refer to the rate of gas exchange between water and CO2 through leaf stomata. Conductance can be a function of stomatal diameter, stomatal size, and / or stomatal density. Methods for measuring stomatal conductance are well known in the art. For example, stomatal conductance can be determined using an infrared open gas exchange system. For example, stomatal conductance can be measured using a leaf stomatometer. For example, stomatal conductance can be measured using methods based on thermal imaging, thermal imaging, and / or infrared temperature sensing. For example, stomatal conductance can also be estimated by measuring sap flow. For example, stomatal conductance can also be measured at the canopy level using techniques such as multispectral imaging and / or remote sensing. For example, stomatal conductance can also be measured at the canopy level using techniques such as eddy covariance measurements of latent heat flux. Those skilled in the art will recognize that various techniques are available for measuring stomatal conductance at multiple scales (individual leaf, whole plant, canopy, etc.).

[0233] Stomatal density is understood in the art to refer to the number of stomata per unit area of ​​a leaf. Stomatal density can refer to either the abaxial or adaxial surface of the leaf. It is well known that an increase in stomatal density leads to an increase in stomatal conductance, while a decrease in stomatal conductance leads to a decrease in stomatal conductance. Methods for measuring stomatal density are well known in the art. For example, measuring stomatal density preferably includes obtaining an epidermal impression from the abaxial or adaxial surface of a plant leaf, observing the epidermal impression under a suitable microscope, and determining stomatal density counts on multiple independent regions of each leaf.

[0234] In the context of the method of this invention, water retention preferably refers to reducing water loss due to transpiration. Therefore, increased water retention may result from reduced stomatal conductance and / or reduced stomatal density.

[0235] Preferably, any of the methods described herein may include growing the plant under water-scarce conditions. Growth may preferably include cultivating the plant under water-scarce conditions. Water-scarce conditions are known to those skilled in the art, and exemplary water-scarce conditions are described herein.

[0236] In the methods described herein, when the phenotypic effect is described, for example, as an increase or decrease, it should be understood that this preferably refers to an increase or decrease relative to a control unmodified plant grown under the same conditions.

[0237] Technicians can modify the heritable genetic material of a plant in any suitable manner to cause the water-forming oxidase to be expressed in at least one mesophyll cell of the plant. For example, technicians can use any method for producing the transgenic plants described herein, such as any suitable transformation method described herein.

[0238] The modification may include the conversion of C3 or C4 plants with the following substances: i. a polynucleotide according to the invention; or ii. a vector according to the invention; or iii. a polynucleotide comprising a promoter element operatively linked to a nucleotide sequence encoding a water-forming oxidase.

[0239] iii. The polynucleotide promoter element can be any promoter element capable of driving operatively linked gene expression, and is not limited to plant cell promoter elements. The promoter element may include, be derived from, or consist of any suitable promoter known to those skilled in the art. The promoter element may also be defined according to the polynucleotide promoter elements provided herein.

[0240] The plant can be any plant described herein, with C3 or C4 plants being the most preferred.

[0241] The water-forming oxidase may be any water-forming oxidase described herein, but is preferably NADPH water-forming oxidase, NADH water-forming oxidase or bifunctional NAD(P)H water-forming oxidase.

[0242] Plant products

[0243] This document provides plant parts, plant tissues, plant organs, plant cells, plant protoplasts, embryos, callus cultures, pollen grains, or seeds derived from plants according to the invention or from plants produced by methods for increasing plant growth and yield as described herein, optionally wherein said plant parts contain nucleic acid sequences corresponding to water-forming oxidases as defined according to the invention.

[0244] In some cases, the plant, plant part, plant tissue, plant organ, plant cell, plant protoplast, embryo, callus culture, pollen grain or seed is not obtained solely through basic biological processes.

[0245] use

[0246] This article provides an application of a water-forming oxidase, which is used for:

[0247] (i) Reduce the stomatal density of plants; and / or

[0248] (ii) Reduce the stomatal conductance of plants; and / or

[0249] (iii) Increase the water retention capacity of plants,

[0250] Preferably, the use comprises cultivating plants containing nucleotide sequences corresponding to water-forming oxidases as defined according to the present invention.

