Transgenic plants with increased photosynthetic efficiency and growth

Abstract

The present disclosure provides a transgenic plant comprising one or more nucleotide sequences encoding a polypeptide selected from the group consisting of photosynthetic system II subunit S (PsbS), zeaxanthin epoxidase (ZEP), and violaxanthin deepoxidase (VDE), which nucleotide sequences are operably linked to at least one expression control sequence. Also provided are expression vectors for making transgenic plants, as well as methods for increasing biomass production and / or carbon fixation and / or growth in plants, comprising increasing expression of at least one of PsbS, ZEP and VDE polypeptides.

Description

[0001] This application is a divisional application of Chinese patent application No. 201780045867.0, entitled “Transgenic plants with increased photosynthetic efficiency and growth”, filed on May 26, 2017.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 342,248, filed May 27, 2016, which is hereby incorporated by reference in its entirety.

[0004] Submit a sequence listing as an ASCII text file

[0005] The following content, submitted as an ASCII text file, is incorporated herein by reference in its entirety: Computer Readable Form (CRF) of the Sequence Listing (File Name: 335032000840SEQLIST.txt, Record Date: May 26, 2017, Size: 246KB). Technical Field

[0006] The present invention relates to a method for increasing the photosynthetic efficiency and growth of plants. Background Art

[0007] Light intensity in plant canopies is very dynamic, and leaves often experience dramatic fluctuations in absorbed irradiance levels. When light intensity is too high or increases too rapidly to allow photochemistry to utilize the absorbed energy, several photoprotective mechanisms are induced to protect the photosynthetic antenna complexes from overexcitation. Excess excitation energy in the photosystem II (PSII) antenna complexes is harmlessly dissipated as heat by an inducible protective process that is observable and generally referred to as non-photochemical quenching of chlorophyll fluorescence (NPQ; et al., Plant Physiol. Vol. 125, 1558-1566, 2000). NPQ changes can be fast but not instantaneous, and therefore lag behind fluctuations in absorbed irradiance. The rate of NPQ relaxation is much slower than the induction rate, and this asymmetry is exacerbated by prolonged or repeated exposure to excessive light conditions. This relatively slow recovery rate of the PSII antenna from the quenched state to the non-quenched state may mean that the photosynthetic quantum yield and the associated CO2 fixation are instantaneously limited by NPQ when changing from high light intensity to low light intensity. When this hypothesis was tested in model simulations and integrated on the crop canopy, the corresponding loss of CO2 fixation was estimated to be in the range of 7.5%-30% (Zhu et al., J. Exp. Bot. Vol. 55, 1167-1175, 2004). Based on these calculations, increasing the relaxation rate of NPQ has proposed a possible strategy to improve photosynthetic efficiency, but experimental evidence is lacking to date.

[0008] Although the exact site of NPQ quenching and the nature of the quenching mechanisms involved remain to be elucidated, it is clear that in order for NPQ to occur, the PSII-associated antenna needs to undergo a conformational change to a quenched state, which can be induced by a number of different mechanisms with contrasting time constants. The major and ubiquitous mechanism of NPQ in higher plants is the so-called energy-dependent quenching (qE). The induction of qE requires low thylakoid lumen pH and is greatly aided by the presence of photosystem II subunit S (PsbS) and the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin via the reversible xanthophyll pigment cycle.

[0009] Overexpression of PsbS strongly affects the amplitude of qE formation and leads to increased induction and relaxation rates of qE, but can compete with photosynthetic quantum yield under less stressful conditions. Therefore, while qE enhancement via PsbS overexpression can provide increased photoprotection under conditions of strong light or rapid fluctuations, the positive effects of PsbS overexpression alone on CO2 fixation and plant growth will greatly depend on the prevailing light environment. Another NPQ manipulation pathway is to modify the reversible xanthophyll pigment cycle. A schematic diagram of the pathway for the biosynthesis of carotenoids (carotenes and luteins) from lycopene is shown in Fig.17 . Zeaxanthin accumulation is associated with several NPQ components (qE, qZ, and qI). The conversion of violaxanthin to zeaxanthin under excess light is catalyzed by the enzyme violaxanthin de-epoxidase (VDE). The conversion of zeaxanthin to violaxanthin is catalyzed by the enzyme zeaxanthin epoxidase (ZEP). Arabidopsis mutants with increased xanthophyll pigment pool size were shown to have slower NPQ formation and relaxation rates, while the amplitude of NPQ was unaffected. Interestingly, the rates of NPQ formation and relaxation in these mutants and wild-type control plants appear to be primarily controlled by the de-epoxidation state of the xanthophyll pigment pool. Nilkens et al., Biochimica et Biophysica Acta 1797;466–475 (2010) showed that, specifically, the kinetics of zeaxanthin epoxidation are strongly correlated with the NPQ relaxation rate. Therefore, the rate of regulation of the xanthophyll cycle balance also controls the rate of NPQ formation and relaxation and appears to be influenced by the size of the xanthophyll pool in terms of the rate of conversion by violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP).

[0010] It is not known whether NPQ can be manipulated to exploit photosynthetic quantum yield to reduce instantaneous competition at low light intensities while maintaining photoprotection at high light intensities. Plants with improved quantum yield and CO2 fixation under fluctuating light conditions could provide improved plant growth and crop yields. Summary of the invention

[0011] One aspect of the present disclosure relates to a transgenic plant having one or more heterologous nucleotide sequences encoding PsbS, ZEP and / or VDE. In some embodiments, the nucleotide sequence is derived from a dicot. In some embodiments, the nucleotide sequence is derived from Arabidopsis thaliana. In some embodiments, the transgenic plant has one or more heterologous nucleotide sequences encoding PsbS, ZEP and VDE. In some embodiments, PsbS is encoded by the nucleotide sequence of SEQ ID NO: 1. In some embodiments, ZEP is encoded by the nucleotide sequence of SEQ ID NO: 2. In some embodiments, VDE is encoded by the nucleotide sequence of SEQ ID NO: 3. In some embodiments, PsbS is encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 1. In some embodiments, ZEP is encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 2. In some embodiments, VDE is encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 3. In some embodiments, PsbS is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:1. In some embodiments, ZEP is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:2. In some embodiments, VDE is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:3. In some embodiments, PsbS has an amino acid sequence of SEQ ID NO:4. In some embodiments, ZEP has an amino acid sequence of SEQ ID NO:5. In some embodiments, VDE has an amino acid sequence of SEQ ID NO:6. In some embodiments, PsbS has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4. In some embodiments, ZEP has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5. In some embodiments, VDE has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:6. In some embodiments, PsbS has an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4. In some embodiments, the ZEP has an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 5. In some embodiments, the VDE has an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6. In some embodiments, the PsbS further comprises the conserved domain of SEQ ID NO: 7. In some embodiments, the ZEP further comprises the conserved domain of SEQ ID NO: 8.In some embodiments, the VDE further comprises a conserved domain of SEQ ID NO: 9. In some embodiments of the foregoing, the plant is a crop plant, a model plant, a monocotyledon, a dicotyledon, a plant with crassulacean acid metabolism (CAM) photosynthesis, a plant with C3 photosynthesis, a plant with C4 photosynthesis, an annual plant, a greenhouse plant, a horticultural flowering plant, a perennial plant, a switchgrass plant, a corn plant, a biomass plant, or a sugarcane plant. In some embodiments of the above, the plant is switchgrass, miscanthus, alfalfa, sweet sorghum, grain sorghum, sugarcane, energy sugarcane, elephant grass, corn, cassava, cowpea, wheat, barley, oats, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, camelina, Brassicanapus, Brassica carinata, Brassica juncea, pearl millet, foxtail millet, other cereals, rice, oilseeds, vegetable crops, forage crops, industrial crops, woody crops, or biomass crops. In some embodiments, the plant is tobacco (Nicotiana tabacum). In some embodiments, the plant is corn (Zea mays). In some embodiments, the plant is rice (Oryza sativa). In some embodiments, the plant is sorghum (Sorghum bicolor). In some embodiments, the plant is soybean (Glycinemax). In some embodiments, the plant is cowpea (Vigna unguiculata). In some embodiments, the plant is a species of Populus (Populus spp). In some embodiments, the plant is a species of Eucalyptus (Eucalyptus spp). In some embodiments, the plant is cassava (Manihot esculenta). In some embodiments, the plant is barley (Hordeum vulgare). In some embodiments, the plant is potato (Solanum tuberosum). In some embodiments, the plant is a species of Saccharum (Saccharum spp). In some embodiments, the plant is Medicago sativa (Medicago sativa). In some embodiments above, the plant has increased growth under fluctuating light conditions compared to control plants under fluctuating light conditions. In some embodiments above, the plant has increased photosynthetic efficiency under fluctuating light conditions compared to control plants under fluctuating light conditions. In some embodiments above, the plant has enhanced light protection efficiency under fluctuating light conditions compared to control plants under fluctuating light conditions.In some embodiments of the above, the plant has increased quantum yield and CO2 fixation under fluctuating light conditions compared to control plants under fluctuating light conditions. In some embodiments of the above, the plant is an elite line or strain. In some embodiments of the above, the plant further comprises expression of at least one additional polypeptide that provides herbicide resistance, insect or pest resistance, disease resistance, modified fatty acid metabolism and / or modified carbohydrate metabolism.

[0012] Another aspect of the present disclosure relates to an expression vector having one or more heterologous nucleotide sequences encoding PsbS, ZEP and / or VDE. In some embodiments, the vector contains promoters Rbcs1A, GAPA-1 or FBA2. In some embodiments, the Rbcs1A promoter drives the expression of ZEP, the GAPA-1 promoter drives the expression of PsbS, and the FBA2 promoter drives the expression of VDE. In some embodiments, the vector is T-DNA. In some embodiments, the vector has a nucleotide sequence encoding a polypeptide that provides antibiotic resistance. In some embodiments, the vector has a left border (LB) domain and a right border (RB) domain flanking the expression control sequence and the nucleotide sequence encoding the PsbS, ZEP and VDE polypeptides. In some embodiments, PsbS is encoded by the nucleotide sequence of SEQ ID NO: 1. In some embodiments, ZEP is encoded by the nucleotide sequence of SEQ ID NO: 2. In some embodiments, VDE is encoded by the nucleotide sequence of SEQ ID NO: 3. In some embodiments, PsbS is encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:1. In some embodiments, ZEP is encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:2. In some embodiments, VDE is encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:3. In some embodiments, PsbS is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:1. In some embodiments, ZEP is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:2. In some embodiments, VDE is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:3. In some embodiments, PsbS has the amino acid sequence of SEQ ID NO:4. In some embodiments, ZEP has the amino acid sequence of SEQ ID NO:5. In some embodiments, VDE has the amino acid sequence of SEQ ID NO:6. In some embodiments, PsbS has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4. In some embodiments, the ZEP has an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, the VDE has an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the PsbS has an amino acid sequence that has at least 70% sequence identity to SEQ ID NO: 4. In some embodiments, the ZEP has an amino acid sequence that has at least 70% sequence identity to SEQ ID NO: 5.In some embodiments, the VDE has an amino acid sequence that has at least 70% sequence identity to SEQ ID NO:6.

[0013] In some embodiments, PsbS further comprises the conserved domain of SEQ ID NO:7. In some embodiments, ZEP further comprises the conserved domain of SEQ ID NO:8. In some embodiments, VDE further comprises the conserved domain of SEQ ID NO:9. In some embodiments, the expression vector is in a bacterial cell. In some embodiments of the above, the expression vector is in an Agrobacterium cell. In some embodiments of the above, the expression vector is used to produce a transgenic plant. In some embodiments of the above, the transgenic plant produces seeds. In some embodiments of the above, the seeds also produce progeny plants.

[0014] Other aspects of the disclosure relate to methods of increasing photosynthesis and growth of plants, the methods comprising increasing the expression of two or more polypeptides described herein in the plants. In one aspect, the disclosure relates to a method of increasing the growth of plants under fluctuating light conditions, comprising increasing the expression of at least two polypeptides from PsbS, ZEP and VDE in the plants, thereby producing plants having increased expression of the two or more polypeptides compared to control plants. In one aspect, the disclosure relates to a method of increasing the photosynthetic efficiency of plants under fluctuating light conditions, comprising increasing the expression of at least two polypeptides from PsbS, ZEP and VDE in the plants, thereby producing plants having increased expression of the two or more polypeptides compared to control plants. In one aspect, the disclosure relates to a method of increasing the photoprotection efficiency of plants under fluctuating light conditions, comprising increasing the expression of at least two polypeptides from PsbS, ZEP and VDE in the plants, thereby producing plants having increased expression of the two or more polypeptides compared to control plants. In one aspect, the present disclosure relates to a method of increasing the quantum yield and CO2 of a plant under fluctuating light conditions, comprising increasing the expression of at least two polypeptides from PsbS, ZEP and VDE in a plant, thereby producing a plant having increased expression of the two or more polypeptides compared to a control plant. In one aspect, the present invention relates to a method of increasing the relaxation rate of non-photochemical quenching (NPQ) in a plant, comprising increasing the expression of at least two polypeptides from PsbS, ZEP and VDE in a plant, thereby producing a plant having increased expression of the two or more polypeptides compared to a control plant. In some embodiments, the expression of PsbS and ZEP is increased. In some embodiments, the expression of PsbS and VDE is increased. In some embodiments, the expression of VDE and ZEP is increased. In some embodiments, the expression of PsbS, ZEP and VDE is increased. In some embodiments, the expression of PsbS, ZEP and VDE is increased. In some embodiments, the expression of PsbS, ZEP and VDE is increased. In some embodiments, the expression of PsbS, ZEP and VDE is increased. In some embodiments, the expression of PsbS, ZEP and / or VDE is increased by expressing one or more heterologous nucleotide sequences encoding PsbS, ZEP and / or VDE. In some embodiments, the expression of PsbS, ZEP and / or VDE is increased by modifying the promoter region of PsbS, ZEP and / or VDE. In some embodiments, promoter modification is achieved by a genome editing system. In some embodiments, the genome editing system is CRISPR.

[0015] Another aspect of the present disclosure relates to a method for selecting plants with improved growth characteristics under fluctuating light conditions, comprising the following steps: providing a plant population; modifying the plant population to increase the activity of any of PsbS, ZEP and VDE; detecting the level of non-photochemical quenching (NPQ) in plants under fluctuating light conditions; comparing the NPQ level in plants under fluctuating light conditions with the control NPQ level under fluctuating light conditions; and selecting plants with increased NPQ relaxation rates when the plants are converted from high light intensity to low light intensity. In some embodiments, the control NPQ level is the lowest NPQ level in the population. In some embodiments, the control NPQ level is the median NPQ level in the population. In some embodiments, the control NPQ level is the average NPQ level in the population. In some embodiments, the control NPQ level is the NPQ level in the control plant. In some embodiments, the plant is modified by inducing one or more mutations in PsbS, ZEP and / or VDE with a mutagen. In some embodiments, the mutagen is ethyl methanesulfonate (EMS). In some embodiments, plants are modified by introducing heterologous PsbS, ZEP and / VDE using transgenic technology. In some embodiments, plants are modified by modifying the corresponding natural promoters of PsbS, ZEP and / VDE using a genome editing system. In some embodiments, the genome editing system is CRISPR. Another aspect of the present disclosure relates to a method for screening nucleotide sequence polymorphisms associated with improved growth characteristics under fluctuating light conditions, comprising the following steps: providing a plant population; obtaining a nucleotide sequence that regulates and / or encodes any of PsbS, ZEP and VDE in the plant population; obtaining one or more polymorphisms in a nucleotide sequence that regulates and / or encodes any of PsbS, ZEP and VDE in the plant population; detecting the relaxation rate of non-photochemical quenching (NPQ) of the plant population when it is converted from high light intensity to low light intensity; performing statistical analysis to determine the association between the polymorphism in the plant population and the NPQ relaxation rate; and selecting a polymorphism that has a statistically significant correlation with the NPQ relaxation rate. In some embodiments, the polymorphism is a single nucleotide polymorphism (SNP). In some embodiments, the polymorphism is located in the promoter of PsbS, ZEP and / or VDE. In some embodiments, the polymorphism is detected by sequencing. In some embodiments, the polymorphism is detected by gel electrophoresis. In some embodiments, the polymorphism is also used to screen plant populations to select plants with improved growth characteristics under fluctuating light conditions. In some embodiments, the polymorphism is also used as a target for genome editing in PsbS, ZEP and / or VDE to improve the growth characteristics of plants under fluctuating light conditions.In some embodiments of the above, the improved growth characteristic is improved growth, improved photosynthetic efficiency, improved photoprotection efficiency, improved quantum yield and / or improved CO2 fixation. In some embodiments of the above, NPQ in plants is detected by measuring chlorophyll fluorescence.

[0016] In some of the above embodiments, the improved growth characteristic is improved growth. In some embodiments, the improved growth characteristic is improved photosynthetic efficiency. In some embodiments, the improved growth characteristic is improved light protection efficiency. In some embodiments, the improved growth characteristic is improved quantum yield and CO2 fixation. In some embodiments, the improved growth characteristic is increased non-photochemical quenching (NPQ) relaxation rate. In some embodiments, NPQ is detected using chlorophyll fluorescence imaging.

[0017] In some embodiments that can be combined with any of the preceding embodiments, PsbS is encoded by the nucleotide sequence of SEQ ID NO:1. In some embodiments that can be combined with any of the preceding embodiments, ZEP is encoded by the nucleotide sequence of SEQ ID NO:2. In some embodiments that can be combined with any of the preceding embodiments, VDE is encoded by the nucleotide sequence of SEQ ID NO:3. In some embodiments that can be combined with any of the preceding embodiments, PsbS is encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:1. In some embodiments that can be combined with any of the preceding embodiments, ZEP is encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:2. In some embodiments that can be combined with any of the preceding embodiments, VDE is encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:3. In some embodiments that can be combined with any of the preceding embodiments, PsbS is encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO:1. In some embodiments that can be combined with any of the preceding embodiments, ZEP is encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO:2. In some embodiments that can be combined with any of the preceding embodiments, the VDE is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:3. In some embodiments that can be combined with any of the preceding embodiments, the PsbS has SEQ ID NO:4. In some embodiments that can be combined with any of the preceding embodiments, the ZEP has SEQ ID NO:5. In some embodiments that can be combined with any of the preceding embodiments, the VDE has an amino acid sequence of SEQ ID NO:6. In some embodiments that can be combined with any of the preceding embodiments, the PsbS has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4. In some embodiments that can be combined with any of the preceding embodiments, the ZEP has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5. In some embodiments that can be combined with any of the preceding embodiments, the VDE has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:6. In some embodiments that can be combined with any of the preceding embodiments, the PsbS has an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4. In some embodiments that may be combined with any of the preceding embodiments, the ZEP has an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 5. In some embodiments that may be combined with any of the preceding embodiments, the VDE has an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6.In some embodiments that may be combined with any of the preceding embodiments, the PsbS further comprises the conserved domain of SEQ ID NO: 7. In some embodiments that may be combined with any of the preceding embodiments, the ZEP further comprises the conserved domain of SEQ ID NO: 8. In some embodiments that may be combined with any of the preceding embodiments, the VDE further comprises the conserved domain of SEQ ID NO: 9. In some embodiments that may be combined with any of the preceding embodiments, the plant is a crop plant, a model plant, a monocot, a dicot, a plant with crassulacean acid metabolism (CAM) photosynthesis, a plant with C3 photosynthesis, a plant with C4 photosynthesis, an annual plant, a greenhouse plant, a horticultural flowering plant, a perennial plant, a switchgrass plant, a corn plant, a biomass plant, or a sugarcane plant. In some embodiments that can be combined with any of the preceding embodiments, the plant is switchgrass, Miscanthus, alfalfa, sweet sorghum, sorghum, sugarcane, energy sugarcane, elephant grass, corn, wheat, barley, oats, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, camelina (Camelina), Brassica napus, Ethiopian mustard (Brassica carinata), mustard, pearl millet, millet, other cereals, rice, oilseeds, vegetable crops, fodder crops, industrial crops, woody crops or biomass crops. In some embodiments that can be combined with any of the preceding embodiments, the plant is tobacco (Nicotiana tabacum). In some embodiments that can be combined with any of the preceding embodiments, the plant is corn (Zea mays). In some embodiments that can be combined with any of the preceding embodiments, the plant is rice (Oryza sativa). In some embodiments that can be combined with any of the preceding embodiments, the plant is sorghum (Sorghum bicolor). In some embodiments that can be combined with any of the preceding embodiments, the plant is soybean (Glycine max). In some embodiments that may be combined with any of the preceding embodiments, the plant is cowpea (Vigna unguiculata). In some embodiments that may be combined with any of the preceding embodiments, the plant is a Populus species (Populus spp). In some embodiments that may be combined with any of the preceding embodiments, the plant is a Eucalyptus species (Eucalyptus spp). In some embodiments that may be combined with any of the preceding embodiments, the plant is cassava (Manihot esculenta). In some embodiments that may be combined with any of the preceding embodiments, the plant is barley (Hordeum vulgare).In some embodiments that may be combined with any of the preceding embodiments, the plant is Solanum tuberosum. In some embodiments that may be combined with any of the preceding embodiments, the plant is Saccharum spp. In some embodiments that may be combined with any of the preceding embodiments, the plant is Medicago sativa.

[0018] In some embodiments that may be combined with any of the preceding embodiments, the transcript level of VDE in the plant is increased by 3 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the transcript level of PsbS in the plant is increased by 3 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the transcript level of ZEP in the plant is increased by 8 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the transcript level of VDE in the plant is increased by 10 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the transcript level of PsbS in the plant is increased by 3 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the transcript level of ZEP in the plant is increased by 6 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the transcript level of VDE in the plant is increased by 4 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the transcript level of PsbS in the plant is increased by 1.2 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the transcript level of ZEP in the plant is increased by 7 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the protein level of VDE in the plant is increased by 16 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the protein level of PsbS in the plant is increased by 2 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the protein level of ZEP in the plant is increased by 80 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the protein level of VDE in the plant is increased by 30 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the protein level of PsbS in the plant is increased by 4 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the protein level of ZEP in the plant is increased by 74 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the protein level of VDE in the plant is increased by 47 times compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the protein level of PsbS in the plant is increased 3-fold compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the protein level of ZEP in the plant is increased 75-fold compared to a control plant. In some embodiments that may be combined with any of the preceding embodiments, the increase in transcript levels in the plant has a ratio of 3:3:8, 10:3:6, or 4:1.2:7 between VDE, PsbS, and ZEP compared to a control plant.In some embodiments that may be combined with any of the preceding embodiments, the increase in protein levels in the plant compared to a control plant has a ratio of 16:2:80, 30:4:74, or 47:3:75 between VDE, PsbS, and ZEP. In some embodiments that may be combined with any of the preceding embodiments, the increase in transcript levels of VDE in the plant compared to a control plant is in the range of 3-fold to 10-fold. In some embodiments that may be combined with any of the preceding embodiments, the increase in transcript levels of PsbS in the plant compared to a control plant is about 1.2-fold to about 3-fold. In some embodiments that may be combined with any of the preceding embodiments, the increase in transcript levels of ZEP in the plant compared to a control plant is about 6-fold to about 8-fold. In some embodiments that may be combined with any of the preceding embodiments, the increase in protein levels of VDE in the plant compared to a control plant is in the range of 16-fold to 47-fold. In some embodiments that may be combined with any of the preceding embodiments, the increase in protein levels of PsbS in the plant compared to a control plant is about 2-fold to about 4-fold. In some embodiments that may be combined with any of the preceding embodiments, the increase in protein level of ZEP in the plant is about 74-fold to about 80-fold compared to control plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Features, objects and advantages other than those described above will become more apparent when considering the following detailed description. Such detailed description refers to the following drawings, in which:

[0020] Figure 1 .(A) When 1000 μmol m -2 s -1 Non-photochemical quenching in seedlings of wild-type and three (VDE-PsbS-ZEP) VPZ overexpressing lines during 10 min PFD irradiation followed by 10 min dark relaxation. -2 s -1 PFD was irradiated for 3 min and then treated with 200 μmol m -2 s -1 (B) Non-photochemical quenching and (C) PSII efficiency in seedlings during repeated cycles of 2 min PFD illumination. Error bars indicate ± se (n=18), asterisks indicate significant differences between VPZ lines and wild type (α=0.05).

