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WO2018102728A1 - Procédé d'amélioration de fonction chloroplastique et d'augmentation du rendement des semis - Google Patents

Procédé d'amélioration de fonction chloroplastique et d'augmentation du rendement des semis Download PDF

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WO2018102728A1
WO2018102728A1 PCT/US2017/064286 US2017064286W WO2018102728A1 WO 2018102728 A1 WO2018102728 A1 WO 2018102728A1 US 2017064286 W US2017064286 W US 2017064286W WO 2018102728 A1 WO2018102728 A1 WO 2018102728A1
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plant
gnl
asa
lines
plants
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Argelia Lorence
Jessica Patricia YACTAYO-CHANG
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Arkansas State University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8269—Photosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • Vitamin C (a.k.a. L-ascorbic acid, AsA) is the most abundant water-soluble antioxidant found in plants (Smirnoff, 2000), In the late 1750's it was established that citrus fruits contained a "cure” for scurvy a.k.a. "sea plague”. However it was 1928 when Albert Szent-Gyoryi isolated ascorbate and identified it as the anti- scorbutic component present in citrus and other fruits. Since then, a significant body of evidence has been accumulated about the importance of vitamin C for human and animal health (Drouin et al, 201 1 ; Padayatty, 2016).
  • Ascorbate is a non-enzymatic antioxidant with a simple molecular structure. As is true for many other antioxidants, vitamin C is present in plants in two forms, the reduced and most active form called ascorbate and the oxidized form named dehydroascorbate.
  • Ascorbate belongs to the family of six carbon sugars with a conjugated pi enediol system at carbons 2 and 3.
  • Ascorbate is a very effective antioxidant due to its ability to donate a pair of electrons and stabilize the subsequent charge using the two oxygen atoms at C2 and C3 in the oxidized form of the molecule (Lee et al., 2006). Ascorbate eliminates reactive oxygen species (ROS) from both inside and outside cells, because it is able to pass through the cell membrane in its oxidized form where it reduces the lipophilic a-tocopherol (vitamin E) which is membrane bound in its oxidized state. This makes ascorbate an important part of the enzymatic antioxidant cycle in recycling other important antioxidants which would otherwise he lost to the cell
  • ROS reactive oxygen species
  • vitamin C In animals, vitamin C is involved in the synthesis of collagen, an important component of the skin, scar tissue, tendons, ligaments, and blood vessels (Levine et al. 1995; Davey et al, 2000). Another essential role of vitamin C is related to its function in redox homeostasis; this means when the production of ROS increases, the body's response will increase the activity of the endogenous antioxidant system through redox signaling (Figueroa-Mendez and Rivas Arancibia, 2015). Vitamin C functions in oxidative protein folding and in the maintenance of the intraluminal oxidative environment, which suggests that it has a particular role in endoplasmic reticulum related processes (Mandl et al, 2009).
  • vitamin C In animals, vitamin C interacts enzymaticallv and non-enzymatically with ROS. In humans vitamin C is essential in preventing pathological conditions including cardiovascular disease, cancer, hepatitis, bacterial infections, fungal infection, and allergies (Cathcart, 1981 ; Padayatty et al, 2006). The two-time Nobel Prize-winner, Linus Pauling, demonstrated that cancer patients treated with high doses of vitamin C had an increased survival rate (Cameron and Pauling, 1976). Recently Yun et al, (2015) showed that high levels of vitamin C killed human colorectal cancer cells. This effect is due to increased uptake of dehydroascorbate, the oxidized form of the molecule. Vitamin C has a uricosuric effect in humans and decreases uric acid levels, exerting a protective effect on gout (Stamp et al, 2013).
  • ascorbate In plants, ascorbate (AsA) has a wide variety of physiological roles. It functions as an enzyme cofactor, as a radical scavenger, and as donor/acceptor of electron transport in the chioroplast (Conklin and Barth, 2004; Ishikawa et al, 2006). Ascorbate can protect tissues against damage caused by ROS produced through normal oxygenic metabolism or generated from biotic and abiotic stress, and is strongly associated with photosynthesis and respiration. Reactive oxygen species include molecules such as superoxide and hydrogen peroxide.
  • Chioroplasts as well as mitochondria produce ROS as byproducts of normal cellular metabolism, but this production is enhanced by a variety of environmental stresses (Conklin et al., 1996, Conklin and Barth, 2004).