[0251] Preferably, in the use of the invention described herein, the plant is a transgenic plant that, compared to a control unmodified plant grown under the same conditions, has:

[0252] (i) Reduced porosity; and / or

[0253] (ii) Reduced pore density; and / or

[0254] (iii) Increased water retention

[0255] Preferably, the plant is a C3 or C4 plant, and preferably, the condition is a water-deficient condition.

[0256] The features of the present invention can be defined by any polynucleotide according to the present invention.

[0257] The uses may also include any steps defined according to the methods of the invention described herein. For example, the uses may include the step of producing transgenic plants by transformation, such that the plants express water-forming oxidases as defined by the polynucleotides according to the invention.

[0258] Example

[0259] The invention will now be described with reference to specific embodiments, which should not be construed as limiting the invention in any way.

[0260] Example 1: Water-forming NADH or NADPH oxidases from various species can be expressed in plant cells.

[0261] To demonstrate that water-forming NADH or NADPH oxidases from multiple different species can be expressed in plants, codon optimization was performed on genes from *Streptococcus mutans* SmNOX [SEQ ID NO: 19], *Lactobacillus brevis* LbNOX [SEQ ID NO: 15 and SEQ ID NO: 17] for expression in plants [nucleotide sequences defined by SEQ ID NO: 18, SEQ ID NO: 14 and SEQ ID NO: 16, respectively], and cloned into the pICH47742 vector (Weber E et al., 2011 PLoSOne. 6(2):e16765). Therefore, each water-forming oxidase gene cloned into this vector was expressed by fusion with a C-terminal GFP [SEQ ID NO: 4 and SEQ ID NO: 5] under the control of the 35S CaMV promoter [SEQ ID NO: 2]. Figure 1A). The vector was introduced into Arabidopsis leaf protoplasts using polyethylene glycol-mediated protoplast transformation. In each case, expression was readily detected using confocal microscopy, confirming the expression of the introduced transgenes (LbNOX and SmNOX variants). Figure 2 ).

[0262] Example 2: Engineered water-forming NADH or NADPH oxidases can be expressed in plant cells.

[0263] To demonstrate that the engineered water-forming NADH or NADPH oxidase can be expressed in plants, the engineered water-forming NADH or NADPH oxidase from *Streptococcus mutans* SmNOX [SEQ ID NO: 19] was codon-optimized for expression in plants [SEQ ID NO: 18] and cloned into the pICH47742 vector. As described above, the gene is expressed as a C-terminal GFP fusion under the control of the 35S CaMV promoter [SEQ ID NO: 2] [SEQ ID NO: 4 and SEQ ID NO: 5]. Figure 1 A). The vector was introduced into Arabidopsis leaf protoplasts using polyethylene glycol-mediated protoplast transformation. Expression was readily detected using confocal microscopy, confirming the expression of the introduced transgene (SmNOX variant). Figure 2 ).

[0264] Example 3: Water-forming NADH or NADPH oxidases can target different subcellular locations in plant cells.

[0265] To demonstrate that water-forming oxidases can target different subcellular locations in plant cells, a bifunctional water-forming NAD(P)H oxidase from Streptococcus mutans SmNOX was fused to several different protein sequences at its N-terminus via translation, and to GFP at its C-terminus via translation [SEQ ID NO: 4 and SEQ ID NO: 5]. Figure 1B). These proteins include chloroplast transport peptides from the oxygen-evolving protein 16 gene [SEQ ID NO: 8 and SEQ ID NO: 9]. Another chloroplast transport peptide from the oxygen-evolving protein 23 gene [SEQ ID NO: 10 and SEQ ID NO: 11]. The rubisco small subunit gene [SEQ ID NO: 6 and SEQ ID NO: 7]. The outer envelope protein 9 gene [SEQ ID NO: 12 and SEQ ID NO: 13]. In each case, expression was readily detected using confocal microscopy, confirming the expression of the introduced transgene in the correct location. Specifically, SmNOX is fused with a sequence encoding a chloroplast transport peptide from oxygen-evolving protein 16 [SEQ ID NO: 36 and SEQ ID NO: 37] or a sequence fused with a chloroplast transport peptide from oxygen-evolving protein 23 [SEQ ID NO: 38 and SEQ ID NO: 39] (CpSmNOX, Figure 2 ), or fuse with the rubisco small subunit [SEQ ID NO: 34 and SEQ ID NO: 35], localizing to the chloroplast (RbcS-SmNOX, Figure 2 SmNOX fuses with outer membrane protein 9 [SEQ ID NO: 40 and SEQ ID NO: 41] and localizes to the chloroplast membrane (CIMS SmNOX, Figure 2 ).