[0021] Figure 2. mRNA and protein expression of native (Nt) and transgenic (At) violaxanthin de-epoxidase (VDE), photosystem II subunit S (PsbS), and zeaxanthin epoxidase (ZEP). (AC) mRNA levels relative to actin and tubulin; (DF) protein levels relative to wild type as determined by densitometry on western blots, error bars indicate ± se (n = 5), asterisks indicate significant differences between the VPZ line and wild type (α = 0.05); (G) Examples of western blots of VDE, PsbS, and ZEP.

[0022] Figure 3 Non-photochemical quenching of gas exchange as a function of absorbed light intensity in fully expanded leaves of wild-type and VPZ-overexpressing lines. Light intensity was either (A) increased from low to high PFD while waiting for steady state at each step or (B) treated with 2000 μmol m for 4 min before each light intensity change. -2 s -1 Shift from high to low PFD. Error bars indicate ± se (n=6), asterisks indicate significant differences between VPZ strains and wild type (α=0.05).

[0023] Figure 4 (AB) Linear electron transfer (J) and net assimilation rate (An) as a function of light intensity, and the initial slope of the corresponding parameter fit (CD). Before each light intensity change, 2000 μmol m -2 s -1 PFD changes light intensity from high PFD to low PFD. Error bars indicate ± se (n=6), asterisks indicate significant differences between VPZ strains and wild type (α=0.05).

[0024] Figure 5 After exposure to 2000 μmol m -2 s -1 PFD(λ 最大 =470 nm) Photoprotection index in seedlings of VPZ overexpressing lines and wild type after (A) one hour or (B) two hours. Index values ​​less than 1 indicate the occurrence of photoinhibition. (C) Photoprotection index in seedlings of VPZ overexpressing lines and wild type after exposure to 2000 μmol m -2 s -1 Plot of PSII efficiency as a function of residual NPQ in seedlings after one hour (upward triangles - VPZ, circles - WT) or two hours (downward triangles - VPZ, squares - WT) and subsequent dark relaxation of 10 min. Error bars indicate ± se (n = 18), asterisks indicate significant differences between VPZ lines and wild type (α = 0.05).

[0025] Figure 6Final plant size and weight relative to WT in greenhouse experiments. (A) Total dry weight per plant, (B) leaf area per plant, (C) plant height, (D) leaf dry weight per plant, (E) stem dry weight per plant, (F) root dry weight per plant. Error bars indicate ± SE (n = 20 and n = 19 for Experiment 1 and Experiment 2), and asterisks indicate significant differences between VPZ lines and wild type (α = 0.05).

[0026] Figure 7 Linear electron transfer and net assimilation rates as a function of light intensity. Before each change in light intensity, 2000 μmol m -2 s -1 PFD changes light intensity from high PFD to low PFD. (AB) Linear electron transfer (J) and net assimilation rate (An), and corresponding parameter fits initial slope (CD), convexity (EF) and asymptote (GH). Error bars indicate ± se (n=6).

[0027] Figure 8 Convexity (AB) and asymptote (CD) parameters were fitted to the linear electron transfer (J) and net assimilation rate (An) as a function of light intensity. Before each light intensity change, 2000 μmol m -2 s -1 PFD varies light intensity from high PFD to low PFD. Error bars indicate ± se (n=6).

[0028] Fig. 9 .Plasmid map of the VDE-PsbS-ZEP construct.

[0029] Fig.10 The intended goal of increasing the speed of photoprotection in response to changes in light intensity and the roles of VDE, PsbS and ZEP in this process. The blue line represents the transgenic plants compared to the orange line (wild type).

[0030] Fig.11 Rapid high-throughput screening of phenotypes. Chlorophyll fluorescence of leaf discs (left) and seedlings (right).

[0031] Fig.12 Growth experiments comparing VPZ-23 and VPZ-34 transgenic plants with wild-type plants.

[0032] Fig.13 Greenhouse experiments showed significantly increased growth in all lines.

[0033] Fig.14 Leaves were previously exposed to 2000 μmol m -2 s -1Quantum yield and CO2 fixation results at various light intensities after PFD.

[0034] Fig.15 .NPQ dynamics under fluctuating illumination.

[0035] Fig.16 .Time constant of NPQ in the first induction / relaxation.

[0036] Fig.17 NPQ components (qE, qZ, and qI) associated with the conversion of violaxanthin to zeaxanthin

[0037] Fig.18 .Transient overexpression of NPQ-related genes in Nicotiana benthamiana. The upper left figure shows the transient overexpression of NPQ-related genes in Nicotiana benthamiana at 600 μmol photons m -2 s -1 NPQ measurements on leaf spots overexpressing FLAG-tagged PsbS, VDE, ZEP and GUS (as negative control) during 13 min of illumination (white bars) followed by 10 min of darkness (black bars). Error bars indicate standard deviation (n=6). The upper right panel shows a false color image of NPQ of leaves expressing PSBS, VDE, ZEP and GUS after 10 min of high light exposure. The rainbow bar indicates the relative amount of NPQ. The lower panel shows an immunoblot analysis of tissue collected from the leaves in the upper right panel and probed with anti-FLAG.

[0038] Fig.19 . Transient co-overexpression of VDE and ZEP in N. benthamiana accelerates NPQ induction and relaxation. Error bars represent standard error (n=4).

[0039] Fig. 20 NPQ kinetics of transgenic T1 progeny. NPQ measurements using DUAL PAM were performed at 600 μmol photons m -2 s -1 During 10 min of illumination followed by 10 min of darkness, performed on the youngest fully developed leaves of T1 adults of three different lines: a wild-type segregant (Null), a ZEP overexpressing (ZEP), and a ZEP and VDE overexpressing (ZEP-VDE). Each curve corresponds to the mean NPQ measurements of three different plants; error bars indicate standard error (n=3).

[0040] Fig.21 .Photosystem II quantum yield (YII) of stable transgenic T1 plants of tobacco cv. Petite Havana.

[0041] Fig. 22.Growth experiment in greenhouse. Four groups of plants are shown, one for each transgenic line. Each group contains 36 plants.

[0042] Fig.23 .Transient overexpression of NbPsbS and Rbcs1aAtZEP+Gapa1AtPsbS+Fba2AtVDE constructs in N. benthamiana.

[0043] FIG24 . Amino acid alignment of NPQ genes for (A) PsbS, (B) ZEP, and (C) VDE in representative plant species.

[0044] Fig.25 mRNA and protein levels of VDE, PsbS and ZEP in leaves of transgenic tobacco plants grown under greenhouse conditions. (A, C and E) mRNA levels relative to actin and tubulin. (B, D and F) Protein levels relative to wild type (WT) as determined by densitometry on immunoblots. Error bars indicate standard error of measurement (SEM) (n=5 biological replicates) and asterisks indicate significant differences between VPZ lines and WT (α=0.05). (G) Representative immunoblots of VDE, PsbS and ZEP.

[0045] Fig.26 mRNA and protein levels of VDE, PsbS and ZEP in leaves of transgenic tobacco plants grown under field conditions. (A, C, E) mRNA levels relative to actin and tubulin. (B, D, F) Protein levels relative to wild type (WT) as determined by densitometry on immunoblots. Error bars indicate SEM (n=4) and asterisks indicate significant differences (α=0.05) between VPZ lines and WT.

[0046] Fig. 27 NPQ relaxation kinetics of transgenic cowpea 1643B1. The X-axis is time in seconds. The Y-axis is normalized NPQ.

[0047] Fig.28 .NPQ induction and relaxation kinetics in transgenic cowpea 1643B1. X is time. Y axis is NPQ divided by 4.

[0048] Fig.29 Normalized NPQ induction and relaxation kinetics of transgenic cowpea 1643B1 under fluctuating light. X is time. Y axis is NPQ divided by 4. Data are normalized to the highest NPQ within each group.

[0049] Fig.30 . NPQ induction and relaxation kinetics in transgenic cowpea CP472A. X is time. Y axis is NPQ divided by 4.

[0050] Fig.31 .NPQ induction and relaxation kinetics in nine transgenic rice lines.

[0051] Fig.32 .Average NPQ induction and relaxation kinetics in transgenic rice.

[0052] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. DETAILED DESCRIPTION

[0053] Plants, vectors, and methods will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the invention are shown. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

[0054] Likewise, those skilled in the art to which the present invention pertains, having benefited from the teachings presented in the previous description and the associated drawings, will appreciate many modifications and other embodiments of the plants, vectors, and methods described herein. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, and that such modifications and other embodiments are also included within the scope of the appended claims. Although specific terms are employed herein, they are used in a general and descriptive sense only and not for limiting purposes.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described herein.

[0056] definition

[0057] Unless otherwise indicated, technical terms are used in accordance with conventional usage. Definitions of common terms in molecular biology can be found in Lewin, Genes VII, 2001 (Oxford University Press), The Encyclopedia of Molecular Biology, Kendrew et al., ed., 1999 (Wiley-Interscience) and Molecular Biology and Biotechnology, a Comprehensive Desk Reference, Robert A. Meyers, ed., 1995 (VCH Publishers. Inc), Current Protocols In Molecular Biology, FM Ausubel et al., ed., 1987 (Green Publishing), Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, 2001.

[0058] The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid", "gene" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment; (one or more) loci defined by linkage analysis; exons; introns; messenger RNA (mRNA); transfer RNA; ribosomal RNA; short interfering RNA (siRNA); short hairpin RNA (shRNA); micro RNA (miRNA); ribozymes; cDNA; recombinant polynucleotides; branched polynucleotides; plasmids; vectors; isolated DNA of any sequence; isolated RNA of any sequence, nucleic acid probes; and primers. A polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if any, may be imparted before or after polymer assembly. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component.

[0059] As used herein, a "vector" is a replicon, such as a plasmid, phage or cosmid, into which another DNA segment can be inserted to bring about the replication of the inserted segment. The vector described herein may be an expression vector.

[0060] As used herein, an "expression vector" is a vector that includes one or more expression control sequences.

[0061] As used herein, an "expression control sequence" or "expression cassette" is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence. The expression control sequence may comprise a heterologous or non-heterologous promoter.

[0062] As used herein, "operably linked" means incorporated into a genetic construct such that the expression control sequence effectively controls the expression of a coding sequence of interest.

[0063] As used herein, "transformed" and "transfected" encompass the introduction of a nucleic acid (eg, a vector) into a cell by a variety of techniques known in the art.

[0064] "Plasmid" is represented by a lower case letter "p" preceded and / or followed by upper case letters and / or numbers.

[0065] As used herein, the term "expression level" refers to the measurable expression level of a given nucleic acid or polypeptide. The expression level of a given nucleic acid or polypeptide is determined by methods well known in the art. The term "differentially expressed" or "differentially expressed" refers to an increase or decrease in the measurable expression level of a given nucleic acid or polypeptide. "Differentially expressed" or "differentially expressed" means a difference of 1-fold or more, up to and including 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or more in the expression level of a given nucleic acid or polypeptide in two samples for comparison. If one of the two samples does not contain detectable expression of a given nucleic acid or polypeptide, then the given nucleic acid or polypeptide is also said to be "differentially expressed" in the two samples.

[0066] Polymorphism refers to a nucleotide sequence variation that may or may not have a functional outcome in the genome. These variations can be developed as genetic markers and used in all aspects of genetic research, including the analysis that genetic differences are associated with the variation of a trait of interest. As used herein, the term "polymorphism" includes but is not limited to single nucleotide polymorphism (SNP), insertion / deletion (InDel), simple sequence repeat (SSR), presence / absence variation (PAV) and copy number variation (CNV). Polymorphism can be naturally occurring or artificially induced. The method for inducing and detecting polymorphism is well known in the art.

[0067] As used herein, the term "promoter" refers to a DNA sequence that can control the expression of a coding sequence or functional RNA. The promoter sequence is composed of a proximal upstream element and a more distal upstream element, and the latter element is generally referred to as an enhancer. An "enhancer" is a DNA sequence that can stimulate promoter activity, and can be an intrinsic element of a promoter or an inserted heterologous element to enhance the level or tissue specificity of a promoter. Promoters can be derived from natural genes as a whole, or can be composed of different elements derived from naturally occurring different promoters, and / or comprise synthetic DNA segments. It will be appreciated by those skilled in the art that different promoters can instruct genes to be expressed in different tissues or cell types, or to be expressed at different developmental stages, or to be expressed in response to different environmental conditions. It should be further recognized that, since the exact boundaries of regulatory sequences have not yet been fully determined in most cases, DNA fragments with some variations can have the same promoter activity. As is well known in the art, promoters can be classified according to their strength and / or the conditions under which they are active, such as constitutive promoters, strong promoters, weak promoters, inducible / repressible promoters, tissue-specific / developmentally regulated promoters, cell cycle-dependent promoters, etc.

[0068] As used herein, the term "genome editing" is a genetic engineering process in which DNA is inserted into, replaced, or removed from a genome using artificially engineered nucleases or "molecular scissors". It is a useful tool for clarifying the function and effect of genes in a sequence-specific manner and making changes within the genome to produce desired phenotypic changes. Genome editing systems include, but are not limited to, large-range nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPRs).

[0069] The term "plant" refers to any of the various photosynthetic eukaryotic multicellular organisms in the plant kingdom, characterized in that they produce embryos, contain chloroplasts, have cellulose cell walls and lack movement. As used herein, "plant" includes any plant or plant part at any stage of development, including seeds, suspension cultures, plant cells, embryos, meristem regions, callus, leaves, roots, buds, gametophytes, sporophytes, pollen, microspores and their offspring. Also included are cuttings, cell or tissue cultures. As used in conjunction with the present disclosure, plant tissues include, for example, whole plants, plant cells, plant organs (e.g., leaves, stems, roots), meristems, plant seeds, protoplasts, callus, cell cultures, and any plant cell groups organized into structural and / or functional units.

[0070] The term "plant" is used in its broadest sense. It includes, but is not limited to, any woody species, ornamental or decorative species, crop or cereal species, fruiting plant species, and algae (e.g., Chlamydomonas reinhardtii). It also refers to a variety of plant cells, most of which are differentiated into structures that are present at any stage of plant development. Such structures include, but are not limited to, fruits, buds, stems, leaves, petals, etc.

[0071] As used herein, the term "control plant" or "wild type" refers to a plant cell, explant, seed, plant component, plant tissue, plant organ or whole plant for comparing with a transgenic or genetically modified plant for identifying an enhanced phenotype or desired trait purpose in a transgenic or genetically modified plant. In some cases, a "control plant" can be a transgenic plant line that comprises an empty vector or marker gene but does not contain the recombinant polynucleotide of interest present in the transgenic or genetically modified plant being assessed. The control plant can be a plant of the same line or variety as the transgenic or genetically modified plant tested, or it can be another line or variety, such as a plant known to have a specific phenotype, feature or known genotype. Suitable control plants will include genetically unaltered or non-transgenic plants of the parental line for producing the transgenic plant herein.

[0072] The term "plant tissue" includes differentiated and undifferentiated tissues of plants, including those present in roots, shoots, leaves, inflorescences, anthers, pollen, ovaries, seeds, and tumors; and cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissues can be in planta in situ, in organ culture, in tissue culture, or in cell culture.

[0073] The term "plant part" as used herein refers to a plant structure, plant organ or plant tissue.

[0074] "Non-naturally occurring plants" refers to plants that do not exist in nature without human intervention. Non-naturally occurring plants include transgenic plants, plants produced by genetic engineering, and plants produced by non-transgenic means (such as traditional or market-assisted plant breeding).

[0075] The term "plant cell" refers to a plant structural and physiological unit including a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell or a cultured cell, or as part of a higher organizational unit such as a plant tissue, a plant organ, or a whole plant. The term "plant cell culture" refers to a culture of plant units such as protoplasts, cells and cell clusters in liquid or solid culture medium, cells in plant tissues and organs, microspores and pollen, pollen tubes, anthers, ovules, embryo sacs, fertilized eggs, and embryos at different stages of development.

[0076] The term "plant material" refers to leaves, stems, roots, inflorescences and flowers or flower parts, fruits, pollen, anthers, egg cells, fertilized eggs, seeds, cuttings, cell or tissue cultures, or any other part of a plant or plant product.

[0077] "Plant organ" refers to a distinct and visible structured and differentiated part of a plant, such as a root, stem, leaf, flower bud, inflorescence, spikelet, floret, seed or embryo.

[0078] The term "crop plants" refers in particular to: monocotyledonous plants, such as cereals (wheat, millet, sorghum, rye, triticale, oats, barley, teff, spelt, buckwheat, fonio and quinoa), rice, corn (maize) and / or sugar cane; or dicotyledonous crops, such as beets (such as sugar beet or fodder beet); fruit (such as pome, stone fruit or soft fruit, for example apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries or blackberries); leguminous plants (such as beans, lentils, peas or soybeans); oil plants (such as Rapeseed, mustard, olive, sunflower, coconut, castor oil plant, cocoa bean or groundnut); Cucurbitaceae (such as zucchini, cucumber or melon); fiber plants (such as cotton, flax or jute); citrus fruits (such as orange, lemon, grapefruit or mandarin); vegetables (such as spinach, lettuce, cabbage, carrot, tomato, potato, gourd or red pepper); Lauraceae (such as avocado, cinnamon or camphor); tobacco; nuts; coffee; tea; vines; hops; durian; bananas; natural rubber plants; and ornamental plants (such as flowers, shrubs, broadleaf trees or evergreen plants such as conifers). This list does not represent any limitation.

[0079] The term "woody crop" or "woody plant" refers to a plant that produces xylem as its structural tissue. Woody crops include trees, shrubs or vines. Examples of woody crops include, but are not limited to, thornless locust, hybrid chestnut, black walnut, Japanese maple, eucalyptus, casuarina, spruce, fir, pine (e.g., Pinus radiata and Pinus caribaea) and Aquilegia multiflora.

[0080] The term "improved growth" or "increased growth" is used herein in its broadest sense. It includes any improvement or enhancement in plant growth and development processes. Examples of improved growth include, but are not limited to, increased photosynthetic efficiency, increased biomass, increased yield, increased seed number, increased seed weight, increased stem height, increased leaf area, and increased plant dry weight,

[0081] "Quantum yield" refers to the number of moles of CO2 fixed per mole of absorbed quanta (photons), or the efficiency with which light is converted into fixed carbon. The quantum yield of photosynthesis is derived from measurements of light intensity and photosynthesis rate. Therefore, quantum yield is a measure of the efficiency with which the absorbed light produces a specific effect. The amount of photosynthesis carried out in a plant cell or plant can be indirectly detected by measuring the amount of starch produced by a transgenic plant or plant cell. The amount of photosynthesis in a plant cell culture or plant can also be detected using a CO2 detector (e.g., a reduction or consumption of CO2 indicates an increase in the level of photosynthesis) or an O2 detector (e.g., an increase in the level of O2 indicates an increase in the level of photosynthesis) (see, e.g., Silva et al., Aquatic Biology 7:127-141, 2009; and Bai et al., Biotechnol. Lett. 33:1675-1681, 2011). Photosynthesis can also be measured using radiolabeled CO2 (e.g., 14CO2 and H 14 CO3-) (see, for example, the methods described in Silva et al., Aquatic Biology 7:127-141, 2009 and references cited therein). Photosynthesis can also be measured by detecting chlorophyll fluorescence (e.g., Silva et al., Aquatic Biology 7:127-141, 2009 and references cited therein). Other methods for detecting photosynthesis in plants are described in Zhang et al., Mol. Biol. Rep. 38:4369-4379, 2011".

[0082] In physical science, the term "relaxation" means the return of a perturbed system to equilibrium, typically from a high energy level to a low energy level. As used herein, the term "non-photochemical quenching relaxation" or "NPQ relaxation" refers to the process by which NPQ levels decrease when transitioning from high to low light intensity.

[0083] References herein to "about" a value or parameter refer to the usual error range for the corresponding value that is readily known to those skilled in the art. References herein to "about" a value or parameter include (and describe) all aspects of the value or parameter itself. For example, a description of "about X" includes a description of "X".

[0084] Overview

[0085] In the face of a rapidly growing world population, further increases in food production are a prerequisite for global political and social stability, and it is therefore estimated that by 2050, crop yields will need to triple to meet this demand. Therefore, a better understanding of the fundamental physiological processes of important crop traits (such as photosynthesis) is the key to improving the world's food security crisis. Photosynthesis is a process used by plants and green algae to convert light energy into chemical energy, which is subsequently released to provide fuel for the activities of the organism, during which atmospheric carbon dioxide (CO2) is assimilated and oxygen is released. The ratio of the amount of fixed or assimilated CO2 to the amount of absorbed photons (quanta), also known as quantum yield, is commonly used as a measure of plant photosynthetic efficiency.

[0086] Although light is necessary for photosynthesis, damage occurs when leaves are exposed to high light intensities. To avoid this, plants have developed several photoprotection mechanisms. Non-photochemical quenching (NPQ) is one of these mechanisms, which allows excess absorbed irradiance to be dissipated as heat. However, when plants are shifted from high to low light intensities, the quantum yield of photosynthesis is temporarily reduced due to the fact that NPQ inhibits CO2 fixation. Furthermore, NPQ turns on quickly (induction) under high light intensities, but turns off more slowly (relaxation) when returning to limited irradiance. Therefore, under fluctuating light conditions common under natural field conditions, plant photosynthetic efficiency and growth are impaired.

[0087] The present disclosure provides a method for accelerating NPQ relaxation after a plant transitions from high light intensity to low light intensity, thereby allowing faster recovery of photosynthetic quantum yield of CO2 fixation. The method comprises increasing the expression of one or more nucleotide sequences encoding photosystem II subunit S (PsbS), zeaxanthin epoxidase (ZEP) and violaxanthin de-epoxidase (VDE). Since this is achieved without reducing the NPQ amplitude, normal light protection under high light intensity is not affected. Under fluctuating light conditions, plants often undergo a transition from high light intensity to low light intensity, and this method results in improved light protection efficiency, and in turn, improved photosynthetic efficiency and plant growth.

[0088] The present disclosure also provides a method for genetically engineering plants to improve photosynthesis and growth. Expression vectors containing nucleotide sequences encoding PsbS, ZEP and VDE can be introduced into plants by currently available methods, including but not limited to protoplast transformation, Agrobacterium-mediated transformation, electroporation, and microprojectile bombardment. The method can be used to produce transgenic plants with improved photosynthesis and growth in plant species including but not limited to tobacco, wheat, corn, rice, soybean, sorghum, cassava, cowpea, poplar and eucalyptus.

[0089] It is well known in the art that the underlying mechanisms of the NPQ response and the associated xanthophyll cycle are highly conserved among plants and green algae. See e.g. Niyogi KK, Truong TB (2013). Evolution of flexible non-photochemical quenching mechanisms that regulate light harvesting in oxygenicphotosynthesis. Curr Op Plant Biol 16:307-314, Koziol AG, Borza T, Ishida KI, Keeling P, Lee RW, Durnford DG (2007). Tracing the evolution of the light-harvesting antennae in chlorophyll a / b-containing organisms.Plant Physiol143:1802-1816,Engelken J,Brinkmann H,Adamska I(2010).Taxonomic distribution and origins of the extended LHC(light-harvesting complex)antenna proteinsuperfamily.BMC Evol Biol 10:233,Brooks MD,Jansson S,Niyogi KK(2014).PsbS-dependent non-photochemical quenching.In: Non-photochemical quenching and energy dissipation in plants, algae and cyanobacteria. Demmig-Adams B, Garab G, Adams WW III, Govindjee editor (Dordrecht: Springer), pages 297-314, Kasajima I, EbanaK, Yamamoto T, Takahara K, Yano M, Kawai-Yamada M, Uchimiya H(2011).Moleculardistinction in genetic regulation of nonphotochemical quenching in rice.Proc Natl Acad Sci USA 108:13835-13840, Alboresi A, Gerotto C, Giacometti GM, Bassi R, Morosinotto T (2010). Physcomitrella patens mutants affected on heatdissipation clarify the evolution of photoprotection mechanisms upon landcolonization. Proc Natl Acad Sci USA 107:11128-11133, and Goss R, Lepetit B (2015). Biodiversity of NPQ. Journal of Plant Physiology, 172, 13-32. Therefore, the method disclosed in the present invention can be applied to all plants and green algae. .

[0090] Unless otherwise indicated, the present disclosure encompasses all conventional techniques of plant transformation, plant breeding, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, 2001; Current Protocols in Molecular Biology, FM Ausubel et al., ed., 1987; Plant Breeding: Principles and Prospects, MD Hayward et al., 1993; Current Protocols in Protein Science, Coligan et al., ed., 1995, (John Wiley & Sons, Inc.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach, MJ MacPherson, BD Hames and GR Taylor, ed., 1995.