  • Another essential role of AsA is the modulation of processes such as lignifi cation, cell division, ceil elongation, the hypersensitive response, tolerance to stresses, and senescence in plants (Smirnoff and Wheeler, 2000; Barth et al, 2004; Pavet et al, 2005).
  • AsA controls flowering time through phytohormones (Barth et al, 2004).
  • Ascorbate can accumulate at millimolar concentrations in both photosynthetic and non-photosynthetic tissues (Foyer et al, 1983), This accumulation in such high quantities suggests that AsA is important for the plant as a major antioxidant. 1.4.
  • D-glucose which is converted to GDP-D-mannose followed by L-galactose that leads to AsA production.
  • D-Glucose-6-P is then converted to GDP-D-mannose by a series of steps catalyzed by fruciose-6-P, D-mannose-6-P, and D-mannose-l-P, and that include the vtcl mutant locus (GDP-mannose pyrophosphorylase).
  • the conversion of GDP-D-mannose to AsA comprises four steps: GDP-L-gaiactose, L- galactose-l -P (Laing et al, 2004), L-galactose (Gatzek et al., 2002) and L-galactono-1,4- lactone (Imai et al, 1998).
  • the GDP-D-mannose to GDP-L-galactose reaction is catalyzed by GDP-D-mannose-3 ',5'-epimerase a.k.a.
  • the vtc2 enzyme and its close homolog vtc5 convert GDP-L-galactose into L-galactose- 1-P (Smirnoff et al, 2001; Dowdle et al, 2007, Linster et al, 2007).
  • the L-galactose-1- phosphate phosphatase (locus vtcA in Arabidopsis) converts L-galactose-l-P into L- galactose (Conklin et al, 2006).
  • L-Galactose is oxidized at the CI -position by an L- galactose dehydrogenase (GaIDH) present in the cytosol to L-gal actono- 1 ,4-Iactone (Wheeler et al, 1998; Gatzek et al, 2002). All enzymes in this pathway are cytosolic except the last step involving the oxidation of gal actono- 1-44 act one to AsA that is carried out by the mitochondrial L-galactono-l,4-lactone dehydrogenase (GLDH), (0stergaard et al, 1997; Xmai et al, 1998).
  • GLDH mitochondrial L-galactono-l,4-lactone dehydrogenase
  • the L-gulose pathway uses a similar precursor as the D-mannose/L-galactose to the branch point at GDP-D-mannose. GDP-D-mannose is then converted by the GME enzyme into GDP -L-gulose, It is proposed that GDP-L-gulose is converted to L-gulono-l,4-lactone and to AsA in three subsequent steps catalyzed by GDP-L-gulose pyrophosphatase, L- gulose-1 -phosphate phosphatase, and L-gulose dehydrogenase respectively (Wolucka and Van Montagu, 2003). The only enzyme that has been characterized in this pathway is GME and it is known to be cytosolic.
  • This pathway involves four enzymes, starting from the oxidation of Twyo-inositol to D-glucuronic acid and further reduction to L-gulonic acid and to L-gulono-l,4-lactone, and further oxidation to AsA.
  • MIOX wyo-inositol oxygenase
  • GlcUR glucuronate reductase
  • GNL gluconolactonase
  • GLO L-gulono-l,4-lactone oxidase
  • the first two enzymes have already characterized by the Lorence Laboratory.
  • Arabidopsis tkaliana lines over-expressing MIOX and GuILO are tolerant to multiple abiotic stresses such as salt, cold, heat, and pyrene (Lisko et al, 2013).
  • AsA plants exhibit increased growth and biomass accumulation (Lorence et al., 2004; Lorence and Nessler, 2007; Lisko et al, 2013).
  • Transgenic rice overexpressing MIOX showed improved growth performance when grown in the presence of 200 niM mannitol and presented higher survival rates compared to wild type plants treated with polyethylene glycol (Duan et al., 2012).
  • MIOX proteins are present in almost all multicellular eukaryotes and are highly conserved across phyla. It has been reported that the role of MIOX and D-GlcUA for As A biosynthesis in plants is a major plant antioxidant to counterbalance oxidative damage (Shao et al, 2008).
  • the first two enzymes in the inositol pathway to AsA are cytosoiic.
  • the Lorence laboratory has evidence indicating that some isoforms of the last two enzymes in this pathway reside in the chloroplast and the endoplasmic reticulum (ER).