[0266] Example 4: Compared with unmodified plants, the expression of water-forming NADH or NADPH oxidases in plants led to enhanced growth.

[0267] To demonstrate the effect of water-forming oxidase expression on plant growth, the bifunctional water-forming NAD(P)H oxidase from *Streptococcus mutans* [SEQ ID NO: 18] was cloned into the pAGM37443 vector (Grützner R et al., *Plant Communications*, 2020, 100135). In this vector, the bifunctional water-forming NAD(P)H oxidase gene was expressed under the control of the chlorophyll a / b binding protein 3 (CAB3) promoter [SEQ ID NO: 1]. Figure 1 C). This produced a vector for expressing SmNOX in the cytosol of plant cells, labeled as cytosol SmNOX [SEQ ID NO: 18 and SEQ ID NO: 19]. A series of composite forms were also produced in which the SmNOX gene is fused to a number of different protein sequences at its N-terminus in a translational manner ( Figure 1D). This collection includes: a modified version of water-forming oxidase that is translated and fused with the oxygen-evolving protein 16 transport peptide (SEQ ID NO: 8 and SEQ ID NO: 9) and targets chloroplasts (Cp SmNOX SEQ ID NO: 22 and SEQ ID NO: 23); a modified version of water-forming oxidase that is translated and fused with the rubisco small subunit (SEQ ID NO: 6 and SEQ ID NO: 7) and targets chloroplasts (RbcS-SmNOX SEQ ID NO: 20 and SEQ ID NO: 21); and a modified version of water-forming oxidase that is translated and fused with the outer membrane protein 9 (SEQ ID NO: 12 and SEQ ID NO: 13) and targets the chloroplast membrane intermembrane space (CIMS SmNOX SEQ ID NO: 26 and SEQ ID NO: 27). Arabidopsis plants were transformed with these vectors using the floral-dip method. Individual insertion events were isolated, and the growth rate, biomass accumulation, plant height, bolting time, and flowering time of transgenic plants were analyzed and compared with unmodified control plants.

[0268] Plants were allowed to grow for 20 days, and the growth rates of the transgenic plants on days 10 and 20 were compared with those of the unmodified control plants. This revealed that all plant lines expressing water-forming oxidase grew faster than the unmodified control plants. Figure 3 A). Specifically, plants expressing CIMS SmNOX [SEQ ID NO: 26 and SEQ ID NO: 27] grew the fastest, 100% faster than the unmodified control plants (p < 0.001, one-way ANOVA). Plants expressing Cp SmNOX [SEQ ID NO: 22 and SEQ ID NO: 23] grew 80% faster than the unmodified control plants (p < 0.001, one-way ANOVA). Plants expressing cytosol SmNOX [SEQ ID NO: 18 and SEQ ID NO: 19] also grew 80% faster than the unmodified control plants (p < 0.001, one-way ANOVA). Plants expressing RbcS-SmNOX [SEQ ID NO: 20 and SEQ ID NO: 21] grew 20% faster than the unmodified control plants (p < 0.05, one-way ANOVA).

[0269] Example 5: Compared with unmodified plants, the expression of water-forming NADH or NADPH oxidases in plants resulted in enhanced biomass.