[0091] In one aspect, a transgenic plant, or plant part, or plant material, or plant seed, or plant cell is provided, the transgenic plant, or plant part, or plant material, or plant seed, or plant cell comprising one or more heterologous nucleotide sequences operably linked to an expression control sequence, the heterologous nucleotide sequence encoding a polypeptide selected from PsbS, ZEP, and VDE. In one embodiment, the PsbS polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 1, the ZEP polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 2, and the VDE polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 3. In another embodiment, the transgenic plant comprises a nucleotide sequence encoding PsbS, ZEP, and VDE. The transgenic plant may comprise any combination of at least two of PsbS, ZEP, and VDE, or only one of PsbS, ZEP, and VDE. The nucleotide sequence may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2 and / or SEQ ID NO: 3. In another embodiment, the PsbS polypeptide has the amino acid sequence of SEQ ID NO: 4, the ZEP polypeptide has the amino acid sequence of SEQ ID NO: 5, and the VDE polypeptide has the amino acid sequence of SEQ ID NO: 6. The polypeptide may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% amino acid sequence identity to SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. Homologs of Arabidopsis PsbS, Zep and VDE nucleotides and polypeptides encoded by the nucleotide sequences are present in most plant species and the plants listed below and can be used in place of the Arabidopsis genes.

[0092] Enzymes with similar activities to PsbS, ZEP and VDE, or those with conserved domains, may be used instead, including but not limited to homologs in switchgrass, miscanthus, alfalfa, sweet sorghum, sorghum, sugarcane, energy cane, elephant grass, corn, wheat, barley, oats, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, camelina, Brassica napus, mustard, mustard, pearl millet, millet, other cereals, rice, oilseeds, vegetable crops, forage crops, industrial crops, woody crops and biomass crops. PsbS has a conserved Chloroa_b-binding domain (SEQ ID NO: 7), ZEP contains NADB_Rossman and FHA superfamily domains (SEQ ID NO: 8), and VDE has a Lipocalin domain (SEQ ID NO: 9). Homologs with these domains may also be used.

[0093] In another embodiment, the transgenic plant, or plant part, or plant material, or plant seed, or plant cell is a crop plant, a model plant, a monocot, a dicot, a plant with crassulacean acid metabolism (CAM) photosynthesis, a plant with C3 photosynthesis, a plant with C4 photosynthesis, an annual plant, a greenhouse plant, a horticultural flowering plant, a perennial plant, a switchgrass plant, a corn plant, a biomass plant, or a sugarcane plant. In another embodiment, the plant is selected from switchgrass, Miscanthus, alfalfa, sweet sorghum, sorghum, sugarcane, energy sugarcane, elephant grass, corn, wheat, barley, oats, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, Camelina, Brassica napus, Brassica carinata, mustard, pearl millet, millet, other cereals, rice, oilseeds, vegetable crops, feed crops, industrial crops, woody crops, and biomass crops. In another embodiment, the transgenic plant is tobacco. In another embodiment, the plant has improved quantum yield and / or CO2 fixation, and / or improved growth under fluctuating light conditions. The balance of gene expression levels can play a role in plant improvement. In one embodiment, the VDE polypeptide and the ZEP polypeptide are expressed at relatively similar levels.

[0094] In another embodiment, the transgenic plant, or plant part, or plant material, or plant seed, or plant cell has additional characteristics, such as herbicide resistance, insect or pest resistance, disease resistance, modified fatty acid metabolism and / or modified carbohydrate metabolism.

[0095] In another aspect, an expression vector is provided, the expression vector comprising at least one expression control sequence, the expression control sequence being operably linked to at least one nucleotide sequence encoding one or more polypeptides selected from PsbS, ZEP and VDE. In one embodiment, the vector comprises at least one expression control sequence, the expression control sequence comprising a promoter, the promoter being capable of driving expression of a nucleotide sequence encoding one or more polypeptides selected from PsbS, ZEP and VDE in a plant, plant part or plant material, plant seed or plant cell. In another embodiment, the promoter is selected from Rbcs1A, GAPA-1 and FBA2. In another embodiment, the vector comprises an Rbcs1A promoter driving expression of ZEP, a GAPA-1 promoter driving expression of PsbS, and an FBA2 promoter driving expression of VDE. In another embodiment, the vector is a T-DNA. In another embodiment, the vector comprises as Fig. 9In another embodiment, the vector can express the nucleotide sequence encoding PsbS, ZEP and VDE polypeptides in a crop plant, a model plant, a monocotyledon, a dicotyledon, a plant with crassulacean acid metabolism (CAM) photosynthesis, a plant with C3 photosynthesis, a plant with C4 photosynthesis, an annual plant, a greenhouse plant, a horticultural flowering plant, a perennial plant, a switchgrass plant, a corn plant, a biomass plant or a sugarcane plant, a plant part, or a plant material, or a plant seed, or a plant cell. In another embodiment, the plant is selected from switchgrass, miscanthus, alfalfa, sweet sorghum, sorghum, sugarcane, energy cane, elephant grass, corn, wheat, barley, oats, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, Camelina, Brassica napus, Brassica carinata, mustard, pearl millet, millet, other cereals, rice, oilseeds, vegetable crops, forage crops, industrial crops, tree crops and biomass crops.

[0096] In another aspect, a transgenic plant, or plant part, or plant material, or plant seed is provided, comprising a recombinant vector as described herein.

[0097] In another aspect, a method of increasing biomass production and / or carbon fixation and / or growth in a plant, or plant part, or plant material, or plant seed or plant cell is provided, the method comprising introducing into the genome of a plant, plant tissue, plant seed or plant cell one or more nucleotide sequences encoding a polypeptide selected from PsbS, ZEP and VDE operably linked to one or more expression control sequences. It was found that incorporation of a polypeptide encoded by nucleotides SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 increased quantum yield, CO2 fixation, and improved plant growth under fluctuating light conditions. In one embodiment, the method comprises a recombinant vector as described herein.

[0098] Transgenic plants of the present disclosure

[0099] In one aspect, the present invention provides a transgenic plant having one or more heterologous nucleotide sequences encoding one or more polypeptides PsbS, ZEP or VDE. In some embodiments, the transgenic plant has one or more heterologous nucleotide sequences encoding PsbS. In some embodiments, the transgenic plant has one or more heterologous nucleotide sequences encoding ZEP. In some embodiments, the transgenic plant has one or more heterologous nucleotide sequences encoding VDE. In some embodiments, the transgenic plant has one or more heterologous nucleotide sequences encoding PsbS and ZEP. In some embodiments, the transgenic plant has one or more heterologous nucleotide sequences encoding PsbS and VDE. In some embodiments, the transgenic plant has one or more heterologous nucleotide sequences encoding ZEP and VDE. In some embodiments, the transgenic plant has one or more heterologous nucleotide sequences encoding PsbS, ZEP and VDE.

[0100] In some embodiments described above, the one or more heterologous nucleotide sequences are derived from dicotyledons. In some embodiments, the one or more heterologous nucleotide sequences are derived from monocotyledons. In some embodiments, the one or more heterologous nucleotide sequences are derived from Arabidopsis thaliana. In some embodiments, the one or more heterologous nucleotide sequences are derived from corn. In some embodiments, the one or more heterologous nucleotide sequences are derived from rice. In some embodiments, the one or more heterologous nucleotide sequences are derived from sorghum. In some embodiments, the one or more heterologous nucleotide sequences are derived from soybean. In some embodiments, the one or more heterologous nucleotide sequences are derived from cowpea. In some embodiments, the one or more heterologous nucleotide sequences are derived from poplar species. In some embodiments, the one or more heterologous nucleotide sequences are derived from eucalyptus species. In some embodiments, the one or more heterologous nucleotide sequences are derived from cassava. In some embodiments, the one or more heterologous nucleotide sequences are derived from barley (Hordeum vulgare). In some embodiments, the one or more heterologous nucleotide sequences are derived from potatoes. In some embodiments, the one or more heterologous nucleotide sequences are derived from Saccharum species. In some embodiments, the one or more heterologous nucleotide sequences are derived from alfalfa. In some embodiments, the one or more heterologous nucleotide sequences are derived from switchgrass, miscanthus, alfalfa, sweet sorghum, sorghum, sugarcane, energy cane, elephant grass, corn, cassava, cowpea, wheat, barley, oats, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, Camelina, Brassica napus, Brassica carinata, mustard, pearl millet, millet, other cereals, rice, oilseeds, vegetable crops, forage crops, industrial crops, tree crops, or biomass crops.

[0101] In some embodiments of the foregoing, the transcript level of any of VDE, PsbS, or ZEP is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, or at least about 30 fold compared to control plants. In some embodiments, the protein level of any of VDE, PsbS, or ZEP is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, or at least about 100 fold compared to control plants.

[0102] Photoprotection mechanisms are highly conservative in higher plants. The degree of conservation or homology can be analyzed by comparing the nucleotide sequence or amino acid sequence of the gene of interest. As used herein, "sequence identity" refers to the percentage of identical residues in the same position in the sequence being analyzed. Sequence alignment methods for comparison are well known to those skilled in the art, and include but are not limited to manual alignment and computer-assisted sequence alignment and analysis. Due to the increased throughput provided by computer-assisted methods, the latter method is a preferred method in the present disclosure. Mathematical algorithms can be used to achieve the determination of the sequence identity percentage between any two sequences. Examples of such mathematical algorithms are the algorithm of Myers and Miller, CABIOS 4:11-17 (1988); the local homology algorithm of Smith et al., Adv. Appl. Math. 2:482 (1981); the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); the search-for-similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444-2448 (1988); the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 88:2364-2368 (1991). 90:5873-5877 (1993) modified. Computer implementations of these mathematical algorithms can be used to compare sequences to determine sequence identity and / or similarity. Such implementations include, for example: CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, Calif.); GAP, BESTFIT, BLAST, FASTA, and TFASTA in the AlignX program, version 10.3.0 (Invitrogen, Carlsbad, CA) and Wisconsin Genetics Software Package, version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using default parameters.The CLUSTAL program is well described by Higgins et al., Gene 73:237-244 (1988); Higgins et al., CABIOS 5:151-153 (1989); Corpet et al., Nucleic Acids Res. 16:10881-90 (1988); Huang et al., CABIOS 8:155-65 (1992); and Pearson et al., Meth. Mol. Biol. 24:307-331 (1994). The BLAST program of Altschul et al., J. Mol. Biol. 215:403-410 (1990) is based on the algorithm of Karlin and Altschul (1990) supra.

[0103] In some embodiments of the foregoing, PsbS is encoded by a nucleotide sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, ZEP is encoded by a nucleotide sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the VDE is encoded by a nucleotide sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99%, or 100% identical to SEQ ID NO: 3. In some embodiments, the PsbS has an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, the ZEP has an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the VDE has an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, PsbS further comprises the conserved domain of SEQ ID NO: 7. In some embodiments, ZEP further comprises the conserved domain of SEQ ID NO:8.In some embodiments, the VDE further comprises the conserved domain of SEQ ID NO:9.

[0104] In some embodiments of the above, the transgenic plant contains an expression vector, wherein the expression vector contains one or more nucleotide sequences described herein. In some embodiments, the transgenic plant produces seeds containing an expression vector having one or more heterologous nucleotide sequences encoding any of PsbS, ZEP or VDE. In some embodiments, seeds derived from the transgenic plant further produce progeny plants.

[0105] In some of the above embodiments, the transgenic plant has increased growth under fluctuating light conditions compared to a control plant under fluctuating light conditions. In some embodiments, the transgenic plant has increased photosynthetic efficiency under fluctuating light conditions compared to a control plant under fluctuating light conditions. In some embodiments, the transgenic plant has improved photoprotection efficiency under fluctuating light conditions compared to a control plant under fluctuating light conditions. In some embodiments, the transgenic plant has improved quantum yield and CO2 fixation under fluctuating light conditions compared to a control plant under fluctuating light conditions. In some embodiments, the transgenic plant is an elite strain or an elite line. In some embodiments, the transgenic plant also comprises expression of at least one additional polypeptide that provides herbicide resistance, insect or pest resistance, disease resistance, modified fatty acid metabolism, and / or modified carbohydrate metabolism.

[0106] In some embodiments of the above, the transcript level of VDE in the plant increases 3-fold compared to a control plant. In some embodiments of the above, the transcript level of PsbS in the plant increases 3-fold compared to a control plant. In some embodiments of the above, the transcript level of ZEP in the plant increases 8-fold compared to a control plant. In some embodiments of the above, the transcript level of VDE in the plant increases 10-fold compared to a control plant. In some embodiments of the above, the transcript level of PsbS in the plant increases 3-fold compared to a control plant. In some embodiments of the above, the transcript level of ZEP in the plant increases 6-fold compared to a control plant. In some embodiments of the above, the transcript level of VDE in the plant increases 4-fold compared to a control plant. In some embodiments of the above, the transcript level of PsbS in the plant increases 1.2-fold compared to a control plant. In some embodiments of the above, the transcript level of ZEP in the plant increases 7-fold compared to a control plant. In some embodiments of the above, the protein level of VDE in the plant increases 16-fold compared to a control plant. In some embodiments of the above, the protein level of PsbS in the plant increases by 2 times compared to a control plant. In some embodiments of the above, the protein level of ZEP in the plant increases by 80 times compared to a control plant. In some embodiments of the above, the protein level of VDE in the plant increases by 30 times compared to a control plant. In some embodiments of the above, the protein level of PsbS in the plant increases by 4 times compared to a control plant. In some embodiments of the above, the protein level of ZEP in the plant increases by 74 times compared to a control plant. In some embodiments of the above, the protein level of VDE in the plant increases by 47 times compared to a control plant. In some embodiments of the above, the protein level of PsbS in the plant increases by 3 times compared to a control plant. In some embodiments of the above, the protein level of ZEP in the plant increases by 75 times compared to a control plant. In some embodiments of the above, the increase in transcript levels in the plant has a ratio of 3:3:8, 10:3:6, or 4:1.2:7 between VDE, PsbS, and ZEP compared to a control plant. In some embodiments of the above, the increase in protein levels in the plant has a ratio of 16:2:80, 30:4:74, or 47:3:75 between VDE, PsbS, and ZEP compared to a control plant. In some embodiments of the above, the increase in transcript levels of VDE in the plant is in the range of 3-fold to 10-fold compared to a control plant. In some embodiments of the above, the increase in transcript levels of PsbS in the plant is about 1.2-fold to about 3-fold compared to a control plant.In some embodiments of the above, the increase in transcript levels of ZEP in the plant is about 6-fold to about 8-fold compared to control plants. In some embodiments of the above, the increase in protein levels of VDE in the plant is in the range of 16-fold to 47-fold compared to control plants. In some embodiments of the above, the increase in protein levels of PsbS in the plant is about 2-fold to about 4-fold compared to control plants. In some embodiments of the above, the increase in protein levels of ZEP in the plant is about 74-fold to about 80-fold compared to control plants.

[0107] Expression vectors disclosed herein

[0108] In another aspect, the present invention relates to an expression vector having one or more nucleotide sequences encoding any one of PsbS, ZEP and VDE. In some embodiments, the expression vector has one or more nucleotide sequences encoding PsbS. In some embodiments, the expression vector has one or more nucleotide sequences encoding ZEP. In some embodiments, the expression vector has one or more nucleotide sequences encoding VDE. In some embodiments, the expression vector has one or more nucleotide sequences encoding PsbS and ZEP. In some embodiments, the expression vector has one or more nucleotide sequences encoding PsbS and VDE. In some embodiments, the expression vector has one or more nucleotide sequences encoding ZEP and VDE. In some embodiments, the expression vector has one or more nucleotide sequences encoding PsbS, ZEP and VDE.

[0109] In some embodiments of the foregoing, PsbS is encoded by a nucleotide sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, ZEP is encoded by a nucleotide sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the VDE is encoded by a nucleotide sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99%, or 100% identical to SEQ ID NO: 3. In some embodiments, the PsbS has an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, the ZEP has an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the VDE has an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, PsbS further comprises the conserved domain of SEQ ID NO: 7. In some embodiments, ZEP further comprises the conserved domain of SEQ ID NO:8.In some embodiments, the VDE further comprises the conserved domain of SEQ ID NO:9.

[0110] In some of the above embodiments, the vector includes one or more expression control sequences having a promoter capable of driving the expression of the nucleotide sequence of PsbS, ZEP or VDE in the plant. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a weak promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is a seed and / or embryo-specific promoter. In some embodiments, the promoter is a leaf-specific promoter. In some embodiments, the promoter is a time-specific promoter. In some embodiments, the promoter is an anther and / or pollen-specific promoter. In some embodiments, the promoter is a flower-specific promoter. In some embodiments, a combination of promoters is used in the expression vector.

[0111] In some of the above embodiments, the promoter is Rbcs1A, GAPA-1 or FBA2. In some embodiments, the Rbcs1A promoter drives the expression of ZEP, the GAPA-1 promoter drives the expression of PsbS, and the FBA2 promoter drives the expression of VDE. In some embodiments, the vector is T-DNA. In some embodiments, the expression vector further comprises a nucleotide sequence encoding a polypeptide that provides antibiotic resistance. In some embodiments, the expression vector further comprises a left border (LB) domain and a right border (RB) domain flanking the expression control sequence and the nucleotide sequence encoding the PsbS, ZEP and VDE polypeptides. In some embodiments, the expression vector is in a bacterial cell. In some embodiments, the expression vector is in an Agrobacterium cell.

[0112] Methods of the present disclosure

[0113] In certain other aspects, the present disclosure relates to methods of increasing photosynthesis and growth of a plant, comprising increasing expression of one or more polypeptides described herein in the plant.

[0114] In some embodiments, increased expression is achieved by introducing one or more heterologous nucleotide sequences encoding any of PsbS, ZEP, and VDE into the plant. In some embodiments, increased expression is achieved by modifying the expression of endogenous nucleotide sequences encoding any of PsbS, ZEP, and VDE. In some embodiments, increased expression is achieved by modifying the promoter of the endogenous nucleotide sequence encoding any of PsbS, ZEP, and VDE. In some embodiments, increased expression is achieved by modifying transcription factors that regulate the transcription efficiency of any of PsbS, ZEP, and VDE. In some embodiments, increased expression is achieved by increasing the stability of mRNA of any of PsbS, ZEP, and VDE. In some embodiments, increased expression is achieved by optimizing the codon usage of any of PsbS, ZEP, and VDE in the target plant. In some embodiments, increased expression is achieved by changing epigenetics in the plant. In some embodiments, increased expression is achieved by changing DNA methylation in the plant. In some embodiments, increased expression is achieved by changing histone modifications in the plant. In some embodiments, increased expression is achieved by altering small RNA (sRNA) in the plant. In some embodiments, increased expression is achieved by increasing the translation efficiency of any of PsbS, ZEP, and VDE. In some embodiments, genome editing technologies including but not limited to ZFN, TALEN, and CRISPR are used to modify the nucleotide sequence that regulates the expression of any of PsbS, ZEP, and VDE.

[0115] In some embodiments, the transcript level of VDE in the plant increases by 3 times compared to a control plant. In some embodiments, the transcript level of PsbS in the plant increases by 3 times compared to a control plant. In some embodiments, the transcript level of ZEP in the plant increases by 8 times compared to a control plant. In some embodiments, the transcript level of VDE in the plant increases by 10 times compared to a control plant. In some embodiments, the transcript level of PsbS in the plant increases by 3 times compared to a control plant. In some embodiments, the transcript level of ZEP in the plant increases by 6 times compared to a control plant. In some embodiments, the transcript level of VDE in the plant increases by 4 times compared to a control plant. In some embodiments, the transcript level of PsbS in the plant increases by 1.2 times compared to a control plant. In some embodiments, the transcript level of ZEP in the plant increases by 7 times compared to a control plant. In some embodiments, the protein level of VDE in the plant increases by 16 times compared to a control plant. In some embodiments, the protein level of PsbS in the plant increases by 2 times compared to a control plant. In some embodiments, the protein level of ZEP in the plant increases by 80 times compared to a control plant. In some embodiments, the protein level of VDE in the plant increases by 30 times compared to a control plant. In some embodiments, the protein level of PsbS in the plant increases by 4 times compared to a control plant. In some embodiments, the protein level of ZEP in the plant increases by 74 times compared to a control plant. In some embodiments, the protein level of VDE in the plant increases by 47 times compared to a control plant. In some embodiments, the protein level of PsbS in the plant increases by 3 times compared to a control plant. In some embodiments, the protein level of ZEP in the plant increases by 75 times compared to a control plant. In some embodiments, the increase in transcript levels in the plant has a ratio of 3:3:8, 10:3:6, or 4:1.2:7 between VDE, PsbS, and ZEP compared to a control plant. In some embodiments, the increase in protein levels in the plant compared to a control plant has a ratio of 16:2:80, 30:4:74, or 47:3:75 between VDE, PsbS, and ZEP. In some embodiments, the increase in transcript levels of VDE in the plant is in the range of 3-fold to 10-fold compared to a control plant. In some embodiments, the increase in transcript levels of PsbS in the plant is about 1.2-fold to about 3-fold compared to a control plant. In some embodiments, the increase in transcript levels of ZEP in the plant is about 6-fold to about 8-fold compared to a control plant. In some embodiments, the increase in protein levels of VDE in the plant is in the range of 16-fold to 47-fold compared to a control plant.In some embodiments, the protein level of PsbS in the plant increases by about 2-fold to about 4-fold compared to control plants. In some embodiments, the protein level of ZEP in the plant increases by about 74-fold to about 80-fold compared to control plants.

[0116] Methods for increasing growth under fluctuating light conditions

[0117] In one aspect, the present invention provides a method for increasing the growth of plants under fluctuating light conditions, including increasing the expression of PsbS, ZEP and / or VDE in plants, thereby producing plants with increased expression of the one or more polypeptides compared to control plants. In some embodiments, the increased expression is in the form of increased transcript levels. In some embodiments, any transcript level in VDE, PsbS or ZEP increases at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, or at least about 30 times compared to control plants. In some embodiments, the increased expression is in the form of increased protein levels. In some embodiments, the protein level of any of VDE, PsbS, or ZEP is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, or at least about 100 fold compared to a control plant. In some embodiments, the method comprises increasing the expression of PsbS. In some embodiments, the method comprises increasing the expression of ZEP. In some embodiments, the method comprises increasing the expression of VDE. In some embodiments, the method comprises increasing the expression of PsbS and ZEP. In some embodiments, the method comprises increasing the expression of PsbS and VDE. In some embodiments, the method comprises increasing the expression of ZEP and VDE. In some embodiments, the methods comprise increasing expression of PsbS, ZEP, and VDE.

[0118] In some embodiments of the foregoing, PsbS is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, ZEP is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the VDE is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO:3.

[0119] In some embodiments of the foregoing, PsbS has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, ZEP has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the VDE has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% identical to SEQ ID NO: 6. In some embodiments, the PsbS further comprises the conserved domain of SEQ ID NO: 7. In some embodiments, the ZEP further comprises the conserved domain of SEQ ID NO: 8. In some embodiments, the VDE further comprises the conserved domain of SEQ ID NO: 9.

[0120] In some embodiments of the above, the plant is corn (Zea mays). In some embodiments, the plant is rice (Oryza sativa). In some embodiments, the plant is sorghum (Sorghum bicolor). In some embodiments, the plant is soybean (Glycine max). In some embodiments, the plant is cowpea (Vigna unguiculata). In some embodiments, the plant is a poplar species (Populus spp). In some embodiments, the plant is a eucalyptus species (Eucalyptus spp). In some embodiments, the plant is cassava (Manihot esculenta). In some embodiments, the plant is barley (Hordeum vulgare). In some embodiments, the plant is potato (Solanum tuberosum). In some embodiments, the plant is a sugarcane species (Saccharum spp). In some embodiments, the plant is Medicago sativa. In some embodiments, the plant is switchgrass, miscanthus, alfalfa, sweet sorghum, sorghum, sugarcane, energy cane, elephant grass, corn, cassava, cowpea, wheat, barley, oats, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, Camelina, Brassica napus, Brassica carinata, mustard, pearl millet, millet, other cereals, rice, oilseeds, vegetable crops, forage crops, industrial crops, tree crops, or biomass crops.