  • Chloroplasts are the organelles responsible for photosynthesis, a process that is essential for plant growth and development (Rustchow et al., 2008; Venkatasalam, 2012). Key metabolites in the photosynthetic process are NADPH and ATP. Although photosynthesis is an essential process, light absorption creates oxidative stress due to the formation ROS, such as singlet oxygen ( ! 0?_), superoxide (()? " ) and hydrogen peroxide (H2O2) (Oelze et al., 2008).
  • ROS such as singlet oxygen ( ! 0?_), superoxide (()? " ) and hydrogen peroxide (H2O2) (Oelze et al., 2008).
  • Ascorbate is essential to detoxify H2O2 produced during the Mehler reaction, which is formed by dismutation of O2 " and can be regenerated via the AsA-glutathione cycle to counteract 0 2 " (Halliwell and Foyer, 1976; Foyer and Noctor, 2000, Munne-Bosch and Alegre, 2002; Talla et al, 2011).
  • FIG. 3 shows the Anti -oxidation of reactive oxygen species. Reactive oxygen is generated when electrons (e-) not utilized in photosynthesis are donated to oxygen, thus creating superoxide (02,-) that can he converted to hydrogen peroxide (H202) by superoxide dismutase (SOD).
  • the H202 is further converted to H20 by ascorbate peroxidase (APX) utilizing ascorbate (AsA) as an electron donor that, in turn, becomes oxidized ascorbate (ox-AsA). Additional electrons are consumed via the conversion of ox-AsA back to AsA or the conversion of double ox-AsA back to AsA using glutathione. The resulting oxidized glutathione is reduced by electrons from electron transport by means of glutathione reductase (GR). Source: Demmig-Adams et a!., 2012.
  • Ascorbate is present in all plants although its concentration varies greatly and has been identified in various compartments of the ceil. Ascorbate occurs inside as well as outside the chloroplast (Constable, 1963; Hall and Rao, .1999, Habermann, 20.13), where it has been shown to accumulate at concentrations up to 50 mM (Hall and Rao, 1999); this represents about 25-30% of the total AsA in the plant cell (Horemans et al., 2000). All known AsA biosynthetic enzymes reside in compartments other than the chloroplast, and therefore it is currently unknown how this organelle is able to accumulate such high concentrations of this antioxidant.
  • Ascorbate was at one time considered to be a necessary component of the photosynthetic phosphorylation system (Arnon, 1959) however is now considered important in providing a protective role in preventing inactivation of essential components of the chloroplasts (Pinto-Marijuan and Munne-Bosch, 2014).
  • Chloroplasts alter their distribution within cells depending on the external light conditions, Chloroplasts can be observed to move to positions that maximize photon absorption under low-influence light and, conversely, to move to positions that minimize photon absorption under high light. The movement away from areas of strong light is believed to offer areas of strong light protection against photo-oxidative damage (Eckardt, 2003).
  • MIOX /wyo-inositol oxygenase
  • GlcUR glucuronate reductase
  • lilg56500 encodes a functional giucuronolactonase (GNL) that resides in the chloroplast, then this protein will protect this organelle and green tissues, and will counteract reactive oxygen species formed under light stress. When over-expressed in plants this enzyme will confer plants enhanced photosynthetic efficiency.
  • GNL giucuronolactonase
  • Aim 1 Characterize the ⁇ tGNL ( iilg56500) functional enzyme.
  • Aim 2 Establish the role of the AtGNL under low, normal, and high light conditions.
  • Aim 3 Characterize the phenotype and photosynthetic efficiency of Arabidopsis lines with low, normal, and high AtGNL expression.
  • Chloroplasts the organelles responsible for photosynthesis, are essential for plant growth and development, and are involved in the metabolism of carbon, nitrogen, and sulfur (Venkatasalam, 2012, Rustchow et al., 2008).
  • chloroplasts synthesize amino acids, fatty acids, purine, and pyrimidine bases, isoprenoids, tetrapyrroles, and the lipid components of their own membranes, followed by processing, folding, and assembly by various chaperone systems (Peltier et al., 2006).
  • the chloroplasts need considerable protein import from the cytosol. Chloroplasts control nuclear gene expression indirectly by metabolites, ROS and other cellular processes (Pogson et al., 2008; Pfannschmidtm, 2010).
  • Chloroplasts alter their distribution within cells depending on the external light conditions. Chloroplasts can be obseived to move to positions that maximize photon absorption under low light and, conversely, to move to positions that minimize photon absorption under high light. The movement away from areas of strong light is believed to offer protection against photo- oxidative damage (Eckardt, 2003).