[0270] To demonstrate the effect of water-forming oxidase expression on plant biomass accumulation, the transgenic plants were grown under identical conditions, and the total rosette leaf area of ​​each plant was measured on day 20. Consistent with the growth rate data above, plants expressing water-forming oxidase accumulated more biomass than the unmodified control plants over the same time period. Figure 3 B). Specifically, plants expressing CIMS SmNOX [SEQ ID NO: 26 and SEQ ID NO: 27] produced 100% more leaf area than control plants (p < 0.001, one-way ANOVA). Plants expressing Cp SmNOX [SEQ ID NO: 22 and SEQ ID NO: 23] produced 80% more leaf area than control plants (p < 0.001, one-way ANOVA). Plants expressing cytosol SmNOX [SEQ ID NO: 18 and SEQ ID NO: 19] produced 80% more leaf area than control plants (p < 0.001, one-way ANOVA). Plants expressing RbcS-SmNOX [SEQ ID NO: 20 and SEQ ID NO: 21] produced 20% more leaf area than control plants (p < 0.05, one-way ANOVA).

[0271] Example 6: Compared with unmodified plants, the expression of water-forming NADH or NADPH oxidases in plants resulted in enhanced inflorescence size.

[0272] To further demonstrate the effect of water-forming oxidase expression on plant yield, the transgenic plants were grown under identical conditions, and the inflorescence height of each plant was measured on day 32. Consistent with the growth rate and biomass data above, plants expressing water-forming oxidase produced larger inflorescences than unmodified control plants over the same time period. Figure 3C). Specifically, plants expressing CIMS SmNOX [SEQ ID NO: 26 and SEQ ID NO: 27] produced 20% more main inflorescences than control plants (p < 0.001, one-way ANOVA). Plants expressing Cp SmNOX [SEQ ID NO: 22 and SEQ ID NO: 23] produced 15% more main inflorescences than control plants (p < 0.001, one-way ANOVA). Plants expressing cytosol SmNOX [SEQ ID NO: 18 and SEQ ID NO: 19] produced 14% more main inflorescences than control plants (p < 0.001, one-way ANOVA). Plants expressing RbcS-SmNOX [SEQ ID NO: 20 and SEQ ID NO: 21] produced no significant difference in main inflorescences compared to control plants (p > 0.05, one-way ANOVA).

[0273] Example 7: Compared with unmodified plants, the expression of water-forming NADH or NADPH oxidases in plants resulted in a shortened flowering time.

[0274] To demonstrate the effect of water-forming oxidase expression on other plant yield components, the transgenic plants were grown under identical conditions, and the time required for each plant to flower was recorded. Consistent with the growth rate, biomass accumulation, and main inflorescence size data above, plants expressing water-forming oxidase flowered earlier than the unmodified control plants. Figure 3 D). Specifically, plants expressing CIMS SmNOX [SEQ ID NO: 26 and SEQ ID NO: 27] flowered on average 1.3 days earlier than unmodified control plants (p < 0.001, one-way ANOVA). Plants expressing Cp SmNOX [SEQ ID NO: 22 and SEQ ID NO: 23] flowered on average 0.9 days earlier than unmodified control plants (p < 0.001, one-way ANOVA). Plants expressing cytosol SmNOX [SEQ ID NO: 18 and SEQ ID NO: 19] flowered on average 0.8 days earlier than unmodified control plants (p < 0.01, one-way ANOVA). Plants expressing RbcS-SmNOX [SEQ ID NO: 20 and SEQ ID NO: 21] flowered on average at the same time as unmodified control plants (p > 0.05, one-way ANOVA).

[0275] Example 8: Compared with unmodified plants, the expression of water-forming NADH or NADPH oxidases in plants led to a decrease in stomatal density.