[0121] Method for increasing photosynthetic efficiency under fluctuating light conditions

[0122] On the other hand, the present invention provides a method for increasing the photosynthetic efficiency of plants under fluctuating light conditions, including increasing the expression of any of PsbS, ZEP or VDE in plants, thereby producing plants with increased expression of the one or more polypeptides compared to control plants. In some embodiments, the increased expression is in the form of increased transcript levels. In some embodiments, compared to control plants, the transcript levels of any of VDE, PsbS or ZEP increase by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, or at least about 30 times. In some embodiments, the increased expression is in the form of increased protein levels. In some embodiments, the protein level of any of VDE, PsbS, or ZEP is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, or at least about 100 fold compared to a control plant. In some embodiments, the method comprises increasing the expression of PsbS. In some embodiments, the method comprises increasing the expression of ZEP. In some embodiments, the method comprises increasing the expression of VDE. In some embodiments, the method comprises increasing the expression of PsbS and ZEP. In some embodiments, the method comprises increasing the expression of PsbS and VDE. In some embodiments, the method comprises increasing the expression of ZEP and VDE. In some embodiments, the methods comprise increasing expression of PsbS, ZEP, and VDE.

[0123] In some embodiments of the foregoing, PsbS is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, ZEP is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the VDE is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO:3.

[0124] In some embodiments of the foregoing, PsbS has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, ZEP has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the VDE has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% identical to SEQ ID NO: 6. In some embodiments, the PsbS further comprises the conserved domain of SEQ ID NO: 7. In some embodiments, the ZEP further comprises the conserved domain of SEQ ID NO: 8. In some embodiments, the VDE further comprises the conserved domain of SEQ ID NO: 9.

[0125] In some embodiments of the above, the plant is corn (Zea mays). In some embodiments, the plant is rice (Oryza sativa). In some embodiments, the plant is sorghum (Sorghum bicolor). In some embodiments, the plant is soybean (Glycine max). In some embodiments, the plant is cowpea (Vigna unguiculata). In some embodiments, the plant is a poplar species (Populus spp). In some embodiments, the plant is a eucalyptus species (Eucalyptus spp). In some embodiments, the plant is cassava (Manihot esculenta). In some embodiments, the plant is barley (Hordeum vulgare). In some embodiments, the plant is potato (Solanum tuberosum). In some embodiments, the plant is a sugarcane species (Saccharum spp). In some embodiments, the plant is Medicago sativa. In some embodiments, the plant is switchgrass, miscanthus, alfalfa, sweet sorghum, sorghum, sugarcane, energy cane, elephant grass, corn, cassava, cowpea, wheat, barley, oats, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, Camelina, Brassica napus, Brassica carinata, mustard, pearl millet, millet, other cereals, rice, oilseeds, vegetable crops, forage crops, industrial crops, tree crops, or biomass crops.

[0126] Method for increasing the efficiency of photoprotection under fluctuating light conditions

[0127] On the other hand, the present invention provides a method for increasing the photoprotection efficiency of plants under fluctuating light conditions, comprising increasing the expression of PsbS, ZEP and / or VDE in plants, thereby producing plants with increased expression of the one or more polypeptides compared to control plants. In some embodiments, the increased expression is in the form of increased transcript levels. In some embodiments, any transcript level in VDE, PsbS or ZEP increases at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, or at least about 30 times compared to control plants. In some embodiments, the increased expression is in the form of increased protein levels. In some embodiments, the protein level of any of VDE, PsbS, or ZEP is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, or at least about 100 fold compared to a control plant. In some embodiments, the method comprises increasing the expression of PsbS. In some embodiments, the method comprises increasing the expression of ZEP. In some embodiments, the method comprises increasing the expression of VDE. In some embodiments, the method comprises increasing the expression of PsbS and ZEP. In some embodiments, the method comprises increasing the expression of PsbS and VDE. In some embodiments, the method comprises increasing the expression of ZEP and VDE. In some embodiments, the methods comprise increasing expression of PsbS, ZEP, and VDE.

[0128] In some embodiments of the foregoing, PsbS is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, ZEP is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the VDE is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO:3.

[0129] In some embodiments of the foregoing, PsbS has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, ZEP has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the VDE has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% identical to SEQ ID NO: 6. In some embodiments, the PsbS further comprises the conserved domain of SEQ ID NO: 7. In some embodiments, the ZEP further comprises the conserved domain of SEQ ID NO: 8. In some embodiments, the VDE further comprises the conserved domain of SEQ ID NO: 9.

[0130] In some embodiments of the above, the plant is corn (Zea mays). In some embodiments, the plant is rice (Oryza sativa). In some embodiments, the plant is sorghum (Sorghum bicolor). In some embodiments, the plant is soybean (Glycine max). In some embodiments, the plant is cowpea (Vigna unguiculata). In some embodiments, the plant is a poplar species (Populus spp). In some embodiments, the plant is a eucalyptus species (Eucalyptus spp). In some embodiments, the plant is cassava (Manihot esculenta). In some embodiments, the plant is barley (Hordeum vulgare). In some embodiments, the plant is potato (Solanum tuberosum). In some embodiments, the plant is a sugarcane species (Saccharum spp). In some embodiments, the plant is Medicago sativa. In some embodiments, the plant is switchgrass, miscanthus, alfalfa, sweet sorghum, sorghum, sugarcane, energy cane, elephant grass, corn, cassava, cowpea, wheat, barley, oats, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, Camelina, Brassica napus, Brassica carinata, mustard, pearl millet, millet, other cereals, rice, oilseeds, vegetable crops, forage crops, industrial crops, tree crops, or biomass crops.

[0131] Methods for increasing quantum yield and CO2 fixation under fluctuating light conditions

[0132] On the other hand, the present invention provides a method for increasing the quantum yield and CO2 fixation of plants under fluctuating light conditions, including increasing the expression of PsbS, ZEP and / or VDE in plants, thereby producing plants with increased expression of the one or more polypeptides compared to control plants. In some embodiments, the increased expression is in the form of increased transcript levels. In some embodiments, compared to control plants, any transcript level in VDE, PsbS or ZEP increases by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, or at least about 30 times. In some embodiments, the increased expression is in the form of increased protein levels. In some embodiments, the protein level of any of VDE, PsbS, or ZEP is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, or at least about 100 fold compared to a control plant. In some embodiments, the method comprises increasing the expression of PsbS. In some embodiments, the method comprises increasing the expression of ZEP. In some embodiments, the method comprises increasing the expression of VDE. In some embodiments, the method comprises increasing the expression of PsbS and ZEP. In some embodiments, the method comprises increasing the expression of PsbS and VDE. In some embodiments, the method comprises increasing the expression of ZEP and VDE. In some embodiments, the methods comprise increasing expression of PsbS, ZEP, and VDE.

[0133] In some embodiments of the foregoing, PsbS is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, ZEP is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the VDE is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO:3.

[0134] In some embodiments of the foregoing, PsbS has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, ZEP has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the VDE has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% identical to SEQ ID NO: 6. In some embodiments, the PsbS further comprises the conserved domain of SEQ ID NO: 7. In some embodiments, the ZEP further comprises the conserved domain of SEQ ID NO: 8. In some embodiments, the VDE further comprises the conserved domain of SEQ ID NO: 9.

[0135] In some embodiments of the above, the plant is corn (Zea mays). In some embodiments, the plant is rice (Oryza sativa). In some embodiments, the plant is sorghum (Sorghum bicolor). In some embodiments, the plant is soybean (Glycine max). In some embodiments, the plant is cowpea (Vigna unguiculata). In some embodiments, the plant is a poplar species (Populus spp). In some embodiments, the plant is a eucalyptus species (Eucalyptus spp). In some embodiments, the plant is cassava (Manihot esculenta). In some embodiments, the plant is barley (Hordeum vulgare). In some embodiments, the plant is potato (Solanum tuberosum). In some embodiments, the plant is a sugarcane species (Saccharum spp). In some embodiments, the plant is Medicago sativa. In some embodiments, the plant is switchgrass, miscanthus, alfalfa, sweet sorghum, sorghum, sugarcane, energy cane, elephant grass, corn, cassava, cowpea, wheat, barley, oats, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, Camelina, Brassica napus, Brassica carinata, mustard, pearl millet, millet, other cereals, rice, oilseeds, vegetable crops, forage crops, industrial crops, tree crops, or biomass crops.

[0136] Method for increasing the relaxation rate of non-photochemical quenching (NPQ) under fluctuating light conditions

[0137] On the other hand, the present invention provides a method for increasing the non-photochemical quenching (NPQ) relaxation rate in plants, comprising increasing the expression of PsbS, ZEP and / or VDE in plants, thereby producing plants with increased expression of the one or more polypeptides compared to control plants. In some embodiments, the increased expression is in the form of increased transcript levels. In some embodiments, compared to control plants, any transcript level in VDE, PsbS or ZEP increases by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, or at least about 30 times. In some embodiments, the increased expression is in the form of increased protein levels. In some embodiments, the protein level of any of VDE, PsbS, or ZEP is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, or at least about 100 fold compared to a control plant. In some embodiments, the method comprises increasing the expression of PsbS. In some embodiments, the method comprises increasing the expression of ZEP. In some embodiments, the method comprises increasing the expression of VDE. In some embodiments, the method comprises increasing the expression of PsbS and ZEP. In some embodiments, the method comprises increasing the expression of PsbS and VDE. In some embodiments, the method comprises increasing the expression of ZEP and VDE. In some embodiments, the methods comprise increasing expression of PsbS, ZEP, and VDE.

[0138] In some embodiments of the foregoing, PsbS is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, ZEP is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the VDE is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO:3.

[0139] In some embodiments of the foregoing, PsbS has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, ZEP has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the VDE has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% identical to SEQ ID NO: 6. In some embodiments, the PsbS further comprises the conserved domain of SEQ ID NO: 7. In some embodiments, the ZEP further comprises the conserved domain of SEQ ID NO: 8. In some embodiments, the VDE further comprises the conserved domain of SEQ ID NO: 9.

[0140] In some embodiments of the above, the plant is corn (Zea mays). In some embodiments, the plant is rice (Oryza sativa). In some embodiments, the plant is sorghum (Sorghum bicolor). In some embodiments, the plant is soybean (Glycine max). In some embodiments, the plant is cowpea (Vigna unguiculata). In some embodiments, the plant is a poplar species (Populus spp). In some embodiments, the plant is a eucalyptus species (Eucalyptus spp). In some embodiments, the plant is cassava (Manihot esculenta). In some embodiments, the plant is barley (Hordeum vulgare). In some embodiments, the plant is potato (Solanum tuberosum). In some embodiments, the plant is a sugarcane species (Saccharum spp). In some embodiments, the plant is Medicago sativa. In some embodiments, the plant is switchgrass, miscanthus, alfalfa, sweet sorghum, sorghum, sugarcane, energy cane, elephant grass, corn, cassava, cowpea, wheat, barley, oats, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, Camelina, Brassica napus, Brassica carinata, mustard, pearl millet, millet, other cereals, rice, oilseeds, vegetable crops, forage crops, industrial crops, tree crops, or biomass crops.

[0141] Method for selecting plants having improved growth characteristics under fluctuating light conditions

[0142] On the other hand, provided herein is a method for selecting plants with improved growth characteristics under fluctuating light conditions, comprising the following steps: providing a plant population; modifying the plant population to increase the activity of any of PsbS, ZEP and VDE; detecting the level of non-photochemical quenching (NPQ) in plants under fluctuating light conditions; comparing the NPQ level in plants under fluctuating light conditions with the control NPQ level under fluctuating light conditions; and selecting plants with increased NPQ relaxation rates when switching from high light intensity to low light intensity. In some embodiments, the control NPQ level is the lowest NPQ level in the population. In some embodiments, the control NPQ level is the median NPQ level in the population. In some embodiments, the control NPQ level is the average NPQ level in the population. In some embodiments, the control NPQ level is the NPQ level in the control plant. In some embodiments, the population includes plants expressing heterologous sequences of PsbS, ZEP and / or VDE, or plants in which the genome has been edited to increase the expression of PsbS, ZEP and / or VDE. In some embodiments, the genome editing technology is ZFN. In some embodiments, the genome editing technology is TALEN. In some embodiments, the genome editing technology is CRISPR. In some embodiments, the promoter of PsbS is modified. In some embodiments, the promoter of ZEP is modified. In some embodiments, the promoter of VDE is modified. In some embodiments, the population includes plants that have been treated to induce mutations in PsbS, ZEP, and / or VDE. In some embodiments, the mutagen is EMS.

[0143] In some embodiments of the foregoing, PsbS is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, ZEP is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the VDE is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO:3.

[0144] In some embodiments of the foregoing, PsbS has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, ZEP has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the VDE has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% identical to SEQ ID NO: 6. In some embodiments, the PsbS further comprises the conserved domain of SEQ ID NO: 7. In some embodiments, the ZEP further comprises the conserved domain of SEQ ID NO: 8. In some embodiments, the VDE further comprises the conserved domain of SEQ ID NO: 9.

[0145] In some embodiments of the above, the plant is corn (Zea mays). In some embodiments, the plant is rice (Oryza sativa). In some embodiments, the plant is sorghum (Sorghum bicolor). In some embodiments, the plant is soybean (Glycine max). In some embodiments, the plant is cowpea (Vigna unguiculata). In some embodiments, the plant is a poplar species (Populus spp). In some embodiments, the plant is a eucalyptus species (Eucalyptus spp). In some embodiments, the plant is cassava (Manihot esculenta). In some embodiments, the plant is barley (Hordeum vulgare). In some embodiments, the plant is potato (Solanum tuberosum). In some embodiments, the plant is a sugarcane species (Saccharum spp). In some embodiments, the plant is Medicago sativa. In some embodiments, the plant is switchgrass, miscanthus, alfalfa, sweet sorghum, sorghum, sugarcane, energy cane, elephant grass, corn, cassava, cowpea, wheat, barley, oats, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, Camelina, Brassica napus, Brassica carinata, mustard, pearl millet, millet, other cereals, rice, oilseeds, vegetable crops, forage crops, industrial crops, tree crops, or biomass crops.

[0146] Methods for screening for polymorphisms associated with improved growth characteristics under fluctuating light conditions

[0147] In another aspect, the present invention provides a method for screening for nucleotide sequence polymorphisms associated with improved growth characteristics under fluctuating light conditions, comprising the following steps: providing a plant population; obtaining a nucleotide sequence that regulates and / or encodes any of PsbS, ZEP, and VDE in the plant population; obtaining one or more polymorphisms in a nucleotide sequence that regulates and / or encodes any of PsbS, ZEP, and VDE in the plant population; detecting the relaxation rate of non-photochemical quenching (NPQ) of the plant population when it is converted from high light intensity to low light intensity; performing a statistical analysis to determine the association of the polymorphism with the NPQ relaxation rate in the plant population; and selecting a polymorphism that has a statistically significant correlation with the NPQ relaxation rate. In some embodiments, the population is a collection of germplasm. In some embodiments, the population includes plants expressing heterologous sequences of PsbS, ZEP, and / or VDE, or plants in which the genome has been edited to increase the expression of PsbS, ZEP, and / or VDE. In some embodiments, the population includes plants that have not been treated to induce mutations. In some embodiments, the colony includes plants that have been treated to induce mutations in PsbS, ZEP and / or VDE. In some embodiments, the polymorphism is a single nucleotide polymorphism (SNP). In some embodiments, the polymorphism is an insertion / deletion (InDel). In some embodiments, the polymorphism is a simple sequence repeat (SSR). In some embodiments, the polymorphism is a presence / absence variation (PAV). In some embodiments, the polymorphism is a copy number variation (CNV). In some embodiments, the polymorphism is located in the promoter of PsbS. In some embodiments, the polymorphism is located in the promoter of ZEP. In some embodiments, the polymorphism is located in the promoter of VDE. In some embodiments, the polymorphism is detected by Sanger sequencing. In some embodiments, the polymorphism is detected by next generation sequencing. In some embodiments, the polymorphism is detected by agarose gel electrophoresis. In some embodiments, the polymorphism is detected by polyacrylamide gel electrophoresis. In some embodiments, the polymorphism is further used to screen a colony different from the colony in which the polymorphism is identified. In some embodiments, the polymorphisms are further used as targets for genome editing to improve growth characteristics of the plant.

[0148] In some of the above embodiments, the improved growth characteristic is improved growth. In some embodiments, the improved growth characteristic is improved photosynthetic efficiency. In some embodiments, the improved growth characteristic is improved light protection efficiency. In some embodiments, the improved growth characteristic is improved quantum yield and CO2 fixation. In some embodiments, the improved growth characteristic is increased non-photochemical quenching (NPQ) relaxation rate. In some embodiments, NPQ is detected using chlorophyll fluorescence imaging.

[0149] In some embodiments of the foregoing, PsbS is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, ZEP is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the VDE is encoded by a nucleotide sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO:3.

[0150] In some embodiments of the foregoing, PsbS has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, ZEP has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the VDE has an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% identical to SEQ ID NO: 6. In some embodiments, the PsbS further comprises the conserved domain of SEQ ID NO: 7. In some embodiments, the ZEP further comprises the conserved domain of SEQ ID NO: 8. In some embodiments, the VDE further comprises the conserved domain of SEQ ID NO: 9.

[0151] In some embodiments of the above, the plant is corn (Zea mays). In some embodiments, the plant is rice (Oryza sativa). In some embodiments, the plant is sorghum (Sorghum bicolor). In some embodiments, the plant is soybean (Glycine max). In some embodiments, the plant is cowpea (Vigna unguiculata). In some embodiments, the plant is a poplar species (Populus spp). In some embodiments, the plant is a eucalyptus species (Eucalyptus spp). In some embodiments, the plant is cassava (Manihot esculenta). In some embodiments, the plant is barley (Hordeum vulgare). In some embodiments, the plant is potato (Solanum tuberosum). In some embodiments, the plant is a sugarcane species (Saccharum spp). In some embodiments, the plant is Medicago sativa. In some embodiments, the plant is switchgrass, miscanthus, alfalfa, sweet sorghum, sorghum, sugarcane, energy cane, elephant grass, corn, cassava, cowpea, wheat, barley, oats, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, Camelina, Brassica napus, Brassica carinata, mustard, pearl millet, millet, other cereals, rice, oilseeds, vegetable crops, forage crops, industrial crops, tree crops, or biomass crops.

[0152] General Methods of Practicing the Embodiments Described Herein

[0153] Transformation of plants with nucleotide sequences of interest

[0154] Conventional techniques can be used to produce transgenic plants to express any nucleotide sequence of interest in plants or plant cells (Methods in Molecular Biology, 2005, Vol. 286, Transgenic Plants: Methods and Protocols, Pena L. ed., Humana Press, Inc. Totowa, NT). Typically, gene transfer or transformation is performed using regenerative explants to produce complete, fertile plants. Typically, the DNA or RNA molecule to be introduced into the organism is part of the transformation vector. A large number of such vector systems known in the art, such as plasmids, can be used. The components of the expression system can be modified, for example, to increase the expression of the introduced nucleic acid. For example, truncated sequences, nucleotide substitutions or other modifications can be used. Expression systems known in the art can be used to transform almost any plant cell under suitable conditions. Transgenics comprising DNA molecules encoding genes of interest are preferably stably transformed and integrated into the genome of the host cell. Transformed cells are preferably regenerated into complete plants. A detailed description of transformation techniques is within the knowledge of those skilled in the art.

[0155] Genetic constructs for transformation

[0156] DNA constructs useful in the methods described herein include transformation vectors capable of introducing a transgene into a plant. As used herein, "transgenic" refers to an organism into which a nucleic acid fragment containing a heterologous nucleotide sequence has been introduced. The transgene in the transgenic organism is preferably stable and heritable. The heterologous nucleic acid fragment may or may not be integrated into the host genome.

[0157] There are several plant transformation vector options available, including those described in Gene Transfer to Plants, 1995, Potrykus et al., ed., Springer-Verlag Berlin Heidelberg New York, Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins, 1996, Owen et al., ed., John Wiley & Sons Ltd. England, and Methods in Plant Molecular Biology: A Laboratory Course Manual, 1995, Maliga et al., ed., Cold Spring Laboratory Press, New York. Plant transformation vectors typically include one or more coding sequences of interest under the transcriptional control of 5' and 3' regulatory sequences (including promoters, transcription termination and / or polyadenylation signals, and selectable or screenable marker genes). In order to express two or more polypeptides from a single transcript, additional RNA processing signals and ribozyme sequences can be engineered into the construct (U.S. Pat. No. 5,519,164). This approach has the advantage of placing multiple transgenes in a single locus, which is advantageous in subsequent plant breeding efforts. In one embodiment, the vector comprises at least one expression control sequence comprising a promoter capable of driving expression of a nucleotide sequence encoding one or more polypeptides selected from the group consisting of PsbS, ZEP and VDE in a plant, plant part or material, plant seed or plant cell. In another embodiment, the promoter is selected from the group consisting of Rbcs1A, GAPA-1 and FBA2. In another embodiment, the Rbcs1A promoter drives expression of ZEP, the GAPA-1 promoter drives expression of PsbS, and the FBA2 promoter drives expression of VDE. In another embodiment, the vector is T-DNA. In another embodiment, the vector is such as Fig. 9 As is known to those skilled in the art, a particular promoter and vector that works in one plant type may not work in another plant type. As described herein, methods for preparing transgenic plants are well known in the art.

[0158] T-DNA

[0159] The method of introducing transgenic into plant by Agrobacterium-mediated transformation method generally relates to T-DNA (transfer DNA), and the T-DNA mixes at least one genetically modified genetic element and transfers these genetic elements into the genome of plant.(One or more) transgenic is usually constructed in DNA plasmid vector, and usually flanks Agrobacterium Ti plasmid right border DNA region (RB) and left border DNA region (LB).In the transformation process mediated by Agrobacterium, DNA plasmid is cut by endonuclease VirD2 at right border region and left border region.The single-stranded DNA (called T chain) from the incision is transferred to plant cell from Agrobacterium cell.The sequence corresponding to T-DNA region is inserted into plant genome.

[0160] T-DNA is integrated into the plant genome and usually starts at RB and continues to the T-DNA end (LB). However, endonucleases sometimes do not cut equally at two borders. When this happens, the T-DNA inserted into the plant genome usually contains some or all of the plasmid vector DNA. This phenomenon is referred to as "read-through". The desired method is usually to only transfer (one or more) transgenics (described T-DNA) between the right border region and the left border region into the plant genome without transferring any adjacent plasmid vector DNA (vector backbone). The vector backbone DNA contains various plasmid maintenance elements, including for example replication origin, bacterial selection marker genes and other DNA fragments that are not needed for expressing desired (one or more) traits in plants.

[0161] Engineered minichromosomes can also be used to express one or more genes in plant cells. The cloned telomeric repeats introduced into the cell can shorten the distal part of the chromosome by forming new telomeres at the integration site. Using this method, vectors for gene transfer can be prepared by trimming the arms of natural plant chromosomes and adding insertion sites for large inserts (Yu et al., 2006, Proc. Natl. Acad. Sci. USA 103: 17331-17336; Yu et al., 2007, Proc. Natl. Acad. Sci. USA 104: 8924-8929).

[0162] An alternative method for chromosome engineering in plants involves the in vivo assembly of autonomous plant minichromosomes (Carlson et al., 2007, PLoS Genet. 3: 1965-74). Plant cells can be transformed with centromere sequences and screened for plants with de novo assembled autonomous chromosomes. Useful constructs combine selectable marker genes with genomic DNA fragments containing centromere satellite and retroviral element sequences and / or other repetitive sequences.

[0163] Another method that can be used for the invention is the engineered trait locus ("ETL") technology (U.S. Pat. No. 6,077,697; US2006 / 0143732). This system targets DNA to heterochromatic regions of plant chromosomes, such as inter-arm heterochromatin in the short arm of acrocentric chromosomes. The targeting sequence may include ribosomal DNA (rDNA) or lambda phage DNA. The inter-arm rDNA region supports stable insertion, low recombination, and high levels of gene expression. This technology can also be used to stack multiple traits in plants (US2006 / 0246586).

[0164] Zinc finger nucleases (ZFNs), TALENs and CRISPR-Cas9 can also be used to practice the present invention because they allow double-stranded DNA cleavage at specific sites on plant chromosomes, allowing for target gene insertion or deletion (Shukla et al., 2009, Nature 459:437-441; Townsend et al., 2009, Nature 459:442-445, WO2015089427A1).

[0165] Tissue culture-based nuclear transformation method

[0166] Transformation protocols, as well as protocols for introducing nucleotide sequences into plants, can vary depending on the type of plant or plant cell (ie, monocot or dicot) targeted for transformation.

[0167] Suitable methods for introducing nucleotide sequences into plant cells and subsequently inserting them into the plant genome are described in US2010 / 0229256 Al to Somleva & Ali and US2012 / 0060413 to Somleva et al.