  • the photoreceptors responsible for light induced chloroplast movement in higher plants are phototropins.
  • the phototropins PHOTl and PHOT2 are involved in blue light mediated chloroplast relocation, stomatal opening and phototropism (Briggs and Christie, 2002).
  • PHOTl is the primary photoreceptor that controls phototropism in low light (Huala et al, 1997), whereas PHOT2 is responsible for the light-avoidance relocation of chloroplast under high light (Kagawa et al., 2001).
  • Ascorbate Ascorbate (As A) is found in all plants although its concentrations vary greatly. Within the leaf, ascorbate occurs inside as well as outside the chloroplasts (Constable, 1963; Habermann, 2013). Ascorbate was at one time considered to be a necessary component of the photosynthetic phosphorylation system but recently it has been regarded as having a protective role in preventing inactivation of essential components of the chloropiasts (Anion, 1959; Pinto-Marijuan and Munne-Bosch, 2014).
  • Ascorbate helps detoxify H2O2 produced during the Mehler reaction, (Foyer and Noctor 2000; Talla et al., 2011) and is important for photoprotection (Demmig- Adams et al, 2012). In chloropiasts, high ascorbate levels are required to overcome photoinhibition caused by strong light (Miyaji et a!., 2014).
  • the /w o-inositol pathway is one of the four routes for the production of AsA in plants. This pathway has not been completely elucidated. Three enzymes have been characterized: /wyo-inositol oxygenase (MIOX), glucuronate reductase (GlcUR), and L- gulono-l,4-lactose oxidase (GuILO) (Lorence et al, 2004; Lorence and Nessler, 2007, Lisko et al., 2013; Aboobucker, 2014).
  • gluconolactonase has been characterized in Ratt s norvegicus (Kondo et al., 2006), Euglena gracilis (Ishikawa et al, 2008), Pseudomonas aeruginosa (Tarighi et al, 2008), Xanthomonas campestri (Chen et al, 2008), Homo sapiens (Aizawa et al, 2013), and Gluconobacter oxydans (Shinagawa et al, 2009), but not in plants.
  • FIG. 4 shows the Subcellular localization of enzymes in the ascorbate metabolic network.
  • GLDH the terminal enzyme in the D-mannose/L-galactose pathway is located in the mitochondria, while the terminal enzyme in the royo-inositol and L-gulose routes (GuILO) is known to reside in the endoplasmic reticulum.
  • GNL the localization of the third enzyme
  • FIG. 5 shows the Putative glucuronolactonases (GNLs) in Arabidopsis.
  • the T-DNA knockouts were screened looking for low AsA lines to identify true GNLs in Arabidopsi s.
  • the foliar AsA content in knockout lines corresponding to the GNL Arabidopsis genes were measured to identify low AsA mutants.
  • Bioinformatics analysis of the genes found that one of the SALK lines with low AsA encodes a protein that possesses a chloroplastic signal peptide.
  • FIG. 6 shows the Schematic of the insertion site of the T-DNA in the Atlg565400 gene in SALK lines 026172 and 0 1623 (red squares). Exons are shown as blue boxes.
  • Source TAIR database.
  • Atlg56500 cDNA was amplified and sub-cloned into the pBIB-Kan vector under the control of the cauliflower mosaic virus 35S promoter and the tobacco etch virus (TEV) enhancer.
  • TSV tobacco etch virus
  • a 6X-HIS tag was added at the 5' end of the cDNA to facilitate protein detection by Western blot and purification by nickel affinity chromatography.
  • the Nicotiana benthamiana was infiltrated with an .4 GNL construct using an optimized Agrohaciermm-mediaXed transient transformation method (Medrano et al., 2009).
  • Chloroplasts were isolated from leaves using a chloroplast isolation kit (CP-ISO Sigma).
  • FIG. 7 shows the The /ifGNL protein resides in the chloropiast.
  • Chioroplasts were isolated from leaves of Nicotiana benthamiana plants infiltrated with the AtGNL construct using a chloropiast isolation kit (CP-ISO, Sigma). Western blot was done using an anti-HIS antibody.
  • M molecular weight marker, lane 1 empty vector fraction, lane 2 non chloroplastic fraction, lane 3 chloroplastic fraction, lane 4 chloropiast fraction after purification by nickel affinity chromatography.