[0276] To demonstrate the effect of water-forming oxidase expression on plant stomatal development, the transgenic plants were grown under identical conditions, and stomatal density was measured from epidermal imprints on the abaxial surface of the leaves. The leaves of plants expressing water-forming oxidase exhibited lower stomatal density compared to age-matched unmodified control plants. Figure 4 A). Specifically, plants expressing cytosol SmNOX [SEQ ID NO: 18 and SEQ ID NO: 19] showed a 30.6% lower stomatal density than control plants (p < 0.001, two-factor ANOVA and Fisher LSD post-hoc analysis). Plants expressing Cp SmNOX [SEQ ID NO: 22 and SEQ ID NO: 23] showed a 22.8% lower stomatal density than control plants (p < 0.001, two-factor ANOVA and Fisher LSD post-hoc analysis). Plants expressing CIMS SmNOX [SEQ ID NO: 26 and SEQ ID NO: 27] showed a 20.5% lower stomatal density than control plants (p < 0.001, two-factor ANOVA and Fisher LSD post-hoc analysis).

[0277] Example 9: Compared with unmodified plants, the expression of water-forming NADH or NADPH oxidases in plants resulted in decreased stomatal conductance.

[0278] To demonstrate the effect of water-forming oxidase expression on leaf gas exchange, the transgenic plants were grown under identical conditions, and the light saturation rate of stomatal conductance was measured. Consistent with the decrease in stomatal density, plants expressing water-forming oxidase exhibited reduced stomatal conductance compared to unmodified control plants. Figure 4 B). Specifically, plants expressing CIMSSmNOX [SEQ ID NO: 26 and SEQ ID NO: 27] showed 27.4% lower stomatal conductance than control plants (p < 0.001, two-factor ANOVA and Fisher LSD post-hoc analysis). Plants expressing cytosol SmNOX [SEQ ID NO: 18 and SEQ ID NO: 19] showed 24.5% lower stomatal conductance than control plants (p < 0.001, two-factor ANOVA and Fisher LSD post-hoc analysis). Plants expressing Cp SmNOX [SEQ ID NO: 22 and SEQ ID NO: 23] showed 22.5% lower stomatal conductance than control plants (p < 0.001, two-factor ANOVA and Fisher LSD post-hoc analysis).

[0279] Example 10: Compared with unmodified plants, the expression of water-forming NADH or NADPH oxidases in plants leads to improved photosynthetic water use efficiency.

[0280] To demonstrate the effect of water-forming oxidase expression on photosynthetic water use efficiency in plants, the aforementioned transgenic plants were grown under identical conditions, and the intrinsic photosynthetic water use efficiency at light saturation, i.e., the ratio of photosynthetic CO2 assimilation to stomatal gas conductance for water vapor, was measured. Plants expressing water-forming oxidase exhibited enhanced photosynthetic water use efficiency compared to unmodified control plants. Figure 4 C). Specifically, plants expressing Cp SmNOX [SEQ ID NO: 22 and SEQ ID NO: 23] showed a 17.6% increase in photosynthetic water use efficiency compared to the control plants (p < 0.05, two-factor ANOVA and Fisher LSD post-hoc analysis). Plants expressing CIMS SmNOX [SEQ ID NO: 26 and SEQ ID NO: 27] showed a 16.8% increase in photosynthetic water use efficiency compared to the control plants (p < 0.05, two-factor ANOVA and Fisher LSD post-hoc analysis). Plants expressing cytosol SmNOX [SEQ ID NO: 18 and SEQ ID NO: 19] showed a 12.6% increase in photosynthetic water use efficiency compared to the control plants (p < 0.05, two-factor ANOVA and Fisher LSD post-hoc analysis).

Claims

1. A polynucleotide comprising a plant cell promoter element operatively linked to a nucleotide sequence encoding a water-forming oxidase.

2. The polynucleotide of claim 1, wherein the plant cell promoter element comprises or is derived from a promoter of a gene expressed in photosynthetic cells, preferably specifically expressed in mesophyll cells, and / or the plant cell promoter element is a C3 or C4 plant cell promoter.