[0168] Transformed cells are grown into plants according to conventional techniques. See, e.g., McCormick et al., 1986, Plant Cell Rep. 5:81-84. These plants can then be grown and pollinated with the same transformed variety or a different variety, and the resulting hybrids with constitutive expression of the desired phenotypic characteristic identified. Two or more generations can be grown to ensure stable maintenance and inheritance of the constitutive expression of the desired phenotypic characteristic, and then seeds are harvested to ensure that the constitutive expression of the desired phenotypic characteristic has been achieved.

[0169] In situ transformation method

[0170] The program of plant in situ transformation (in planta transformation) is not complicated. It avoids tissue culture manipulation and possible somatic clone variation, and only requires a short time to obtain transgenic plants. However, the frequency of transformants in the offspring of this inoculation plant is relatively low and variable. At present, in the absence of a regeneration system based on tissue culture, there are few species that can be conventionally transformed. Stable Arabidopsis transformants can be obtained by several plant in situ methods, and the plant in situ methods include vacuum infiltration (Clough & Bent, 1998, The Plant J.16:735-743), the transformation of germinating seeds (Feldmann & Marks, 1987, Mol. Gen. Genet.208:1-9), flower organ dipping method (Clough and Bent, 1998, Plant J.16:735-743) and the transformation method (Chung et al., 2000, Transgenic Res.9:471-476) of flower organ spraying. Other plants that have been successfully transformed by the in planta method include rapeseed and radish (vacuum infiltration, Ian and Hong, 2001, Transgenic Res., 10:363-371; Desfeux et al., 2000, Plant Physiol. 123:895-904), Medicago truncatula (vacuum infiltration, Trieu et al., 2000, Plant J. 22:531-541), Camelina (floral dip method, Nguyen et al. WO / 2009 / 117555), and wheat (floral dip method, Zale et al., 2009, Plant Cell Rep. 28:903-913). In planta methods have also been used to transform germ cells in maize (pollen, Wang et al., 2001, Acta Botanica Sin., 43, 275-279; Zhang et al., 2005, Euphytica, 144, 11-22; pistil, Chumakov et al., 2006, Russian J. Genetics, 42, 893-897; Mamontova et al., 2010, Russian J. Genetics, 46, 501-504) and Sorghum (pollen, Wang et al., 2007, Biotechnol. Appl. Biochem., 48, 79-83).

[0171] Reporter genes and selectable marker genes

[0172] Reporter genes and / or selective marker genes may be included in expression control sequences (expression cassettes), as described in U.S. Patent Applications 20100229256 and 20120060413, which are incorporated herein by reference. Expression cassettes comprising promoter sequences operably linked to heterologous nucleotide sequences of interest can be used to transform any plant by any of the methods described above. Useful selective marker genes and methods for selecting transgenic lines for a range of different crop species are described in the examples herein.

[0173] Nucleotide sequence expression in plants

[0174] Plant promoters can be selected to control the expression of nucleotide sequences in different plant tissues or organelles, all of which are known to those skilled in the art (Gasser & Fraley, 1989, Science 244: 1293-1299).

[0175] The selection of (one or more) promoters that can be used depends on several factors, including but not limited to efficiency, selectivity, inducibility, expected expression level and / or preferred cell expression or tissue expression. For those skilled in the art, it is a routine matter to regulate the expression of the nucleotide sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the nucleotide sequence. Examples of promoters that can be used are known in the art. Promoters that can be used include those promoters present in the plant genome, and promoters from other sources. Some suitable promoters only start transcription in certain cell types or mainly in certain cell types. Methods for identifying and characterizing promoter regions in plant genomic DNA include, for example, those described in Jordano et al., Plant Cell 1:855-866, 1989; Bustos et al., Plant Cell 1:839-854, 1989; Green et al., EMBO J. 7:4035-4044, 1988; Meier et al., Plant Cell 3:309-316, 1991; and Zhang et al., Plant Physiology 110:1069-1079, 1996.

[0176] Additional examples of promoters that can be used include ribulose-1,5-bisphosphate carboxylase (RbcS) promoters, such as the RbcS promoter from Larix laricina, the pine cab6 promoter (Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994), the Cab-1 gene promoter from wheat (Fejes et al., Plant Mol. Biol. 15:921-932, 1990), the CAB-1 promoter from spinach (Lubberstedt et al., Plant Physiol. 104:997-1006, 1994), the cab1R promoter from rice (Luan et al., Plant Cell 4:971-981, 1992), the GAPA-1 promoter from corn, the promoter from Saccharomyces cerevisiae (Saccharomyces cerevisiae) cerevisiae) FBA2 promoter, pyruvate phosphate dikinase (PPDK) promoter from maize (Matsuoka et al., Proc. Natl. Acad. Sci. USA 90:9586-9590, 1993), tobacco Lhcb1*2 promoter (Cerdan et al., Plant Mol. Biol. 33:245-255, 1997), Arabidopsis thaliana SUC2 sucrose-H + Symport promoters (Truernit et al., Planta 196:564-570, 1995) and thylakoid membrane protein promoters from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, and rbcS). Other exemplary promoters that can be used to drive gene transcription in stems, leaves, and green tissues are described in U.S. Patent Application Publication No. 2007 / 0006346, which is incorporated herein by reference in its entirety. Other promoters that result in preferential expression in green plant tissues include those from genes such as Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit (Fischhoff et al., Plant Mol. Biol. 20:81-93, 1992), aldolase, and pyruvate orthophosphate dikinase (PPDK) (Taniguchi et al., Plant Cell Physiol. 41(1):42-48, 2000).

[0177] Inducible promoter

[0178] Chemically regulated promoters can be used to regulate the expression of nucleotide sequences in plants by applying exogenous chemical regulators. Depending on the purpose, the promoter can be a chemically inducible promoter, in which the application of a chemical induces gene expression; or a chemically repressible promoter, in which the application of a chemical inhibits gene expression. Chemically inducible promoters are known in the art and include, but are not limited to, the maize ln2-2 promoter, which is activated by a benzenesulfonamide herbicide safener; the maize GST promoter, which is activated by a hydrophobic electrophilic compound used as a pre-emergence herbicide; and the tobacco PR-1 promoter, which is activated by salicylic acid. Other chemically regulated promoters include steroid-responsive promoters [see, e.g., glucocorticoid-inducible promoters (Schena et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10421-10425; McNellis et al., 1998, Plant 14: 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, e.g., Gatz et al., 1991, Mol. Gen. Genet. 227: 229-237; U.S. Pat. Nos. 5,814,618 and 5,789,156, which are incorporated herein by reference in their entirety). A three-component osmotic-inducible expression system suitable for plant metabolic engineering has recently been reported (Feng et al., 2011, PLoS ONE 6: 1-9).

[0179] constitutive promoter

[0180] Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter disclosed in WO 99 / 43838 and U.S. Pat. No 6,072,050 and other constitutive promoters; the core CaMV 35S promoter (Odell et al., 1985, Nature 313:810-812); rice actin (McElroy et al., 1990, Plant Cell 2:163-171); ubiquitin (Christensen et al., 1989, Plant Mol. Biol. 12:619-632; Christensen et al., 1992, Plant Mol. Biol. 18:675-689); pEMU (Last et al., 1991, Theor. Appl. Genet. 81:581-588); MAS (Velten et al., 1984, EMBO J. 3:2723-2730) and the ALS promoter (U.S. Pat. No. 5,659,026). Other constitutive promoters are described in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

[0181] Weak promoter

[0182] In the case where low-level expression is needed, a weak promoter can be used. Generally, the term "weak promoter" is intended to describe the promoter that drives the expression of a low-level nucleotide sequence. In the case where the promoter is unacceptable with high-level expression, the part of the promoter sequence can be deleted or modified to reduce the expression level. This weak constitutive promoter includes, for example, the core promoter of the Rsyn7 promoter (WO 99 / 43838 and U.S. Patent No 6,072,050).

[0183] Tissue-specific promoter

[0184] "Tissue-preferred" promoters can be used to target gene expression within specific tissues. Developmental and spatially regulated stimuli are less dependent on penetration of plant cells by external factors than chemically inducible systems. Tissue-preferred promoters include those described by Van Ex et al., 2009, Plant Cell Rep. 28:1509-1520; Yamamoto et al., 1997, Plant J. 12:255-265; Kawamata et al., 1997, Plant Cell Physiol. 38:792-803; Hansen et al., 1997, Mol. Gen. Genet. 254:337-343; Russell et al., 199), Transgenic Res. 6:157-168; Rinehart et al., 1996, Plant Physiol. 1 12:1331-1341; Van Camp et al., 1996, Plant Physiol. 1 12:525-535; Canevascini et al., 1996, Plant Physiol. 1 12:513-524; Yamamoto et al., 1994, Plant Cell Physiol. 35:773-778; Lam, 1994, Results Probl. Cell Differ. 20:181-196, Orozco et al., 1993, Plant Mol. Biol. 23:1 129-1 138; Matsuoka et al., 1993, Proc. Natl. Acad. Sci. USA 90:9586-9590, and Guevara-Garcia et al., 1993, Plant J. 4:495-505. If desired, such promoters can be modified for weak expression.

[0185] Seed / embryo specific promoter

[0186] "Seed preferred" promoters include "seed specific" promoters (those active during seed development, such as promoters of seed storage proteins) and "seed germination" promoters (those active during seed germination). See Thompson et al., 1989, BioEssays 10: 108-113, which is incorporated herein by reference. Such seed preferred promoters include, but are not limited to, Ciml (cytokinin-induced messenger), cZ19B l (corn 19 kDa zein), milps (inositol-1-phosphate synthase)) and celA (cellulose synthase). γ-zein is a preferred endosperm-specific promoter. Glob-1 is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, rapeseed protein, β-conglycinin, soybean lectin, cruciferin, etc. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, wax, shrunken 1, shrunken 2, and globulin 1. The stage-specific developmental promoter of the late embryonic abundant protein gene LEA has been successfully used in seed terminator technology to drive a recombination system for excision-mediated lethal gene expression during late embryonic stages (U.S. Pat. No. 5,723,765 to Oliver et al.).

[0187] Leaf-specific promoter

[0188] Leaf-specific promoters are known in the art. See, for example, WO / 2011 / 041499 and U.S. Pat. No. 2011 / 0179511A1 to Thilmony et al., 1997, Plant J. 12: 255-265, Kwon et al., 1994, Plant Physiol. 105: 357-367, Yamamoto et al., 1994, Plant Cell Physiol. 35: 773-778, Gotor et al., 1993, Plant J. 3: 509-518, Orozco et al., 1993, Plant Mol. Biol. 23: 1129-1138, and Matsuoka et al., 1993, Proc. Natl. Acad. Sci. USA 90: 9586-9590.

[0189] Time-specific promoter

[0190] Also contemplated are temporal promoters that can be utilized during a developmental time frame, such as those that turn on after the plant reaches leaf maturity to enhance carbon flow.

[0191] Anther / pollen specific promoter

[0192] Many genes that are specifically expressed in anthers and / or pollen have been identified and their functions in pollen development and fertility have been characterized. The specificity of these genes has been found to be regulated primarily at the transcriptional level by their promoters (Ariizumi et al., 2002, Plant Cell Rep. 21:90-96 and references therein). A large number of anther and / or pollen-specific promoters and their key ds elements from different plant species have been isolated and functionally analyzed.

[0193] Flower-specific promoter

[0194] Flower-preferred promoters include, but are not limited to, CHS (Liu et al., 2011, Plant Cell Rep. 30:2187-2194), OsMADS45 (Bai et al., 2008, Transgenic Res. 17:1035-1043), PSC (Liu et al., 2008, Plant Cell Rep. 27:995-1004), LEAFY, AGAMOUS and API (Van Ex et al., 2009, Plant Cell Rep. 28:1509-1520), API (Verweire et al., 2007, Plant Physiol. 145:1220-1231), PtAGIP (Yang et al., 2011, Plant Mol. Biol. Rep. 29:162-170), Leml (Somleva & Blechl, 2005, Cereal Res. Comm. 33:665-671; Skadsen et al., 2002, Plant Mol. Biol. 45:545-555), Lem2 (Abebe et al., 2005, Plant Biotechnol. J. 4:35-44), AGL6 and AGL13 (Schauer et al., 2009, Plant J. 59:987-1000).

[0195] Promoter combination

[0196] Certain embodiments utilize transgenic plants or plant cells with multigene expression constructs carrying more than one promoter. The promoters can be the same or different.

[0197] Any of the promoters described can be used to control the expression of one or more of the nucleotide sequences of the present invention, their homologues and / or orthologues and any other genes of interest in a defined spatiotemporal manner.

[0198] Maize promoter

[0199] The transgenic DNA construct used to transform plant cells will contain the heterologous nucleotides that are desired to be introduced and a promoter to express the heterologous nucleotides in the host corn cell. As is well known in the art, such constructs may also include elements as needed, such as the following regulatory elements: 3' untranslated regions (such as polyadenylation sites), transit peptides or signal peptides, and marker gene elements, etc. 1. Regulatory elements. Many promoters active in plant cells have been described in the literature, including both constitutive and tissue-specific promoters and inducible promoters. See the description of various promoters that work in plants in the background technology section of U.S. Patent 6,437,217. Such promoters include the nopaline synthase (NOS) and octopine synthase (OCS) promoters, which are carried by the tumor-inducing plasmid of Agrobacterium tumefaciens; caulimovirus promoters, such as the cauliflower mosaic virus (CaMV) 19S and 35S promoters and the figwort mosaic virus (FMV) 35S promoter; the enhanced CaMV 35S promoter (e35S); the light-inducible promoter from the small subunit of ribulose bisphosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide). For example, see U.S. Pat. Nos. 6,437,217, which discloses the maize RS81 promoter; 5,641,876, which discloses the rice actin promoter; 6,426,446, which discloses the maize RS324 promoter; 6,429,362, which discloses the maize PR-1 promoter, 6,232,526, which discloses the maize A3 promoter; and 6,177,611, which discloses a constitutive maize promoter, all of which are incorporated herein by reference.

[0200] Requirements for constructing plant expression cassettes

[0201] First, the nucleotide sequence intended for expression in transgenic plants is assembled behind a suitable promoter active in plants in an expression cassette. The expression cassette may also include any other sequence required or selected for transgenic expression. Such sequences include, but are not limited to, transcription terminators, foreign sequences (such as introns) that enhance expression, vital sequences, and sequences intended for targeting gene products to specific organelles and cell compartments. These expression cassettes may then be transferred to plant transformation vectors as described herein. The following is a description of the various components of a typical expression cassette.

[0202] Transcription terminator

[0203] A variety of transcription terminators can be used in the expression cassette. These transcription terminators are responsible for terminating transcription after the transgene and correct polyadenylation of the transcript. Suitable transcription terminators are terminators known to function in plants and include the CaMV 35S terminator, the tm1 terminator, the nopaline synthase terminator, and the pea rbcS E9 terminator. These terminators are used in both monocots and dicots.

[0204] Sequences used to enhance or regulate expression

[0205] Many sequences have been found to enhance gene expression from within the transcription unit, and these sequences can be used with genes to increase their expression in transgenic plants. For example, various intron sequences (such as the introns of the maize Adhl gene) have been shown to enhance expression, particularly in cells of monocots. In addition, many non-translated leader sequences derived from viruses are also known to enhance expression, and these non-translated leader sequences are particularly effective in cells of dicots.

[0206] Coding sequence optimization

[0207] The coding sequence of a selected gene can be genetically engineered by altering the coding sequence for optimal expression in the crop species of interest. Methods for modifying coding sequences for optimal expression in specific crop species are well known (Perlak et al., 1991, Proc. Natl. Acad. Sci. USA 88:3324 and Koziel et al., 1993, Biotechnology 11:194-200).

[0208] Construction of plant transformation vectors

[0209] Many transformation vectors that can be used for plant transformation are known to those of ordinary skill in the art of plant transformation. The genes associated with the present disclosure can be used with any such vectors. The choice of vector depends on the selected transformation technique and target species.

[0210] Many vectors can be used for transformation using Agrobacterium tumefaciens. These vectors usually carry at least one T-DNA sequence and include vectors such as pBIN19. Typical vectors suitable for Agrobacterium transformation include binary vectors pCIB200 and pCIB2001, and binary vector pCIB10 and its hygromycin selection derivatives. (See, e.g., U.S. Patent No. 5,639,949).

[0211] Transformation without Agrobacterium tumefaciens avoids the requirement for T-DNA sequences in the selected transformation vector, so in addition to vectors such as those containing T-DNA sequences as described above, vectors lacking these sequences are also utilized. The selection of vectors for transformation techniques that are independent of Agrobacterium depends largely on the preferred selection of the species being transformed. Typical vectors suitable for non-Agrobacterium transformation include pCIB3064, pSOG 19, and pSOG35. (See, e.g., U.S. Patent No. 5,639,949).

[0212] Transformation and selection of cultures and plants

[0213] Plant cultures may be transformed and selected using one or more of the above methods well known to those skilled in the art.

[0214] Manipulation of endogenous promoters

[0215] Zinc finger nucleases (ZFNs), TALENs, and CRISPR-Cas9 can also be used to practice the present invention because they allow double-stranded DNA cleavage at specific sites on plant chromosomes, allowing for target gene insertion or deletion (Shukla et al., 2009, Nature 459:437-441; Townsend et al., 2009, Nature 459:442-445). This method can be particularly useful in the present invention to modify the promoter of endogenous genes to modify the expression of genes homologous to PsbS, ZEP, and VDE, which are present in the genome of the plant of interest. In this case, ZFNs, TALENs, or CRISPR / Cas9 can be used to alter the sequence that regulates the expression of the TF of interest to increase expression or change the timing of expression, exceeding the expression present in unengineered or wild-type plants.

[0216] Example

[0217] The present disclosure will be more fully understood by reference to the following examples. However, it should not be construed as limiting the scope of the present disclosure. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and the various modifications or changes made to the examples and embodiments will be suggested to those skilled in the art, and are included in the spirit and authority of the present application and within the scope of the appended claims.

[0218] Example 1. Transgenic tobacco

[0219] Tobacco (Nicotiana tabacum) plants were transformed with a T-DNA cassette containing three Arabidopsis (Arabidopsis thaliana) genes. Arabidopsis ZEP was overexpressed to increase the rate of lutein epoxidation and the corresponding NPQ relaxation rate, and Arabidopsis PsbS was overexpressed to stimulate the amplitude of qE formation, and Arabidopsis VDE was used to maintain the critical level of zeaxanthin for ROS scavenging. The resulting transgenic plants were shown to have modified NPQ dynamics, resulting in higher quantum yields and CO2 fixation without losing photoprotection efficiency under fluctuating light intensities, and increased growth in two independent greenhouse experiments. These results confirm that photosynthetic efficiency is subject to instantaneous NPQ limitations under fluctuating light intensities, and provide the first evidence of the principle of improving photosynthetic efficiency and crop yields through changes in NPQ dynamics.

[0220] Example 2. NPQ and PSII operational efficiency in seedlings

[0221] In the case of 1000 μmol of -2 s -1 The instantaneous NPQ was determined based on chlorophyll fluorescence imaging of the T1 progeny of 20 independent transformation events using the vde-psbs-zep (VPZ) construct after 10 minutes of irradiation followed by a 10-minute dark relaxation period. Since the goal was to maintain similar non-photochemical quenching capabilities, strains with maximum NPQ levels similar to WT were selected for further studies. This resulted in eight strains, from which two strains with a single T-DNA copy (VPZ-34 and 56) and one strain with two T-DNA insertions (VPZ-23) were included in the present work. All results for homozygous T2 progeny are reported. The NPQ kinetic behavior showed considerable differences between the three VPZ strains and the WT ( Figure 1 A). NPQ in the VPZ lines increased rapidly, reaching maximum NPQ levels between two and four minutes, after which NPQ levels stabilized (VPZ-23) or even decreased slightly (VPZ-34 and VPZ-56). In contrast, NPQ in the WT control continued to increase for seven minutes, after which it remained at maximum levels for the remaining three minutes. As a result of these contrasting induction patterns, NPQ was significantly higher in all three transgenic lines during the first five minutes of induction, but not in the last five minutes ( Figure 1 A) After turning off the lights, NPQ relaxation is very rapid and very similar in both WT and VPZ strains.

[0222] At 2000 (3 min) and 200 (2 min) μmol m -2 s-1 Repeated cycling of light intensities between 1 and 2 resulted in even more pronounced NPQ differences between the VPZ line and WT ( Figure 1 B). NPQ in the VPZ strain increased rapidly during the high light phase of the first two cycles, reaching maximum levels during the second minute of the second cycle. In contrast, NPQ increased more slowly in WT seedlings, reaching maximum levels only in the third minute of the fourth cycle. Interestingly, NPQ showed the opposite pattern during the low light phase of the cycle. In the first cycle, NPQ levels were higher in the VPZ strain during the low light phase, however, NPQ levels were equal between the VPZ strain and WT in the second cycle, and NPQ levels were significantly lower in the VPZ strain in the last three cycles.

[0223] The photosynthetic system II (PSII) efficiency estimated by NPQ showed no difference between the VPZ strain and the WT in the first cycle ( Figure 1 C) However, during the following five cycles, the VPZ strain showed superior PSII operation efficiency during the low light phase of the cycle, while no differences were found during the high light phase. This pattern was established in the second cycle and repeated for the remainder of the experiment.

[0224] Example 3. Transcription and protein expression

[0225] All three VPZ lines showed significant increases in combined transgenic (At) and native (Nt) transcript levels for VDE (3-fold), PsbS (3-fold), and ZEP (8-fold) relative to wild type ( Figure 2 A. Figure 2 B and Figure 2 C). For PsbS, the increase in transcript levels translated into approximately 2-fold higher PsbS protein levels ( Figure 2 E), as exemplified by two bands of approximately equal density at approximately 22 kDa ( Figure 2 G, VPZ lane), representing native and transgenic proteins. However, for VDE and ZEP, the increase in transcript levels was amplified at the protein level ( Figure 2 G, the marker band of ZEP is about 73 kDa, and the marker band of VDE is 45 kDa), indicating that VDE ( Figure 2 D) and ZEP( Figure 2 F) Protein was significantly increased (16-fold and 80-fold, respectively). The interaction between transgenic transcript and native transcript and protein levels appears to be negligible, as transcript and protein levels of the native protein are similar in the VPZ strain and WT.

[0226] Example 4. NPQ dynamics in seedlings after repeated changes in light intensity

[0227] To compare the kinetics of dynamic NPQ adjustment, the time constant of a biexponential model was fitted to the time series of NPQ in seedlings as the light intensity varied between 2000 and 200 μmol m -2 s -1 The time constant for NPQ induction varied from 49.3 ± 1.9 (VPZ-34) to 91.8 ± 6.2 (VPZ-56) and from 2000 to 200 μmol m -2 s -1 The readjustment time constants were also similar in WT and the three VPZ strains, with an average of 10.2 s for τ1 and 669.9 s for τ2. In the second 2000 / 200 cycle, the effect of VPZ expression on NPQ kinetics became more evident. The rapid phase of NPQ increase in WT was approximately 2.3 times faster than in the VPZ strains, with a τ1 of 5.5 s in WT versus an average of 12.6 s in the VPZ strains (Table 1). -2 s -1 The second NPQ adjustment showed a significant difference in the component with the slowest NPQ decline. The estimated τ2 of the VPZ strain was found to be 1.9 times faster than that of the WT (464.3 s vs. 886.4 s). Thus, repeated changes in light intensity resulted in faster accumulation and slower relaxation of NPQ in the WT, while the time constant in the VPZ strain was relatively unaffected. This same trend was observed in the final 3 min of 2000 μmol m -2 s -1 The rapid phase of NPQ increase in WT seedlings was about 2.4 times faster than that in the VPZ strain (τ1 was 4.3 s vs. 10.3 s), while the final NPQ relaxation during the 10 min dark period was 1.4 times (τ1) and 3.5 times (τ2) faster in the VPZ strain relative to the WT. In addition, the τ1 and τ2 measured for the recovery of PSII operating efficiency were 2.1 times and 4.1 times faster in the VPZ strain compared to the WT.

[0228] Table 1 Time constants for NPQ adjustment to repeated changes in light intensity (values ​​± se). Asterisks indicate significant differences between the VPZ strain and the wild type (α = 0.05).

[0229]

[0230] a The data in stage 1HL did not constrain two time constants, so the model was simplified to a single exponential function and only one time constant was fitted.

[0231] bThe data resolution is insufficient to properly constrain the fast phase, and only the slow phase is fitted.