  • Aim 1 Characterize the /i/GNL ( l/lg5050Q) functional enzyme
  • Aim 2 Establish the role of the ⁇ tGNL under low, normal, and high light conditions
  • Aim 3 Characterize the phenotype and photosvnthetic efficiency of Arabidopsis lines with low, normal, and high ,4 GNL expression.
  • Vitamin C L-ascorbic acid, AsA
  • Ascorbate scavenges free radicals, is an enzyme cofactor, and a donor/ acceptor of electrons in the chioroplast. Ascorbate protects tissues against damage caused by reactive oxygen species (ROS) produced through normal metabolism or generated from stress.
  • ROS reactive oxygen species
  • the inositol route to AsA involves four enzymes: wyo-inositol oxygenase, glucuronate reductase, gluconolactonase (GNL), and L-gulono-l,4-lactone oxidase (GulLO).
  • GNL has been characterized in rat and bacteria but not in plants.
  • AtG L is the first AsA biosynthetic enzyme that resides in chloroplasts.
  • AtGlsL AtGlg56500
  • FIG. 1 is a chemical structure view of one embodiment of the present invention
  • FIG. 2 is a metabolic network view thereof
  • FIG. 3 is a flowchart view thereof
  • FIG. 4 is a metabolic network view thereof
  • FIG. 5 is a table view thereof
  • FIG. 6 is a schematic view thereof
  • FIG. 7 is a blot view thereof
  • FIG. 8 is a construct view thereof
  • FIG. 9 is a table view thereof
  • FIG. 10 is a blot view thereof
  • FIG. 1 1 is a blot view thereof
  • FIG. 12 is an analytical view thereof
  • FIG. 13 is a table view thereof
  • FIG. 14 is a graph view thereof
  • FIG. 15 is a graph view thereof
  • FIG. 16 is a graph view thereof
  • FIG. 17 is a table view thereof
  • FIG. 18 is a graph view thereof
  • FIG. 19 is a photographic and chart view thereof;
  • FIG. 20 is a photographic view thereof;
  • FIG. 21 is a chart view thereof
  • FIG. 22 is a table view thereof
  • FIG. 23 is a chart view thereof
  • FIG. 24 is a chart view thereof
  • FIG. 25 is a metabolic network view thereof
  • FIG. 26 is a chart view thereof
  • FIG. 27 is a table view thereof
  • FIG. 28 is a chart view thereof
  • FIG. 29 is a table view thereof
  • FIG. 30 is a chart view thereof
  • FIG. 3 1 is a table view thereof
  • FIG. 32 is a photographic view thereof
  • FIG. 33 is a phyiogenetic view thereof
  • FIGS. 34 A and 34B are table views thereof
  • FIG. 35 is a chart view of one embodiment of the present invention.
  • FIG. 36 is a genetic sequencing view of one embodiment of the present invention.
  • FIG. 37 is a genetic sequencing view of one embodiment of the present invention. DETAILED DESCRIPTION
  • Arabidopsis thaliana ecotype Columbia wild type seeds (Col-0, stock # CS60000), SALK 026172, and SALK 01 1623 were obtained from the Arabidopsis Biological Resource Center (ABRC, Columbus, OH). Seeds were sterilized with 70% (v/v) ethanol for 10 min followed by 50% (v/v) sodium hypochlorite containing 0.05% (v/v) Tween-20 for 15 min. Next, seeds were washed 6 times with sterile water. Finally, seeds were transferred to a petri dish containing medium which consisted of Murashige-Skoog (MS) salts (Murashige and Skoog, 1962), MS vitamins, and 3% (w/v) sucrose, at pH 5.6.
  • MS Murashige-Skoog
  • the medium was supplemented with 0.04% (w/v) MgS0 4 .7H 2 0.
  • the seeds were vernalized for 3 days at 4°C. Plates were transferred to a growth chamber and incubated at 23°C, 65% humidity, 16:8 h photoperiod and 200 ⁇ 1/ ⁇ 2 /8 light intensity. After establishment seedlings were transferred to PM-15-13 AIS MIX Arabidopsis soil (Lehle-Seeds, Round Rock, TX) in 2 inch pots. Pots were covered with a dome for one week and after that plants were grown until they reached maturity.