3. The polynucleotide according to claim 1 or 2, wherein the plant cell promoter element is: a. A promoter derived from or composed of promoters from photosynthetic cells, preferably wherein the cells are mesophyll cells; or b. Derived from or composed of one of the following gene promoters: Rubisco small subunit (pRBCS1A), chlorophyll a / b binding protein (cab), preferably CAB3 promoter, PEPC, IQD22, AT1G70958, SQE6, XTH6, PAL1, CORI3, YAB3, LHCB2.4, CRR23, SPS4F, ENH1, or COR414-TM1; or c. The promoter of a gene expressed or specifically expressed in plant leaf epidermal cells or composed of promoters of genes expressed or specifically expressed in plant leaf epidermal cells, preferably, wherein the cell is a stomatal cell, more preferably, wherein the cell is a stomatal progenitor cell.

4. The polynucleotide according to any one of claims 1 to 3, wherein the promoter is inducible or generally active.

5. The polynucleotide according to any one of claims 1 to 4, wherein the sequence encoding the water-forming oxidase is codon-optimized for expression in a plant, preferably, wherein the plant is a C3 or C4 plant.

6. The polynucleotide according to any one of claims 1 to 5, wherein the water-forming oxidase is NADPH water-forming oxidase, NADH water-forming oxidase or bifunctional NAD(P)H water-forming oxidase.

7. The polynucleotide of claim 6, wherein the NADPH water-forming oxidase or NADH water-forming oxidase is derived from bacteria.

8. The polynucleotide according to claim 6 or 7, wherein the sequence encoding NADPH water-forming oxidase or NADH water-forming oxidase comprises or consists of the following sequences: Based on the sequence of SEQ ID NO: 14, and / or SEQ ID NO: 16, and / or SEQ ID NO: 18 or its active variants.

9. The polynucleotide of claim 8, wherein the active variant is characterized by comprising a sequence having at least 50% sequence identity with SEQ ID NO:14, or SEQ ID NO:16, or SEQ ID NO:18, or a sequence having at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:14, or SEQ ID NO:16, or SEQ ID NO:

18.

10. The polynucleotide according to any one of claims 1 to 9, wherein the polynucleotide further comprises a terminator.

11. The polynucleotide according to any one of claims 1 to 10, wherein the nucleotide sequence further encodes one or more of the following: i. Proteins that are different from the water-forming oxidases mentioned above; ii. target peptides; and iii. Transmembrane domains Optionally, one or more of i to iii are separated from the water-forming oxidase by a connector.

12. A vector comprising a polynucleotide according to any one of claims 1 to 11.

13. The vector of claim 12, wherein the vector comprises an origin of replication and a right boundary repeat of the T-DNA of the Ti or Ri plasmid, optionally further comprising a left boundary repeat of the Ti or Ri plasmid, and at least one bacterial selectable marker.

14. The carrier according to claim 12 or 13, wherein the carrier further comprises one or more of the following: i. Enhancer; ii. Plant selectable markers; iii. Multiple cloning sites; and iv. Recombination sites.

15. A composition for transforming plant cells, the composition comprising a polynucleotide according to any one of claims 1 to 11 and / or a carrier according to any one of claims 12 to 14, preferably, wherein the composition comprises microparticles complexed with the polynucleotide and / or the carrier.

16. A cell comprising a polynucleotide according to any one of claims 1 to 11, or comprising a vector according to any one of claims 12 to 14.

17. The cell according to claim 16, wherein the cell is a bacterial cell, preferably, wherein the bacterial cell is an Escherichia coli cell, an Agrobacterium species cell, or preferably an Agrobacterium tumefaciens cell.

18. The cell according to claim 16, wherein the cell is a C3 plant cell or a C4 plant cell, preferably, wherein the cell is a plant leaf epidermal cell and / or a cell capable of photosynthesis, preferably, wherein the cell is a stomatal cell, more preferably, wherein the cell is a stomatal progenitor cell.

19. A plant or a part thereof, comprising: i. The cell according to claim 18; or ii. A cell containing a polynucleotide, said polynucleotide comprising a promoter element operatively linked to a nucleotide sequence encoding a water-forming oxidase.