[0232] Example 5. NPQ, linear electron transfer and CO2 absorption in steady state

[0233] To measure steady-state gas exchange and chlorophyll fluorescence in fully expanded leaves, light intensity was varied from low to high, taking great care to allow gas exchange and fluorescence to fully stabilize at each intensity. NPQs were very similar between WT and VPZ lines, especially at light intensities below 400 μmol m -2 s -1 hour( Figure 3 A). The corresponding response curves of linear electron transfer and net assimilation rate as a function of absorbed light intensity do not show significant differences between WT and VPZ strains ( Figure 7 ). In addition, the fitting parameter value V derived from the CO2 response curve cmax , J, TPU and Rd were also similar between WT and VPZ strains (Table 2).

[0234] Table 2. Parameters derived from the CO2 response curve. Maximum carboxylation capacity (Vcmax), maximum linear electron transfer rate (Jmax), mitochondrial respiration rate not related to photorespiration (R d ) and maximum triose phosphate utilization rate (TPU). Values ​​± se, n = 10, no significant differences were found between the wild type and VPZ strains.

[0235]

[0236] Example 6. NPQ, electron transfer and CO2 fixation under fluctuating illumination

[0237] To assess the dynamic effects of VPZ overexpression on the shape of the light response curve, 2000 μmol m -2 s -1 The intermittent steps varied the light intensity from high PFD to low PFD in 4-min steps. NPQ in the VPZ line was similar to that of the WT at high light intensity, but significantly lower than that of the WT at low light intensity ( Figure 3 B). The resulting linear electron transfer rate and net assimilation rate response curves were significantly different between the WT and VPZ strains ( Figure 4 A and Figure 4 B). The fitted convexity and asymptote parameters are similar between WT and VPZ strains ( Figure 8 A to Figure Figure 8 D), but the initial slope is significantly different ( Figure 4 C and Figure 4D). The fluctuation intensity in WT plants reduced ΦPSIImax to 0.541±0.012 ( Figure 4 C), while the VPZ lines maintained a less reduced ΦPSIImax of 0.612±0.021 (VPZ-23), 0.599±0.023 (VPZ-34), and 0.595±0.023 (VPZ-56). Similarly, ΦCO2-max in WT plants decreased to 0.058±0.001 ( Figure 4 D), while ΦCO2-max values ​​in the VPZ lines were less affected by intermittent high light intensity, resulting in 0.069 ± 0.003 for VPZ-23, 0.066 ± 0.003 for VPZ-34, and 0.064 ± 0.003 for VPZ-56. Thus, under these fluctuating conditions, the average ΦPSII-max and ΦCO2-max of the VPZ lines were 11.3% and 14.0% higher than those of the WT.

[0238] Example 7. Lutein cycle de-epoxidation as a function of different light treatments

[0239] To evaluate the effect of VPZ overexpression on the xanthophyll cycle, leaves were subjected to four different light treatments (Table 3). The combined pool sizes of violaxanthin, antheraxanthin, and zeaxanthin were similar between WT and VPZ lines. The xanthophyll pigment pool was completely epoxidized in dark-adapted leaves, and no differences were observed between WT and VPZ. -2 s -1 The PFD of 2000 μmol m -2 s -1 Illumination with PFD of 1% resulted in a large accumulation of antheraxanthin and mainly zeaxanthin. The VPZ line retained significantly more violaxanthin and accumulated less zeaxanthin and antheraxanthin than the WT, which resulted in almost two-fold lower lutein DES in the VPZ line than in the WT (25.5% vs. 46.2%). Fluctuating light treatments showed the same trend as high light exposure, with even less lutein de-epoxidation in the VPZ line relative to the WT (17.8% vs. 52.5%).

[0240] Table 3. The results of the experiments in the dark-adapted state or exposed to a constant concentration of 400 or 2000 μmol m -2 s -1 PFD or 3 cycles of 3 min 2000 / 3 min 200 μmol m -2 s -1Xanthophyll cycle pigment concentration and de-epoxidation state (DES) in fully expanded leaves after PFD. Pigment concentrations (values ​​± s.e., n = 3-5) have been normalized per unit leaf area (g m-2). DES (%) = (Zea + 0.5Ant) / (Zea + Ant + Viola), nd = not detected. Asterisks indicate significant differences between VPZ lines and wild type (α = 0.05).

[0241]

[0242] Example 8. Photoprotection efficiency of non-photochemical quenching

[0243] To evaluate the photoprotection efficiency in the VPZ line relative to the WT, seedlings were exposed to 2000 μmol m -2 s -1 The photoprotection index ( Figure 5 ), where a value of 1 equals complete protection from photoinhibition damage. One hour of high light exposure resulted in significant photoinhibition induction, which was slightly higher in WT seedlings than in the VPZ line ( Figure 5 A). Photoprotection efficiency after two hours ( Figure 5 B) was significantly lower than the photoprotection efficiency after 1 hour, but again VPZ seedlings were found to be less photoinhibited than WT. The residual NPQ in VPZ seedlings after 10 minutes of dark recovery tended to be lower ( Figure 5 C). PSII efficiency in VPZ seedlings also tended to be higher after 10 min of dark recovery due to lower residual NPQ and higher photoprotection index ( Figure 5 C).

[0244] Example 9. Plant growth under greenhouse conditions

[0245] To investigate whether the above differences in photosynthetic efficiency affect growth, biomass accumulation was assessed in two greenhouse experiments. Temperature was similar between the two experiments and varied between 21 and 25 °C. In the first experiment, the peak light intensity exceeded 2000 μmol m -2 s -1 And in the second experiment the peak light intensity exceeded 1600 μmol m -2 s -1 , and the daily light integrals in the first and second experiments averaged 21.3 and 19.3 mol m -2 d -1 However, in the last week of the second experiment, due to the seasonal decrease in light intensity, the peak light intensity and the integrated daylight amount dropped significantly to 1037 μmol m-2 s -1 and 8.9 mol m -2 d -1 Thus, the average plant dry weight was significantly higher in the first experiment, 35.6 g vs. 22.5 g, respectively. In both experiments, plants from the VPZ line showed increased stem height ( Figure 6 C) and leaf area ( Figure 6 B). In addition, the total dry weight per plant was 10% to 21% higher in the VPZ lines ( Figure 6 A), which was mainly due to a large increase in stem weight (14% to 26%, Figure 6 E) and an increase in leaf dry weight (7.5% to 16%, Figure 6 D) and an increase in root dry weight (12.5% ​​to 38.3%).

[0246] Example 10. Additional data

[0247] Fig.12 and Fig.13 , leaf, stem, and root size and growth were increased in transgenic plant lines N2-23 and N2-34 compared to the wild type. Fig.14 The NPQ kinetics, quantum yield, and CO2 fixation were shown to be increased in transgenic lines VPZ-56, VPZ-23, and VPZ-34. These results suggest that the leaves were previously exposed to 2000 μmol m -2 s -1 After PFD, quantum yield and CO2 fixation increased at various light intensities. Figures 1, 2, and 3 show the progression of this increase over time after exposure.

[0248] Fig.16 It is shown that the NPQ time constants in the first induction / relaxation are similar, but subsequent light cycles result in slower accumulation of NPQ in transgenic plants (regardless of amplitude, these are time constants) and faster relaxation, as well as faster recovery of quantum yield in dark relaxation. These comparative results are completely consistent with the reduced accumulation of zeaxanthin in transgenic lines. NPQ levels in the high-light VPZ lines are similar to or higher than WT. The qE levels in the VPZ lines estimated by dA532nm are higher than WT, most likely related to PsbS overexpression. Repeated high-light exposure showed higher time constants for NPQ increase and lower time constants for NPQ relaxation in the VPZ lines. Due to the faster NPQ relaxation rate, the VPZ lines showed a 5-10% increase in quantum yield at low PFD during rapid PFD switching.

[0249] Combined overexpression of VDE, ZEP, and PsbS resulted in modified kinetics of NPQ accumulation and relaxation in seedlings and fully grown plants, higher quantum yields in seedlings and fully grown plants under fluctuating light conditions, no significant differences in steady-state gas exchange in fully grown plants, higher total assimilation rates in fully grown plants under fluctuating conditions, and approximately 10% increased growth in the greenhouse.

[0250] Example 11. Materials and methods

[0251] Conversion

[0252] Tobacco cv.'Petite Havana' was transformed using an Agrobacterium-mediated leaf disc protocol according to Clemente, Agrobacterium Protocols (Wang K. ed.), pp. 143–154. Humana Press Inc., Totowa. The binary plasmid contains the coding sequences of the following three genes from Arabidopsis: violaxanthin de-epoxidase (AtVDE), AtPsbS, and zeaxanthin epoxidase (AtZEP); and the bar gene encoding bialaphos resistance (Thompson et al., Journal of Agricultural and Food Chemistry 35, 361–365, 1987). 20 independent T0 transformants were generated, and the T-DNA copy number was determined using digital droplet PCR (ddPCR) analysis of genomic DNA according to Glowacka et al., Plant Cell and Environment doi: 10.1111 / pce.12693, 2015. Two lines with a single copy of T-DNA (VPZ-34 and VPZ-56) and one line with two copies of T-DNA (VPZ-23) were used to generate T1 progeny, where homozygous plants were identified by ddPCR according to Glowacka et al. and self-pollinated to obtain homozygous T2 progeny for further analysis.

[0253] Propagation of plant material

[0254] T2 seeds of VPZ lines and WT seeds from the same harvest date were grown on growth medium (LC1 Sunshine mix, Sun Gro Horticulture, Agawam, MA, USA) in a controlled environment walk-in growth chamber (Environmental Growth Chambers, Chagrin Falls, Ohio, USA) using a 12 h day (23 °C) / 12 h night (18 °C) cycle at 150 μmol quantum m -2 s -1Five days after germination, seedlings were transplanted to 8×12 potting trays (812 series, Hummert International, Earth City, MO, USA) for chlorophyll fluorescence imaging, or transplanted to 9×4 potting trays (3600 series, Hummert International) and grown to two true leaves. After the first transplant, seedlings to be used for gas exchange and biomass analysis were moved to the greenhouse.

[0255] Transcription and protein expression

[0256] Five leaf disks (2.9 cm2 in total) were sampled from the youngest fully expanded leaves of five plants from each line from leaf positions that also undergo gas exchange. Protein and mRNA were extracted from the same leaf samples (NucleoSpin RNA / Protein Kit, REF740933, Macherey-Nagel). The extracted mRNA was treated with DNase (Turbo DNA-free Kit; AM1907, Thermo Fisher Scientific, Waltham, MA, USA) and transcribed into cDNA using the Superscript III First-Strand Synthesis System for RT-PCR (18080-051; Thermo Fisher Scientific). RT-qPCR was used to quantify the expression levels of transgenic AtZEP, AtPsbS and AtVDE and the natural genes NtZEP, NtPsbS and NtVDE relative to NtActin and NtTubulin (primer sequences are provided in the supplementary material).

[0257] After quantification of total protein concentration (protein quantification method ref740967.50, Macherey-Nagel), 4 μg of protein was separated by SDS-PAGE electrophoresis, transferred to a membrane (Immobilon-P, IPVH00010, Millipore, USA) using semi-dry blotting (Trans-Blot SD, Bio-Rad), and stained with a primary antibody against AtPsbS (AS09533, Agrisera, Sweden), primary antibodies against AtZEP (AS08289, Agrisera) and primary antibodies against AtVDE (AS153091, Agrisera) were used for immunolabeling, followed by incubation with secondary antibodies (Promega W401B). Chemiluminescence was detected using a scanner (ImageQuant LAS-4010, Fuji,) and densitometry was performed using ImageJ (version 1.47v, National Institute of Health, USA) to estimate protein concentrations, which were normalized using wild-type protein concentrations.

[0258] NPQ and PSII operation efficiency in seedlings

[0259] Non-photochemical quenching (NPQ) was measured simultaneously in 18 seedlings using a chlorophyll fluorescence imager (CF Imager, Technologica, Colchester, UK). Seedlings were first dark-adapted for 20 min and then illuminated with 800 ms pulses of saturating light intensity (6000 μmol q m -2 s -1 The seedlings were then subjected to 1000 μmol qm for 10 min. -2 s -1 , followed by 10 min of darkness or six cycles of three min of 2000 μmol Qm -2 s -1 , followed by two minutes of 200 μmol qm -2 s -1 Saturating flashes were delivered at regular intervals to image the variable fluorescence (F') and the maximum fluorescence under the illumination condition (Fm'). The mean NPQ for each seedling was then calculated from these measurements according to Equation 1, assuming the Stern-Volmer quenching model:

[0260] NPQ = Fm / Fm'-1 Equation 1

[0261] The maximum PSII efficiency or operating PSII efficiency was estimated from the fluorescence measurements according to equations 2 and 3 according to Genty et al., Biochimica et Biophysica Acta 990, 87-92 1989, 1989.

[0262] Maximum PSII efficiency = (Fm – Fo) / Fm Equation 2

[0263] PSII operating efficiency = (Fm'–F') / Fm' Equation 3

[0264] Time constant for adjusting NPQ to changes in light intensity

[0265] The seedlings were dark-adapted and chlorophyll fluorescence was measured using a chlorophyll fluorescence imager as described above. The light intensity was varied from 2000 to 200, 2000, 200, 2000 and finally 0 μmol quantum m every 3 min. -2 s -1 , maximum fluorescence was measured every 30 seconds. Final relaxation in the dark lasts for 10 minutes. Correspondingly, three VPZ-transformed strains and wild type of six groups of 18 seedlings were measured, thus generating a 5-second frameshift between fluorescence measurement and light intensity changes between groups. NPQ is calculated according to equation 1, normalized for the highest value in each group, and then all six groups are compiled as a function of time to generate a time series of normalized NPQ with a resolution of 5 seconds. The time constant in the biexponential function for the induction or relaxation of NPQ is fitted with the compiled time series after each light intensity change. For final recovery in the dark, the time constant is also fitted with the PSII operating efficiency (estimated using equation 3).

[0266] Light protection efficiency

[0267] The seedlings were dark-adapted for 20 min, after which the maximum PSII efficiency (Fv / Fm) of dark adaptation was determined using a chlorophyll fluorescence imager as described above. Subsequently, the seedlings were exposed to 2000 μmol Q m -2 s -1 Continue for 60min or 120min. After exposure, the seedlings were allowed to recover in the dark for 10min to relax qE, after which the minimum fluorescence (Fo') and maximum fluorescence (Fm') without complete dark adaptation were measured. The measured values ​​of Fo' were compared with the derived values, which only considered the effect of NPQ on Fo' (Oxborough and Baker, Photosynthesis Research 54: 135-142, 1997). The difference between the PSII efficiency of the measured or derived Fo' was then used to determine the photoprotection efficiency.

[0268] Gas exchange and linear electron transport in fully expanded leaves

[0269] For gas exchange analysis, seedlings were transplanted from trays into 3.8L pots (400C, Hummert International) containing growth medium (LC1 Sunshine mix, supplemented with 10g granular fertilizer (Osmocote Plus 15 / 9 / 12, The Scotts Company LLC, Marysville, Ohio, USA) per pot). The pots were randomized and spaced 30cm apart on a greenhouse table. Plants were watered and plant positions were randomly changed every two days until the fifth leaf was fully expanded. Gas exchange measurements were performed using an open gas exchange system (LI6400XT, LI-COR, Lincoln, Nebraska, USA) equipped with a 2cm2 leaf chamber fluorometer. All gas exchange measurements were corrected for diffusion leakage between the sample pool (cuvette) and the surrounding atmosphere according to Gong et al., Plant, Cell and Environment 38, 2417–2432 2015).

[0270] To determine the light dose response curves for net assimilation rate and linear electron transport in fully expanded leaves, gas exchange and pulse amplitude modulated chlorophyll fluorescence were measured over a range of light intensities. All chlorophyll fluorescence measurements used a multiphase flash routine (Loriaux et al., 2013). The youngest fully expanded leaves (n=6) were clamped in a sample cell, where the block temperature was set at 25°C and [CO2] in the airflow was controlled at 1500 ppm. After 30 min of dark adaptation, the minimum fluorescence (Fo) and maximum fluorescence (Fm) were measured. Subsequently, the light intensity was varied in two different ways. The first experiment consisted of a slow increase from 0 to 50, 80, 110, 140, 170, 200, 400, 600, 800, 1000, 1200, 1500 and 2000 μmol m -2 s -1 The light intensity composition was set to try to keep the NPQ induction at each light intensity to an absolute minimum. When steady state was reached, gas exchange parameters were recorded and baseline fluorescence (F') and light-adapted maximum fluorescence (Fm') were measured to estimate NPQ (Equation 1) and PSII operating efficiency (Equation 3). In the second experiment, leaves were exposed to 2000 μmol m -2 s -1 After that, the light intensity was changed from 2000 to 1500, 1000, 800, 600, 400, 200, 170, 140, 110, 80, and 50, each step lasting 4 min, and each step was preceded by 4 min of 2000 μmol m -2 s-1 At each light intensity, F' and Fm' as well as gas exchange parameters were determined after 60 s, 140 s and 220 s. The average of these three measurements was used for subsequent analysis to reconstruct the light response curve with intermittent high PFD.

[0271] The leaf absorbance of incident irradiance was measured at the same point used for gas exchange analysis using an integrating sphere (LI1800, LI-COR, USA) connected to a spectrometer (USB-2000, Ocean Optics Inc, Dunedin, Florida, USA). The linear electron transfer rate (J) for both experiments was determined according to the following formula:

[0272] J = Leaf absorption rate * PSII operating efficiency * PFD * 0.5 Equation 4

[0273] Light intensity dose response curves for linear electron transport and gas exchange from two experiments were adjusted to constant leaf temperature according to the equations of Sharkey et al. (2007) and fit to a descriptive non-rectangular hyperbolic model (Von Caemmerer, Biochemical models of leaf photosynthesis. Collingwood, Australia: CSIRO Publishing 2000) to produce estimates of initial slope, convexity, and asymptotes.

[0274] To analyze the CO2 dose-response curve of the net assimilation rate, leaves were clamped in a sample cell where the block temperature was controlled at 25 °C and the light intensity was set to 2000 μmol m -2 s -1 The CO2 concentration in the gas stream was controlled at 400, 300, 200, 100, 75, 400, 400, 500, 600, 700, 800, 1200, and 1600 ppm, and gas exchange parameters were recorded when steady state was reached. The leaf photosynthesis model of Farquhar et al. Planta 149, 78–90.1980 assumes infinite mesophyll conductance and is temperature corrected according to Sharkey et al. Plant Cell Environ. 30, 1035–1040,2007. The maximum carboxylation rate (Vcmax), 2000 μmol m -2 s -1 The electron transfer rate (J), triose phosphate utilization rate (TPU) and mitochondrial respiration rate (Rd) independent of photorespiration were analyzed.

[0275] Xanthophyll cycle pigment concentration

[0276] Leaves were clamped in a leaf sample cell of an open gas exchange system and dark-adapted as described above. Subsequently, CO2 and H2O were exchanged at 0, 400, and 2000 μmol m -2 s -1 After reaching steady state or undergoing a series of light intensity changes (3 min 2000 / 3 min 200 μmol m -2 s -1 The pigments were analyzed by HPLC using a Zorbax Eclipse XDB-C8 column (963967-906, Agilent Technologies, Santa Clara, CA, USA) and quantitatively analyzed according to the protocol proposed by Van Heukelem and Thomas (2001).

[0277] Growth and final biomass accumulation

[0278] To evaluate the effect of VPZ overexpression on growth, two independent greenhouse experiments were performed from May 25 to June 29, 2015 (using WT, VPZ-23 and VPZ-34) and from October 9 to November 13, 2015 (using WT, VPZ-23, VPZ-34 and VPZ-56). Seedlings were propagated as described above (plant propagation paragraph) and transplanted from trays to 14.5L pots (2000C, Hummert International) filled with growth medium (LC1 Sunshine mixture, Sun Gro Horticulture) and supplemented with 30g slow-release granular fertilizer (OsmocotePlus 15 / 9 / 12, The Scotts Company LLC) per pot. Pots were randomly arranged and placed on a greenhouse table at 30cm intervals. Plants were watered and plant positions were randomly changed every two days. The light intensity at the leaf level was recorded using a quantum sensor (LI-190R, LI-COR, USA) located at the center of the greenhouse table. The quantum sensor was mounted on a tripod and adjusted every day to keep the position of 10 cm above the youngest leaves. Air temperature, relative humidity, and [CO2] were measured at approximately 1 m above the plant canopy using a temperature and humidity combination sensor (HMP60-L, Vaisala Oyj, Helsinki, Finland) and an infrared gas analyzer (SBA-5, PPsystems, Amesbury, MA, USA). All climate data were recorded every 30 min using a data logger (CR1000, Campbell Scientific Inc, Logan, UT, USA). The temperature in the greenhouse was maintained between 28 ° C (daytime) and 18 ° C (night) using a combination of ventilation, evaporative cooling, and a gas heater. Light intensity varied with incident irradiance, with the peak at noon in the first experiment reaching approximately 1800 μmol m -2 s -1 , and the peak at noon in the second experiment reached about 1000–1500 μmol m -2 s -1 [CO2] was not controlled and varied between 360 ppm (daytime) and 430 ppm (nighttime). After the first flower bloomed, stem length and the number of leaves per plant were determined, and the total leaf area per plant was measured using a conveyor belt scanner (LI-3100C area meter, LI-COR, USA). The plants were then separated into leaf, stem and root parts and dried at 70°C to constant weight.

[0279] Statistical analysis

[0280] All statistical analyses were performed using SAS (version 9.3, SAS Institute Inc., Cary, NC, USA). The data were tested for homogeneity of variance using the Brown-Forsythe test, and for normality using the Shapiro-Wilk test. One-way ANOVA was applied to fitted gas exchange parameters, transcript levels, and protein expression. Data sets for chlorophyll fluorescence imaging of NPQ in seedlings were analyzed by two-way ANOVA (light protection) or repeated measures one-way (10 min on / off) or two-way (high / low light) ANOVA. Two replicate greenhouse experiments were analyzed using a mixed model with two completely randomized blocks. In all cases, Dunnett's multiple comparison test (α=0.05) was performed for line means relative to WT controls after significant effects of ANOVA. Fitted time constants for NPQ induction and relaxation were compared based on 95% confidence intervals.

[0281] Example 12. Transgenic corn (hypothetical example)

[0282] The present invention can also provide a corn strain with improved photosynthesis and growth compared to the phenotype in the parental unit of the corn strain. The corn strain can be created in the following manner: a transgenic plant group is generated, the transgenic plant group comprises a heterologous nucleotide sequence encoding a polypeptide selected from PsbS, ZEP and VDE as described herein. Each transgenic event includes introducing at least one nucleotide construct into the genome of the parental plant, the nucleotide construct comprising a promoter operably connected to a heterologous nucleotide as described herein. A sufficient amount of the nucleotide construct is introduced into the parental genome to produce a transgenic cell, which can be cultured into a transgenic corn plant with the enhanced phenotype. The transgenic cell is cultured into a transgenic plant that produces offspring transgenic seeds. The transgenic plant group is screened for observable phenotypes. Seeds are collected from transgenic plants selected to have an unexpectedly enhanced phenotype. Optionally, the method comprises repeating the following cycle: germinating the transgenic seeds, growing offspring plants from the transgenic seeds, observing the phenotype of the offspring plants, and collecting seeds from offspring plants with an enhanced phenotype. In another aspect, the method screens a large population by employing at least one heterologous nucleotide sequence encoding a polypeptide selected from the group consisting of PsbS, ZEP and VDE. Other preferred aspects of the method employ a nucleotide construct in which the heterologous DNA is operably linked to a selected promoter (e.g., the 5' end of the promoter region). The DNA construct may be introduced into a random location in the genome or into a preselected site in the genome.

[0283] An example of a maize transformation protocol is described herein.

[0284] Start Agrobacterium culture

[0285] 1. AGL1 carrying a simple binary vector (e.g. pZY102) from a -80°C stock was streaked on ABC agar plates containing appropriate antibiotics (100 mg / L spectinomycin and 30 mg / L rifampicin for the vector and strain shown here) to prepare serial dilutions to obtain single colonies. The plates were incubated at 28°C in the dark for 3 days.

[0286] 2. Pick a single colony and streak it on YEP agar plates containing the appropriate antibiotics (100 mg / L spectinomycin and 30 mg / L rifampicin for the vector and strain shown here). Incubate the plates in the dark at 20°C for 3 days.

[0287] 3. Add 5 ml of sterile PHI-A (inoculation medium) into a 15 ml conical centrifuge tube.

[0288] 4. Transfer two full loops of AGL1 from the YEP plate to the test tube prepared in step 3. After 2 to 3 minutes, vortex the test tube to thoroughly suspend the bacterial cells.

[0289] 5. Take out 1 ml of the suspension and place it in a spectrophotometer cuvette to check the optical density at 550 nm (OD550). Adjust the cell suspension to an OD550 of 0.35 (0.5×10 9 cfu / ml).

[0290] 6. Shake the culture on a shaker at 100 rpm for 4 to 5 hours at room temperature (eg, 24°C).

[0291] 7. Aliquot 1 ml of the suspension into 2 ml sterile microcentrifuge tubes.

[0292] Embryo isolation, inoculation and co-cultivation

[0293] 8. Remove the bracts and silks from the spikes harvested 10 to 13 days after pollination (where the embryo size is 1.5 mm; see supporting protocol). Insert a pair of forceps into one end of the spike.

[0294] 9. Completely immerse fresh Hi-II compound spikes in a solution containing 0.5 liter of 30% commercial bleach and a few drops of Tween 20 in a sterile 1 liter jar for 20 minutes.

[0295] 10. Rinse the compound spike three times with sterile water (make sure the compound spike is completely immersed in water each time), and place the compound spike upright on a sterile 150×15 mm culture dish.

[0296] 11. Using a sterile #11 razor blade, remove the top half of the kernels from each ear.

[0297] 12. Isolate 1.5 mm immature embryos from sterile compound spikes using a sterile small spatula and transfer 50 to 100 embryos per 1.7 to 2.0 ml microcentrifuge tube. Wash embryos three times with 1 ml PHI-A solution to remove debris and starch.

[0298] 13. Immediately add 1 ml of the Agrobacterium suspension to the tube containing the immature embryos, let the tube sit in a sterile hood for 5 min, and then pour the entire content including all embryos onto a PHI-B (co-culture) agar plate.

[0299] 14. Use a pipette with a tip to draw up the Agrobacterium suspension, then spread the embryos evenly on the plate and place the embryos on the medium with the cotyledons facing up and the flat side facing down.

[0300] 15. Seal the plate with parafilm and incubate at 20°C in the dark for 3 days.

[0301] Rest

[0302] 16. Use a spatula to transfer the embryos to a PHI-C (resting medium) plate. Avoid damaging the embryos.

[0303] 17. Seal the plates with parafilm and incubate at 28°C in the dark for 7 days.

[0304] choose

[0305] 18. Use a spatula or forceps to transfer the embryos to PHI-D1 (selection medium I) plates. Place 25 embryos per plate and seal the plates. Incubate the embryos in the dark at 28°C for the first 2 weeks of selection.

[0306] 19. Use forceps to transfer the callus from the PHI-D1 plate to a PHI-D2 (selective medium II) plate. Using the incubation conditions in step 18, subculture the callus onto fresh PHI-D2 medium every 2 weeks for a total of 2 months.

[0307] 20. Enlarge the herbicide-resistant calli by growing the herbicide-resistant calli on fresh PHI-D2 medium under the same conditions as in steps 18 and 19 for an additional 2 weeks until the calli are approximately 1.0 cm in diameter.

[0308] Maturation and regeneration

[0309] 21. Using forceps, transfer each intact callus mass containing opaque embryos to a 20 x 100 mm dish of PHI-E (maturation medium) (wrapped with 3M porous tape) and place the culture plates in the dark at 28°C for two cycles of 2 to 3 weeks to allow somatic embryo maturation.

[0310] 22. Transfer the milky white callus onto PHI-F (regeneration medium) and incubate at 25°C under a 16-hour photoperiod until shoots and roots develop.

[0311] 23. Transfer each plantlet to a 25 x 150 mm tube containing PHI-F (regeneration medium) and grow at 25°C under a 16 hr photoperiod for 2 to 3 weeks.

[0312] 24. Transfer the plants to small plastic pots with soil mix (e.g., Promix BX soil) and place in a lighted incubator or chamber at 24°C with an 18 hr / light, 6 hr / dark cycle.

[0313] Example 13. Transgenic sorghum (hypothetical example)

[0314] The present invention can also provide a sorghum strain having improved photosynthesis and growth compared to the phenotype in the parental unit of the sorghum strain. The sorghum strain can be created in the following manner: a transgenic plant population is generated, the transgenic plant population comprising a heterologous nucleotide sequence encoding a polypeptide selected from PsbS, ZEP and VDE as described herein. Each transgenic event includes introducing at least one nucleotide construct into the genome of the parent plant, the nucleotide construct comprising a promoter operably linked to a heterologous nucleotide as described herein. A sufficient amount of the nucleotide construct is introduced into the parental genome to produce a transgenic cell, which can be cultured into a transgenic sorghum plant with the enhanced phenotype. The transgenic cell is cultured into a transgenic plant that produces offspring transgenic seeds. The transgenic plant population is screened for observable phenotypes. Seeds are collected from transgenic plants selected to have an unexpectedly enhanced phenotype. Optionally, the method includes repeating the following cycle: germinating the transgenic seeds, growing offspring plants from the transgenic seeds, observing the phenotype of the offspring plants, and collecting seeds from offspring plants with an enhanced phenotype. In another aspect, the method screens a large population by employing at least one heterologous nucleotide sequence encoding a polypeptide selected from the group consisting of PsbS, ZEP and VDE. Other preferred aspects of the method employ a nucleotide construct in which the heterologous DNA is operably linked to a selected promoter (e.g., the 5' end of the promoter region). The DNA construct may be introduced into a random location in the genome or into a preselected site in the genome.

[0315] Examples of sorghum transformation protocols are described in Guo et al., Methods Mol Biol 1223, 181-188, 2015 and Howe et al., Plant Cell Rep 25(8):784-791, 2006.

[0316] Example 14. Genetically modified soybean (hypothetical example)

[0317] The present invention can also provide a soybean line with improved photosynthesis and growth compared to the phenotype in the parental unit of the soybean line. The soybean line can be created in the following manner: a transgenic plant group is generated, the transgenic plant group comprises a heterologous nucleotide sequence encoding a polypeptide selected from PsbS, ZEP and VDE as described herein. Each transgenic event includes introducing at least one nucleotide construct into the genome of the parent plant, the nucleotide construct comprising a promoter operably connected to a heterologous nucleotide as described herein. A sufficient amount of the nucleotide construct is introduced into the parental genome to produce a transgenic cell, which can be cultured into a transgenic soybean plant with the enhanced phenotype. The transgenic cell is cultured into a transgenic plant that produces offspring transgenic seeds. The transgenic plant group is screened for observable phenotypes. Seeds are collected from transgenic plants selected to have an unexpectedly enhanced phenotype. Optionally, the method includes repeating the following cycle: germinating the transgenic seeds, growing offspring plants from the transgenic seeds, observing the phenotype of the offspring plants, and collecting seeds from offspring plants with an enhanced phenotype. In another aspect, the method screens a large population by employing at least one heterologous nucleotide sequence encoding a polypeptide selected from the group consisting of PsbS, ZEP and VDE. Other preferred aspects of the method employ a nucleotide construct in which the heterologous DNA is operably linked to a selected promoter (e.g., the 5' end of the promoter region). The DNA construct may be introduced into a random location in the genome or into a preselected site in the genome.

[0318] An example of a soybean transformation protocol is described herein.

[0319] Prepare cotyledon explants from 5 day-old soybean seedlings by preparing horizontal slices through the hypocotyl region of about 3-5mm below the cotyledon.Prepare vertical slices between the cotyledons subsequently, and remove hypocotyls.This manipulation produces 2 cotyledon node explants.On the adaxial surface of the explant, approximately 7 to 12 vertical slices are prepared in the region of 3mm above the cotyledon / hypocotyl junction and 1mm below the cotyledon / hypocotyl junction.Use No. 15 surgical blades to carry out explant manipulation.

[0320] The explants were immersed in the Agrobacterium inoculum for 30 min and then co-cultivated on 100×15 mm Petri dishes containing Agrobacterium resuspension medium solidified with 0.5% purified agar (BBL Cat#11853). The co-cultivation plates were covered with a piece of Whatman #1 filter paper (Mullins et al., 1990; Janssen and Gardner, 1993; Zhang et al., 1997). Explants (5 per plate) were placed on the co-cultivation plates covered with filter paper and incubated at 24°C in a 1% CO 2 solution using 80 μmol s -1 m -2 The cells were cultured adaxially downward under 18 / 6 hour light conditions with a light intensity of approximately 100 μm (F17T8 / 750 cool white light bulb, Litetronics) for 3 days. The co-culture plates were wrapped with parafilm.

[0321] After the co-cultivation period, the explants were briefly washed in B5 medium supplemented with 1.67 mg l-1 BAP, 3% sucrose, 500 mg l-1 ticarcillin and 100 mg l-1 cefotaxime. The medium was buffered with 3 mM MES at pH 5.6. Growth regulators, vitamins and antibiotics were filter sterilized after autoclaving. After the washing step, the explants were cultured in 100×20 mm culture dishes (5 per culture plate) containing a wash medium solidified with 0.8% purified agar (BBL Cat#11853) and modified with 3.3 or 5.0 mg l-1 glufosinate (AgrEvo USA), with the explants facing up on the adaxial side and the hypocotyls embedded in the culture medium. This culture medium is called shoot initiation medium (SI). The plate was wrapped with 3M pressure-sensitive tape (ScotchTM, 3M, USA) and cultured under the environmental conditions used during the seed germination step.

[0322] After 2 weeks of cultivation, the hypocotyl region is cut off from each explant, and the remaining explant, i.e. the cotyledon with differentiation node, is cultivated on fresh SI medium subsequently. After another 2 weeks of cultivation on SI medium, the cotyledon is removed from the differentiation node. The differentiation node is subcultured to bud elongation (shoot elongation medium, SE) medium, and the SE medium is composed of the basal salts, B5 vitamins, 1mgl-1 zeatin riboside, 0.5mg l-1GA3 and 0.1mg l-1IAA, 50mg l-1 glutamine, 50mg l-1 asparagine, 3% sucrose and 3mM MES of pH 5.6 of Murashige and Skoog (MS) (1962). The SE medium is improved with 1.7 or 2.0mg l-1 glufosinate. Explants are subcultured to fresh SI medium every two weeks until the bud reaches a length greater than 3cm. Elongated shoots were rooted on Murashige and Skoog salts with B5 vitamins, 1% sucrose, 0.5 mg l-1 NAA without further selection in Magenta boxes or Sundae cups (Industrial Soap Company, St. Louis MO).

[0323] Example 15. Transgenic rice (hypothetical example)

[0324] The present invention can also provide a rice line with improved photosynthesis and growth compared to the phenotype in the parental unit of the rice line. The rice line can be created in the following manner: a transgenic plant group is generated, the transgenic plant group comprises a heterologous nucleotide sequence encoding a polypeptide selected from PsbS, ZEP and VDE as described herein. Each transgenic event includes introducing at least one nucleotide construct into the genome of the parent plant, the nucleotide construct comprising a promoter operably connected to a heterologous nucleotide as described herein. A sufficient amount of the nucleotide construct is introduced into the parental genome to produce a transgenic cell, which can be cultured into a transgenic rice plant with the enhanced phenotype. The transgenic cell is cultured into a transgenic plant that produces offspring transgenic seeds. The transgenic plant group is screened for observable phenotypes. Seeds are collected from transgenic plants selected to have an unexpectedly enhanced phenotype. Optionally, the method comprises repeating the following cycle: germinating the transgenic seeds, growing offspring plants from the transgenic seeds, observing the phenotype of the offspring plants, and collecting seeds from offspring plants with an enhanced phenotype. In another aspect, the method screens a large population by employing at least one heterologous nucleotide sequence encoding a polypeptide selected from the group consisting of PsbS, ZEP and VDE. Other preferred aspects of the method employ a nucleotide construct in which the heterologous DNA is operably linked to a selected promoter (e.g., the 5' end of the promoter region). The DNA construct may be introduced into a random location in the genome or into a preselected site in the genome.

[0325] An example of a rice transformation protocol is described herein.

[0326] Infection and co-culture

[0327] The callus was transferred to a sterile tea strainer and incubated in the Agrobacterium suspension by shaking the strainer very gently and intermittently for 15 min, then blotted on Whatmann 1 sterile filter paper stacked in a sterile petri dish to remove excess bacteria. The callus was transferred to sterile filter paper placed on MSG4K medium and cultured at 25°C in the dark for 48 h.

[0328] Rest and Basta options

[0329] The co-cultivated callus was transferred to a sterile 50 ml test tube, washed 5 times with sterile water and once with a liquid co-cultivation medium containing 200 mg / l Timentin. The callus was blotted dry with sterile Whatman filter paper and transferred to a plate containing MSG2K "resting" medium. The callus plates were cultured in the dark at 25°C for 7 days. The callus was transferred to MSG2K bialaphos selection medium. The plates were cultured in the dark at 25°C for 3 weeks. The process was repeated for another 5 weeks, with subcultures to fresh medium every 3 weeks.

[0330] Callus drying, shoot regeneration and rooting

[0331] After 8 weeks, callus is transferred to sterile culture dish, and described culture dish is stacked with 2 layers of aseptic Whatman#1 filter paper in sterile hood.Culture dish is wrapped with 3M surgical tape and stays in sterile hood, so that culture dish is kept undisturbed in the dark for 24 hours.These flat plates need to be wrapped in aluminum foil to ensure the darkness to callus.This callus part drying step is absolutely necessary for inducing bud in bud regeneration medium.After 48h, callus is transferred to MSG75K bud regeneration medium and hatched in the dark for 3 weeks.The proliferation callus with somatic embryo is transferred to the same medium and hatched in 12 hours / 8 hours dark cycle under about 20 to 30 μ Em-2s-1 low light.Bud will begin to appear after illumination 10 days, and most of callus will turn green.Continue to cultivate green callus along embryo in MSG75K bud regeneration medium under the same light conditions, until obtaining the bud of satisfactory amount. Meanwhile, when the shoots reached more than 5 cm in length, they were cut at the base and transferred to Greiner Bio-One plant culture containers (#968161-82051-508-lid container, 330 ml, sterile, 68 diameter×110 height mm) with MSG100K rooting medium.

[0332] Example 16. Transgenic wheat (hypothetical example)

[0333] The present invention can also provide wheat lines with improved photosynthesis and growth compared to the phenotype in the parental units of the wheat lines. The wheat lines can be created in the following manner: a transgenic plant group is generated, the transgenic plant group comprises a heterologous nucleotide sequence encoding a polypeptide selected from PsbS, ZEP and VDE as described herein. Each transgenic event comprises introducing at least one nucleotide construct into the genome of the parental plant, the nucleotide construct comprising a promoter operably connected to a heterologous nucleotide as described herein. A sufficient amount of the nucleotide construct is introduced into the parental genome to produce a transgenic cell, which can be cultured into a transgenic wheat plant with the enhanced phenotype. The transgenic cell is cultured into a transgenic plant that produces offspring transgenic seeds. The transgenic plant group is screened for observable phenotypes. Seeds are collected from transgenic plants selected to have an unexpectedly enhanced phenotype. Optionally, the method comprises repeating the following cycle: germinating the transgenic seeds, growing offspring plants from the transgenic seeds, observing the phenotype of the offspring plants, and collecting seeds from offspring plants with an enhanced phenotype. In another aspect, the method screens a large population by employing at least one heterologous nucleotide sequence encoding a polypeptide selected from the group consisting of PsbS, ZEP and VDE. Other preferred aspects of the method employ a nucleotide construct in which the heterologous DNA is operably linked to a selected promoter (e.g., the 5' end of the promoter region). The DNA construct may be introduced into a random location in the genome or into a preselected site in the genome.

[0334] An example of a wheat transformation protocol is described in Medvecká E, Harwood WA. Wheat (Triticum aestivum L.) Transformation Using Mature Embryos. Agrobacterium Protocols: Vol. 1, 2015: 199-209.

[0335] Example 17. Transgenic cowpea

[0336] According to the transformation scheme described in Higgins et al., Innovative research along the cowpea valuechain. Ibadan, Nigeria: International Institute of Tropical Agriculture, pp. 133-139, 2013, cowpea plants were transformed with T-DNA constructs containing nucleotide sequences encoding PsbS, ZEP and VDE. In order to compare the dynamics of dynamic NPQ adjustment, the double exponential model was fitted to the NPQ dark relaxation in T0 transgenic cowpea after exposure to fluctuating light. As shown in Table 4, the qE relaxation (τ1) in the transformed strain 164381A was significantly faster than the control, with the measured values ​​of 20.5s and 19.7s versus 35.9s and 29.7s. The qZ phase (τ2) of the NPQ relaxation in the transformed strain 1643B1 was slower than the control, which may be due to the limitation of the measurement to the T0 transgenic plant. As is known in the art, for phenotypic measurements, transgenic plants from T1 or T2 generations are generally preferred to T0. One reason is that in T0 transgenic plants, stress from the transformation and tissue culture process may interfere with normal plant physiology and affect phenotypic measurements. Another reason is that molecular characterization of transgenes cannot be completed until the T1 or T2 generation, and transgenic characteristics such as copy number and insertion position in the genome can have a significant impact on transgenic expression. Fig. 27 , Fig.28 and Fig.29 As shown, NPQ in transformed line 1643B1 relaxes faster than in control plants. Fig.30 As shown, NPQ in transformed line CP472A (orange dots) relaxes more slowly than in control plants (blue dots), which may be due to the limitation that the measurements were performed on TO transgenic plants as described above.

[0337] Table 4. Time constants for NPQ relaxation in transgenic cowpea.

[0338]

[0339] Example 18. Genetically modified cassava (hypothetical example)

[0340] The present invention can also provide cassava strains with improved photosynthesis and growth compared to the phenotype in the parental units of the cassava strains. The cassava strains can be created in the following manner: a transgenic plant population is generated, the transgenic plant population comprises a heterologous nucleotide sequence encoding a polypeptide selected from PsbS, ZEP and VDE as described herein. Each transgenic event comprises introducing at least one nucleotide construct into the genome of the parental plant, the nucleotide construct comprising a promoter operably connected to a heterologous nucleotide as described herein. A sufficient amount of the nucleotide construct is introduced into the parental genome to produce a transgenic cell, which can be cultured into a transgenic cassava plant with the enhanced phenotype. The transgenic cell is cultured into a transgenic plant that produces offspring transgenic seeds. The transgenic plant population is screened for observable phenotypes. Seeds are collected from transgenic plants selected to have an unexpectedly enhanced phenotype. Optionally, the method comprises repeating the following cycle: germinating the transgenic seeds, growing offspring plants from the transgenic seeds, observing the phenotype of the offspring plants, and collecting seeds from offspring plants with an enhanced phenotype. In another aspect, the method screens a large population by employing at least one heterologous nucleotide sequence encoding a polypeptide selected from the group consisting of PsbS, ZEP and VDE. Other preferred aspects of the method employ a nucleotide construct in which the heterologous DNA is operably linked to a selected promoter (e.g., the 5' end of the promoter region). The DNA construct may be introduced into a random location in the genome or into a preselected site in the genome.

[0341] An example of a cassava transformation protocol is described in Chetty et al., New Biotechnology 30.2:136-143, 2013.

[0342] Example 19. Transgenic poplar (hypothetical example)

[0343] The present invention can also provide a poplar strain with improved photosynthesis and growth compared to the phenotype in the parental unit of the poplar strain. The poplar strain can be created in the following manner: a transgenic plant group is generated, the transgenic plant group comprises a heterologous nucleotide sequence encoding a polypeptide selected from PsbS, ZEP and VDE as described herein. Each transgenic event includes introducing at least one nucleotide construct into the genome of the parent plant, the nucleotide construct comprising a promoter operably connected to a heterologous nucleotide as described herein. A sufficient amount of the nucleotide construct is introduced into the parental genome to produce a transgenic cell, which can be cultured into a transgenic poplar plant with the enhanced phenotype. The transgenic cell is cultured into a transgenic plant that produces offspring transgenic seeds. The transgenic plant group is screened for observable phenotypes. Seeds are collected from transgenic plants selected to have an unexpectedly enhanced phenotype. Optionally, the method includes repeating the following cycle: germinating the transgenic seeds, growing offspring plants from the transgenic seeds, observing the phenotype of the offspring plants, and collecting seeds from offspring plants with an enhanced phenotype. In another aspect, the method screens a large population by employing at least one heterologous nucleotide sequence encoding a polypeptide selected from the group consisting of PsbS, ZEP and VDE. Other preferred aspects of the method employ a nucleotide construct in which the heterologous DNA is operably linked to a selected promoter (e.g., the 5' end of the promoter region). The DNA construct may be introduced into a random location in the genome or into a preselected site in the genome.

[0344] An example of a poplar transformation protocol is described in Movahedi et al., International Journal of Molecular Science 15.6: 10780-10793, 2014.

[0345] Example 20. Transgenic Eucalyptus (hypothetical example)

[0346] The present invention can also provide eucalyptus strains with improved photosynthesis and growth compared with the phenotype in the parental unit of the eucalyptus strain. The eucalyptus strain can be created in the following manner: produce a transgenic plant group, the transgenic plant group comprises a heterologous nucleotide sequence of a polypeptide selected from PsbS, ZEP and VDE as described herein. Each transgenic event comprises introducing at least one nucleotide construct into the genome of the parental plant, the nucleotide construct comprising a promoter operably connected to a heterologous nucleotide as described herein. A sufficient amount of the nucleotide construct is introduced into the parental genome to produce a transgenic cell, which can be cultured into a transgenic eucalyptus plant with the enhanced phenotype. The transgenic cell is cultured into a transgenic plant that produces offspring transgenic seeds. Screening for observable phenotypes in the transgenic plant group. Collect seeds from the transgenic plant selected to have an unexpectedly enhanced phenotype. Optionally, the method comprises repeating the following cycle: germinate the transgenic seeds, grow offspring plants from the transgenic seeds, observe the phenotype of the offspring plants, and collect seeds from offspring plants with an enhanced phenotype. In another aspect of the method, the large population is screened by using at least one heterologous nucleotide sequence encoding a polypeptide selected from PsbS, ZEP and VDE. Other preferred aspects of the method employ a nucleotide construct in which the heterologous DNA is operably linked to a selected promoter (e.g., the 5' end of the promoter region). The DNA construct may be introduced into a random location in the genome or into a preselected site in the genome.

[0347] An example of a eucalyptus transformation protocol is described in Diwakar et al., Plant Tissue Culture: Propagation, Conservation and Crop Improvement, 219-244, 2016.

[0348] Example 21. Sequence homology analysis of NPQ gene

[0349] To identify amino acid sequences homologous to Arabidopsis PsbS, ZEP and VDE, BLAST protein searches were performed using the BLASTX program. The percentages of sequence similarity obtained by BLAST are presented in the following tables, Table 4 for PsbS, Table 5 for ZEP, and Table 6 for VDE, which lists the top 100 hits in descending order of percentage sequence identity to the Arabidopsis homologs.

[0350] To compare the sequence identity and / or similarity of amino acid sequences homologous to Arabidopsis PsbS, ZEP and VDE, sequence alignment was performed using the CLUSTAL OMEGA program. Figure 24 shows the amino acid sequence similarity obtained by CLUSTAL O for (A) PsbS, (B) ZEP and (C) VDE, respectively.

[0351] Table 5. Percent sequence identity for PsbS.

[0352]

[0353]

[0354]

[0355]

[0356]

[0357] Table 6. Percent sequence identity for ZEP.

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364] Table 7. Percent sequence identity with respect to VDE.

[0365]

[0366]

[0367]

[0368]

[0369]

[0370] Example 22. Greenhouse NPQ expression experiment

[0371] Tobacco was transformed with coding sequences of Arabidopsis thaliana VDE, ZEP and PsbS for expression in leaves under the control of different promoters. Two transformants with single transfer DNA (T-DNA) integration (VPZ-34 and -56) and one with two T-DNA insertions (VPZ-23) were selected based on seedling NPQ screening and self-pollinated to obtain homozygous T2 progeny for further studies. These plants were then grown in a greenhouse. The mRNA and protein levels of VDE, PsbS and ZEP were measured ( Fig.25 ).

[0372] All three VPZ lines showed an increase in total (transgenic plus native) transcript levels for VDE (10-fold), PsbS (three-fold), and ZEP (six-fold) relative to transcript levels for WT (A, C, and E). For PsbS, the increase in transcript levels translated into approximately four-fold higher PsbS protein levels (D), as exemplified by bands at 21 kDa (AtPsbS) and 24 kDa (NtPsbS) (G). For VDE and ZEP, the increase in transcript levels corresponded to a 30-fold (B and G) increase for VDE (45 kDa) and a 74-fold (F and G) increase for ZEP (73 kDa) relative to WT protein levels.