20. The plant of claim 19, wherein the plant is a transgenic plant with enhanced growth compared to a control unmodified plant grown under the same conditions, preferably wherein the plant is a C3 or C4 plant, preferably wherein the conditions are water-deficient conditions.

21. The plant according to claim 19 or 20, wherein the plant is selected from the C3 group of plants: soybean (Glycine max), cotton (Gossypium hirsutum), rapeseed / canola rape (B. napus subsp. Napus), potato (Solanum tuberosum), tomato (Solanum lycopersicum), wheat (Triticumaestivum), barley (Hordeum vulgare), oats (Avena sativa), rice (Oryza sativa), rye (Secale cereal), pigeon bean (Cajanus cajan), cowpea (Vigna unguiculata), pea (Pisumsativum), sugar beet (Beta vulgaris), cassava (Manihot esculenta), sweet potato (Ipomoea batatas), yam (Dioscorea spp.), peanut (Arachis hypogaea), sunflower (Helianthus annuus), flax (Linum Plants including, but not limited to, common beans (Phaseolus vulgaris), lima beans (Phaseolus lunatus), mung beans (Phaseolus mung), red beans (Phaseolus angularis), chickpeas (Cicerarietinum), buckwheat (Fagopyrum esculentum), tobacco (Nicotiana tabacum), hemp (Cannabissativa), oil palm (Elaeis guineensis), American sycamore (Acer pseudoplatanus), sweet chestnut (Castaneasativa), poplar (Populus spp.), eucalyptus (Sucalyptus spp.), plantain / banana (Musa acuminate), or rubber (Hevea brasiliensis), or plants of the C4 species selected from maize (Zea mays), sorghum (Sorghum bicolor), sugarcane, millet, or thrush.

22. A method for increasing the photosynthetic capacity of a plant, preferably, wherein the plant is a C3 or C4 plant, the method comprising modifying the heritable genetic material of the plant such that a water-forming oxidase is expressed in at least one mesophyll cell of the plant, thereby increasing the photosynthetic capacity of the plant.

23. A method for: (i) Reduce the stomatal density of plants; and / or (ii) Reduce the stomatal conductance of plants; and / or (iii) Increase the water retention capacity of plants, Preferably, the plant is a C3 or C4 plant, and the method includes modifying the heritable genetic material of the plant to express a water-forming oxidase in at least one cell of the plant leaf epidermis. Preferably, the cell is a stomatal cell, and more preferably, the cell is a stomatal progenitor cell.

24. The method according to claim 22 or 23, wherein the water-forming oxidase is NADPH water-forming oxidase, NADH water-forming oxidase or bifunctional NAD(P)H water-forming oxidase.

25. The method according to any one of claims 22 to 24, wherein the modification comprises transforming the plant with a substance: i. The polynucleotide according to any one of claims 1 to 11; ii. The carrier according to any one of claims 12 to 14; or iii. A polynucleotide comprising a promoter element operatively linked to a nucleotide sequence encoding a water-forming oxidase.

26. The method according to any one of claims 22 to 25, wherein the method comprises growing the plant under water-deficient conditions.

27. A plant part, plant tissue, plant organ, plant cell, plant protoplast, embryo, callus culture, pollen grain or seed derived from a plant according to any one of claims 19 to 21 or from a plant produced by the method according to any one of claims 22 to 26, optionally wherein the plant part comprises a nucleotide sequence corresponding to a water-forming oxidase as defined in any one of claims 1 to 11.

28. The use of a water-forming oxidase for: (i) Reduce the stomatal density of plants; and / or (ii) Reduce the stomatal conductance of plants; and / or (iii) Increase the water retention capacity of plants, Preferably, the use comprises cultivating plants containing a nucleotide sequence corresponding to a water-forming oxidase as defined in any one of claims 1 to 11.

29. The use according to claim 28, wherein the plant is a transgenic plant that, compared with a control unmodified plant grown under the same conditions, has: (i) Reduced pore density; and / or (ii) Reduced porosity; and / or (iii) Increased water retention Preferably, the plant is a C3 or C4 plant, and preferably, the condition is a water-deficient condition.

Images