[0373] Example 23. Field NPQ expression experiment

[0374] Tobacco was transformed with coding sequences of Arabidopsis thaliana VDE, ZEP and PsbS for expression in leaves under the control of different promoters. Two transformants with single transfer DNA (T-DNA) integration (VPZ-34 and -56) and one with two T-DNA insertions (VPZ-23) were selected based on seedling NPQ screening and self-pollinated to obtain homozygous T2 progeny for further studies. These plants were then grown in the field. The mRNA and protein levels of VDE, PsbS and ZEP were measured ( Fig.26 ).

[0375] All three VPZ lines showed increases in total (transgenic plus native) transcript levels for VDE (4-fold), PsbS (1.2-fold), and ZEP (7-fold) relative to WT transcript levels. All three VPZ lines also showed increases in total (transgenic plus native) protein levels for VDE (47-fold), PsbS (3-fold), and ZEP (75-fold) relative to WT transcript levels.

[0376] Example 24. Transgenic rice experiment

[0377] Rice plants were transformed with a T-DNA construct containing nucleotide sequences encoding PsbS, ZEP and VDE according to the transformation protocol described in Example 15. The same expression construct used in the transgenic tobacco experiments was used in this experiment. Leaves of nine independent TO transformants and two GUS controls were dark adapted. Subsequently, leaves were exposed to 1000 μmol m -2 s -1 NPQ was measured during a 3-min dark period following light exposure. Fig.31 The results show that 1000 μmol m -2 s -1 NPQ of nine rice transformants (dots) and controls (lines) during 3 min of darkness after light exposure. Fig.32 The results show that 1000 μmol m -2 s -1 Mean values ​​of NPQ of nine rice transformants (blue dots) and controls (orange dots) during 3 min of darkness after light exposure.

[0378] like Fig.31 and Fig.32 As shown, at 1000 μmol m -2 s -1 During the light period, the NPQ amplitude of these nine transformants was lower than that of the control. This result is consistent with the increased expression of ZEP in these transformants, which prevents the formation of zeaxanthin and thus reduces the NPQ amplitude. An alternative explanation is that PsbS overexpression interferes with the expression of native PsbS and thus reduces the NPQ amplitude. The results also show that there is no significant NPQ difference between the transformants and the control during the 3 min relaxation period in the dark, which is due to the possible lack of zeaxanthin accumulation in this experiment.

[0379] The lack of changes in NPQ kinetics in these transgenic rice plants is most reasonably attributed to the following limitations of the experimental design: in this experiment the rice plants were transformed with expression cassettes constructed for tobacco transformation containing promoters designed for optimal gene expression in dicots. It is known in the art that dicot promoters do not work well in monocots. Therefore, the expression of PsbS, ZEP and VDE in these transgenic rice plants may not have been optimally increased to levels that would have been beneficial for increasing the NPQ relaxation rate.

[0380] Example 25. Additional Experiments

[0381] Transient overexpression of NPQ-related genes was performed in Nicotiana benthamiana. NPQ measurements were obtained on leaf points that served as negative controls at 600 μmol photons m -2 s -1Overexpression of FLAG-tagged PsbS, VDE, ZEP, and GUS was performed during 13 min of irradiation followed by 10 min of darkness. Fig.18 As shown, the results show that overexpression of PsbS increased NPQ capacity relative to the GUS control. Overexpression of VDE accelerated NPQ induction. Overexpression of ZEP accelerated NPQ relaxation, but negatively affected NPQ induction and capacity.

[0382] Transient co-overexpression of VDE and ZEP was performed in N. benthamiana. The results showed an increase in the rates of NPQ induction and relaxation, as Fig.19 Co-overexpression of VDE was shown to be necessary to balance the overexpression of ZEP and prevent the negative effects of ZEP overexpression on NPQ induction and competence.

[0383] Fig. 20 NPQ kinetics of stable transgenic T1 plants of tobacco cv. Petite Havana are shown. NPQ measurements using DUALPAM were performed at 600 μmol photons m -2 s -1 The experiments were performed on the youngest fully developed leaves of T1 plants from three different lines: a wild-type segregant (Null), a line overexpressing ZEP (ZEP), and a line overexpressing ZEP and VDE (ZEP-VDE) during 10 min of illumination followed by 10 min of darkness. The ZEP-VDE line showed faster NPQ induction and relaxation.

[0384] Fig.21 The quantum yield of photosynthetic system II (YII) of stable transgenic T1 plants of tobacco cv. Petite Havana is shown. Fig. 20 The YII measurements performed simultaneously with the NPQ measurements described in were performed on the same plants and under the same conditions. During the dark recovery period, the YII was higher in lines overexpressing ZEP-VDE compared to lines overexpressing ZEP or Null.

[0385] Fig. 22 Shown is a growth experiment in a greenhouse. The results show that the size and biomass of the stable transgenic T1 plants of tobacco cv. Petite Havana overexpressing ZEP and VDE (ZEP-VDE) increased compared to the wild type (Null). The strains overexpressing PsbS (PsbS) or ZEP and PSBS (ZEP-PsbS) showed biomass similar to the wild type. Four groups of plants are shown, one group for each transgenic strain. Each group includes 36 plants. After 19 days of growth, the total wet weight and total dry weight of the 36 plants harvested were used to measure the aboveground biomass of each group. The data represent the results of a single experiment.

Claims

1. A method for producing a transgenic plant comprising transforming the plant with one or more nucleotide sequences encoding Photosystem II subunit S (PsbS), zeaxanthin epoxidase (ZEP), and violaxanthin de-epoxidase (VDE); or ZEP and VDE, wherein PsbS is encoded by the nucleotide sequence of SEQ ID NO: 1, ZEP is encoded by the nucleotide sequence of SEQ ID NO: 2, and VDE is encoded by the nucleotide sequence of SEQ ID NO: 3, The nucleotide sequence is operably linked to at least one expression control sequence, wherein transcript levels of at least both ZEP and VDE are increased compared to control plants lacking the transfected nucleotide sequence grown under the same conditions; and Wherein the plants have improved growth under fluctuating light conditions compared to control plants grown under the same fluctuating light conditions.

2. The method of claim 1, wherein the transgenic plant comprises one or more nucleotide sequences encoding PsbS, ZEP and VDE.

3. The method of claim 1, wherein the transcript levels of VDE, PsbS and ZEP are increased in the transgenic plants compared to control plants grown under the same conditions.

4. The method according to any one of claims 1 to 3, wherein the plant is a crop plant, a model plant, a monocotyledon, a dicotyledon, a plant with Crassulacean acid metabolism (CAM) photosynthesis, a plant with C3 photosynthesis, a plant with C4 photosynthesis, an annual plant, a greenhouse plant, a horticultural flowering plant or a perennial plant.

5. The method according to any one of claims 1 to 3, wherein the plant is selected from the group consisting of Miscanthus, alfalfa, sorghum, sugarcane, camelina, cereals, oilseeds, vegetable crops, forage crops, industrial crops, tree crops and biomass crops.

6. The method of any one of claims 1 to 3, wherein the plant is switchgrass, sweet sorghum, energy sugarcane, elephant grass, corn, cassava, cowpea, wheat, barley, oats, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, Brassica napus, Brassica carinata, Brassica juncea, pearl millet, millet, rice or Nicotiana tabacum.

7. The method according to any one of claims 1 to 3, wherein the plant is corn (Zea mays).

8. The method according to any one of claims 1 to 3, wherein the plant is rice (Oryza sativa).

9. The method according to any one of claims 1 to 3, wherein the plant is Sorghum bicolor.

10. The method according to any one of claims 1 to 3, wherein the plant is soybean (Glycine max).

11. The method according to any one of claims 1 to 3, wherein the plant is cowpea (Vigna unguiculata).

12. The method according to any one of claims 1 to 3, wherein the plant is a Populus spp. or a Eucalyptus spp.

13. The method according to any one of claims 1 to 3, wherein the plant is cassava (Manihotesculenta).

14. The method according to any one of claims 1 to 3, wherein the plant is barley (Hordeum vulgare).

15. The method according to any one of claims 1 to 3, wherein the plant is potato (Solanum tuberosum).

16. The method of any one of claims 1 to 3, wherein the plant is Saccharum spp.

17. The method according to any one of claims 1 to 3, wherein the plant is alfalfa (Medicagosativa).

18. The method according to any one of claims 1 to 3, wherein the transcript level of VDE is increased 3-fold compared to control plants, wherein the transcript level of PsbS is increased 3-fold compared to control plants, and wherein the transcript level of ZEP is increased 8-fold compared to control plants, wherein the transcript level of VDE increased 10-fold compared to control plants, wherein the transcript level of PsbS increased 3-fold compared to control plants, and wherein the transcript level of ZEP increased 6-fold compared to control plants, wherein the transcript level of VDE increased 4-fold compared to control plants, wherein the transcript level of PsbS increased 1.2-fold compared to control plants, and wherein the transcript level of ZEP increased 7-fold compared to control plants, The protein level of VDE increased 16-fold compared to the control plants, the protein level of PsbS increased 2-fold compared to the control plants, and the protein level of ZEP increased 80-fold compared to the control plants. The protein level of VDE increased 30-fold compared to the control plants, the protein level of PsbS increased 4-fold compared to the control plants, and the protein level of ZEP increased 74-fold compared to the control plants. The protein level of VDE increased 47-fold compared to the control plants, the protein level of PsbS increased 3-fold compared to the control plants, and the protein level of ZEP increased 75-fold compared to the control plants. wherein the increase in transcript levels compared to control plants has a ratio between VDE, PsbS and ZEP selected from the group consisting of: 3:3:8, 10:3:6 and 4:1.2:7, wherein the increase in protein levels compared to control plants has a ratio between VDE, PsbS and ZEP selected from the group consisting of: 16:2:80, 30:4:74 and 47:3:75, wherein the increase in transcript levels of VDE is about 3-fold to about 10-fold compared to control plants, wherein the increase in transcript levels of PsbS is about 1.2-fold to about 3-fold compared to control plants, and wherein the increase in transcript levels of ZEP is about 6-fold to about 8-fold compared to control plants, or wherein the protein level of VDE increases by about 16-fold to about 47-fold compared to control plants, wherein the protein level of PsbS increases by about 2-fold to about 4-fold compared to control plants, and wherein the protein level of ZEP increases by about 74-fold to about 80-fold compared to control plants.

19. The method of any one of claims 1 to 3, wherein the plant has enhanced growth under fluctuating light conditions compared to control plants under fluctuating light conditions.

20. The method of any one of claims 1 to 3, wherein the plant has enhanced photosynthetic efficiency under fluctuating light conditions compared to control plants under fluctuating light conditions.

21. The method of any one of claims 1 to 3, wherein the plant has improved photoprotection efficiency under fluctuating light conditions compared to control plants under fluctuating light conditions.

22. The method of any one of claims 1 to 3, wherein the plant has improved quantum yield and CO2 fixation under fluctuating light conditions compared to control plants under fluctuating light conditions.

23. The method according to any one of claims 1 to 3, wherein the plant is an elite line or strain.

24. The method of any one of claims 1 to 3, wherein the plant further comprises expression of at least one additional polypeptide that provides herbicide resistance, insect or pest resistance, disease resistance, improved fatty acid metabolism and / or improved carbohydrate metabolism.

25. An expression vector comprising one or more nucleotide sequences encoding PsbS, ZEP and VDE; or ZEP and VDE, wherein PsbS is encoded by the nucleotide sequence of SEQ ID NO: 1, ZEP is encoded by the nucleotide sequence of SEQ ID NO: 2, and VDE is encoded by the nucleotide sequence of SEQ ID NO: 3, The nucleotide sequence is operably linked to at least one expression control sequence, wherein said at least one expression control sequence provides for increased transcript levels of at least both ZEP and VDE when said expression vector transfects a plant, as compared to control plants lacking the transfected nucleotide sequence grown under the same conditions; and Wherein the plants have improved growth under fluctuating light conditions compared to control plants grown under the same fluctuating light conditions.

26. The expression vector of claim 25, wherein the at least one expression control sequence comprises a promoter selected from the group consisting of: Rbcs1A, GAPA-1, and FBA2.

27. The expression vector of claim 25, wherein the Rbcs1A promoter drives the expression of ZEP, the GAPA-1 promoter drives the expression of PsbS, and the FBA2 promoter drives the expression of VDE.

28. The expression vector of claim 25, wherein the expression vector is T-DNA.

29. The expression vector of claim 25, further comprising a nucleotide sequence encoding a polypeptide that provides antibiotic resistance.

30. The expression vector of claim 25, further comprising a left border (LB) domain and a right border (RB) domain flanking said expression control sequence and said nucleotide sequence encoding said PsbS, ZEP and VDE polypeptides.

31. A bacterial cell comprising the expression vector according to any one of claims 25 to 30.

32. An Agrobacterium cell comprising the expression vector according to any one of claims 25 to 30.

33. A method for producing a transgenic plant, comprising transforming a plant with the expression vector of any one of claims 25 to 30.

34. A method for enhancing plant growth under fluctuating light conditions, comprising transforming the plant with one or more nucleotide sequences encoding Photosystem II subunit S (PsbS), zeaxanthin epoxidase (ZEP), and violaxanthin de-epoxidase (VDE); or ZEP and VDE, wherein PsbS is encoded by the nucleotide sequence of SEQ ID NO: 1, ZEP is encoded by the nucleotide sequence of SEQ ID NO: 2, and VDE is encoded by the nucleotide sequence of SEQ ID NO: 3, The nucleotide sequence is operably linked to at least one expression control sequence, Thereby increasing the expression of (i) ZEP and VDE or (ii) PsbS, ZEP and VDE in said plant, wherein the expression of at least ZEP and VDE is increased compared to control plants under the same conditions, thereby producing plants having increased expression of at least ZEP and VDE compared to control plants under the same conditions.

35. A method for increasing the photosynthetic efficiency of a plant under fluctuating light conditions, the method comprising transforming the plant with one or more nucleotide sequences encoding Photosystem II subunit S (PsbS), zeaxanthin epoxidase (ZEP), and violaxanthin de-epoxidase (VDE); or ZEP and VDE, wherein PsbS is encoded by the nucleotide sequence of SEQ ID NO: 1, ZEP is encoded by the nucleotide sequence of SEQ ID NO: 2, and VDE is encoded by the nucleotide sequence of SEQ ID NO: 3, The nucleotide sequence is operably linked to at least one expression control sequence, Thereby increasing the expression of (i) ZEP and VDE or (ii) PsbS, ZEP and VDE in said plant, wherein the expression of at least ZEP and VDE is increased compared to control plants under the same conditions, thereby producing plants having increased expression of at least ZEP and VDE compared to control plants under the same conditions.

36. A method for improving the photoprotection efficiency of a plant under fluctuating light conditions, the method comprising transforming the plant with one or more nucleotide sequences encoding Photosystem II subunit S (PsbS), zeaxanthin epoxidase (ZEP), and violaxanthin de-epoxidase (VDE); or ZEP and VDE, wherein PsbS is encoded by the nucleotide sequence of SEQ ID NO: 1, ZEP is encoded by the nucleotide sequence of SEQ ID NO: 2, and VDE is encoded by the nucleotide sequence of SEQ ID NO: 3, The nucleotide sequence is operably linked to at least one expression control sequence, Thereby increasing the expression of (i) ZEP and VDE or (ii) PsbS, ZEP and VDE in said plant, wherein the expression of at least ZEP and VDE is increased compared to control plants under the same conditions, thereby producing plants having increased expression of at least ZEP and VDE compared to control plants under the same conditions.

37. A method for improving quantum yield and CO2 fixation in plants under fluctuating light conditions, the method comprising transforming the plant with one or more nucleotide sequences encoding Photosystem II subunit S (PsbS), zeaxanthin epoxidase (ZEP), and violaxanthin de-epoxidase (VDE); or ZEP and VDE, wherein PsbS is encoded by the nucleotide sequence of SEQ ID NO: 1, ZEP is encoded by the nucleotide sequence of SEQ ID NO: 2, and VDE is encoded by the nucleotide sequence of SEQ ID NO: 3, The nucleotide sequence is operably linked to at least one expression control sequence, Thereby increasing the expression of (i) ZEP and VDE or (ii) PsbS, ZEP and VDE in said plant, wherein the expression of at least ZEP and VDE is increased compared to control plants under the same conditions, thereby producing plants having increased expression of at least ZEP and VDE compared to control plants under the same conditions.

38. A method for increasing the non-photochemical quenching (NPQ) relaxation rate in a plant, the method comprising transforming the plant with one or more nucleotide sequences encoding Photosystem II subunit S (PsbS), zeaxanthin epoxidase (ZEP), and violaxanthin de-epoxidase (VDE); or ZEP and VDE, wherein PsbS is encoded by the nucleotide sequence of SEQ ID NO: 1, ZEP is encoded by the nucleotide sequence of SEQ ID NO: 2, and VDE is encoded by the nucleotide sequence of SEQ ID NO: 3, The nucleotide sequence is operably linked to at least one expression control sequence, Thus increasing PsbS, ZEP and VDE; or ZEP and VDE expression of said two or more polypeptides in said plants, thereby producing plants having increased expression of said two or more polypeptides compared to control plants, wherein transcript levels of at least both ZEP and VDE are increased compared to control plants lacking the transfected nucleotide sequence grown under the same conditions; and Wherein the plants have improved growth under fluctuating light conditions compared to control plants grown under the same fluctuating light conditions.

39. The method according to any one of claims 34 to 38, wherein transcript levels of all three of PsbS, ZEP and VDE are increased compared to control plants lacking the transfected nucleotide sequences grown under the same conditions.

40. The method of any one of claims 34 to 38, comprising increasing expression of ZEP and VDE.

41. The method of any one of claims 34 to 38, comprising increasing the expression of PsbS, ZEP and VDE.

42. The method of any one of claims 34 to 38, wherein the expression of PsbS, ZEP and / or VDE is increased by expressing one or more heterologous nucleotide sequences encoding PsbS, ZEP and / or VDE.

43. A method for selecting plants having improved growth characteristics under fluctuating light conditions, the method comprising: (a) providing a plant population; (b) modifying the plant to enhance the activity of both ZEP and VDE, or all three of PsbS, ZEP and VDE, by transforming the plant with one or more nucleotide sequences encoding Photosystem II subunit S (PsbS), zeaxanthin epoxidase (ZEP), and violaxanthin de-epoxidase (VDE); or ZEP and VDE, wherein PsbS is encoded by the nucleotide sequence of SEQ ID NO: 1, ZEP is encoded by the nucleotide sequence of SEQ ID NO: 2, and VDE is encoded by the nucleotide sequence of SEQ ID NO: 3, The nucleotide sequence is operably linked to at least one expression control sequence; (c) detecting the level of non-photochemical quenching (NPQ) in the plant under fluctuating light conditions; (d) comparing the NPQ level in the plant under fluctuating light conditions to a NPQ control level under fluctuating light conditions; and (e) selecting plants having an increased NPQ relaxation rate when the plants are switched from high light intensity to low light intensity; thereby selecting plants having improved growth characteristics under fluctuating light conditions.

44. The method of claim 43, wherein the NPQ control level is selected from the group consisting of: the lowest NPQ level in the population; the median NPQ level in the population; the average NPQ level in the population, and the NPQ level in control plants.

45. The method of claim 43, wherein the plant is modified by introducing heterologous PsbS, ZEP and / or VDE using transgenic technology.

46. ​​The method of claim 43, wherein the plant is modified by modifying the promoter of PsbS, ZEP and / or VDE using a genome editing system.

47. The method of claim 46, wherein the genome editing system is CRISPR.

48. The method of any one of claims 34 to 38 and 43, wherein the plant is a crop plant, a model plant, a monocotyledon, a dicotyledon, a plant with Crassulacean acid metabolism (CAM) photosynthesis, a plant with C3 photosynthesis, a plant with C4 photosynthesis, an annual plant, a greenhouse plant, a horticultural flowering plant or a perennial plant.

49. The method of any one of claims 34 to 38 and 43, wherein the plant is selected from the group consisting of Miscanthus, alfalfa, sorghum, sugarcane, camelina, cereals, oilseeds, vegetable crops, forage crops, industrial crops, tree crops, or biomass crops.

50. The method of any one of claims 34 to 38 and 43, wherein the plant is switchgrass, sweet sorghum, energy cane, elephant grass, corn, cassava, cowpea, wheat, barley, oats, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, Brassica napus, mustard, mustard, pearl millet, millet, rice or Nicotiana tabacum.

51. The method of any one of claims 34 to 38 and 43, wherein the plant is corn (Zea mays).

52. The method according to any one of claims 34 to 38 and 43, wherein the plant is rice (Oryza sativa).

53. The method of any one of claims 34 to 38 and 43, wherein the plant is Sorghum bicolor.

54. The method of any one of claims 34 to 38 and 43, wherein the plant is soybean (Glycinemax).

55. The method according to any one of claims 34 to 38 and 43, wherein the plant is cowpea (Vigna unguiculata).

56. The method of any one of claims 34 to 38 and 43, wherein the plant is a Populus spp.

57. The method of any one of claims 34 to 38 and 43, wherein the plant is a Eucalyptus spp.

58. The method of any one of claims 34 to 38 and 43, wherein the plant is cassava (Manihotesculenta).

59. The method according to any one of claims 34 to 38 and 43, wherein the plant is barley (Hordeum vulgare).

60. The method according to any one of claims 34 to 38 and 43, wherein the plant is potato (Solanum tuberosum).

61. The method according to any one of claims 34 to 38 and 43, wherein the plant is sugarcane (Saccharum spp.).

62. The method of any one of claims 34 to 38 and 43, wherein the plant is alfalfa (Medicago sativa).

63. The method of any one of claims 34 to 38 and 43, wherein increasing expression comprises increasing transcript levels of VDE in the plant by 3-fold compared to control plants, wherein increasing expression comprises increasing transcript levels of PsbS in the plant by 3-fold compared to control plants, and wherein increasing expression comprises increasing transcript levels of ZEP in the plant by 8-fold compared to control plants, wherein increasing expression comprises increasing transcript levels of VDE in said plant by 10-fold compared to control plants, wherein increasing expression comprises increasing transcript levels of PsbS in said plant by 3-fold compared to control plants, and wherein increasing expression comprises increasing transcript levels of ZEP in said plant by 6-fold compared to control plants, wherein increasing expression comprises increasing transcript levels of VDE in said plant by 4-fold compared to control plants, wherein increasing expression comprises increasing transcript levels of PsbS in said plant by 1.2-fold compared to control plants, and wherein increasing expression comprises increasing transcript levels of ZEP in said plant by 7-fold compared to control plants, wherein increasing expression comprises increasing the protein level of VDE in the plant by 16-fold compared to a control plant, wherein increasing expression comprises increasing the protein level of PsbS in the plant by 2-fold compared to a control plant, and wherein increasing expression comprises increasing the protein level of ZEP in the plant by 80-fold compared to a control plant, wherein increasing expression comprises increasing the protein level of VDE in the plant by 30-fold compared to a control plant, wherein increasing expression comprises increasing the protein level of PsbS in the plant by 4-fold compared to a control plant, and wherein increasing expression comprises increasing the protein level of ZEP in the plant by 74-fold compared to a control plant, wherein the increased expression comprises a 47-fold increase in the protein level of VDE in the plant compared to a control plant, wherein the increased expression comprises a 3-fold increase in the protein level of PsbS in the plant compared to a control plant, and wherein the increased expression comprises a 75-fold increase in the protein level of ZEP in the plant compared to a control plant, wherein increasing expression comprises increasing transcript levels of VDE, PsbS, and ZEP in the plant compared to a control plant at a ratio selected from the group consisting of: 3:3:8, 10:3:6, and 4:1.2:7, wherein increasing expression comprises increasing the protein levels of VDE, PsbS and ZEP in the plant compared to a control plant at a ratio selected from the group consisting of: 16:2:80, 30:4:74 and 47:3:75, wherein increasing expression comprises increasing transcript levels of VDE in the plant by about 3-fold to about 10-fold compared to control plants, wherein increasing expression comprises increasing transcript levels of PsbS in the plant by about 1.2-fold to about 3-fold compared to control plants, and wherein increasing expression comprises increasing transcript levels of ZEP in the plant by about 6-fold to about 8-fold compared to control plants, or wherein increasing expression comprises increasing the protein level of VDE in the plant by about 16-fold to about 47-fold as compared to a control plant, wherein increasing expression comprises increasing the protein level of PsbS in the plant by about 2-fold to about 4-fold as compared to a control plant, and wherein increasing expression comprises increasing the protein level of ZEP in the plant by about 74-fold to about 80-fold as compared to a control plant.

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