  • Nicotiana benthamiana seeds were obtained from The Department of Plant Pathology, Physiology and Weed Science at Virginia Polytechnic Institute and State University (Blacksburg, VA). Seeds were sown in 4.5 inch pots containing Pro-mix BX soil (Premier Horticulture Ltd, Canada) with fertilizer Osmocote 14-14-14 (Scotts, Canada). Vermicuiite was overlaid on top of the seeds. The pots were covered with a dome for one week. Plants were grown in an environmental control chamber with the following conditions: 25°C (day)/ 21°C (night) temperature, 65% relative humidity, 16:8 h photoperiod, and 150 ⁇ imol/m /s light intensity. 2.2.2, Constructs of interest
  • the first construct is one where the cDNA encoding a putative was placed under the control of the 35S promoter and the tobacco etch virus (TEV) enhancer ( ⁇ GNL-6XHIS:pB IB-Kan), (Becker, 1990). In this construct a histidine tag was added to the C-terminus of the protein of interest to allow detection using antibodies and to facilitate purification.
  • the second construct is one where the putative promoter region of the ⁇ lt NZ (a 1000 bp fragment preceding the ATG) was cloned and fused to the GUS reporter gene to better understand the spatial and temporal expression of this gene (p ⁇ tGNL:pCAMBIAl 305.1).
  • FIG. 8 shows the Constructs of interest.
  • A The / g56500-HIS:pBffi-Kan construct containing counsel4rlg56500 (AtGNL with a six histidine (6X-HIS) tag and adjacent neomycin phosphotransferase II (npilT) selectable marker.
  • B The pAtlg56500:pCAMBIA1305.1 construct containing the ,4 GNL promoter with the GUS-PLUS ' reporter gene and the hygromycin phosphotransferase (hph) selectable marker.
  • NOS-P promoter of nopaline synthase gene
  • 35S-T terminator of the 35S cauliflower mosaic virus gene: TEV: tobacco etch virus transiationai enhancer; LB and RB: left and right T-DNA borders, respectively.
  • over-expresser wild type plus ,-!/G ⁇ ' L ⁇ : and restored lines (knockouts plus ⁇ 4 GNL) Tl plants that were high AsA expressers were identified.
  • the seeds of those plants were sterilized with 70% (v/v) ethanoi for 10 min followed by 50% (v/v) sodium hypochlorite containing 0.05% (v/v) Tween-20 for 15 min. Next, seeds were washed 6 times with sterile water.
  • mice were transferred to petri dishes containing Murashige and Skoog (MS) medium which consists of salts, MS vitamins, and 3% (w/v) sucrose at pH 5.6, The medium was supplemented with 0.04% (w/v) MgS0 4 .7H 2 0, and 50 mg/L kanamycin. Plated seeds were vernalized for 3 days at 4°C. After establishment, seedlings were transferred to soil and grown under the above stated conditions until they reached maturity. This process was repeated until plants with a 100% germination score in the presence of antibiotic selection were developed.
  • MS Murashige and Skoog
  • Reduced AsA was determined by measuring the decrease in absorbance at 265 nm after addition of 0.5 unit of ascorbate oxidase to 300 ⁇ ... of the reaction medium containing the plant extract and 100 mM phosphate buffer at pH 6.9. Oxidized ascorbate was measured in a 300 ⁇ . reaction mixture with 10 ⁇ of 40 mM dithiothreitol (DTT) after incubation in the dark for 20 min at room temperature. The reaction was followed by measuring absorption at 265 nm. Calculations were made based on a standard curve made with pure L-ascorbic acid run in parallel. Ten biological replicates were measured in analytical triplicate and reported as ⁇ per gram fresh weight ( ⁇ / g FW).
  • Recombinant /liGNL-oXHIS was detected using an anti-HIS (C-term)/AP antibody at a 1 :2,000 v/v dilution (invitrogen, Carlsbad, CA) and CDP-start, a chemiluminescent substrate for alkaline phosphatase detection (Roche Diagnostics, Indianapolis, IN).
  • Recombinant /IfGNL protein was purified from N. benthamiana leaves. Five grams of leaf tissue were pulverized in liquid nitrogen and proteins were extracted with 10 mL of buffer A (75 mM sodium phosphate dibasic, 25 mM sodium phosphate monobasic, 150 mM NaCl, 10 mM sodium metabi sulfite, and 0.6% (v/v) protease inhibitor cocktail, pH 7.4). The extract was then centrifuged at 13,000 x g for 5 min. The supernatant obtained after centrifugation was loaded onto a nickel affinity column (HIS60 Ni Superflow) and incubated for 1 h at 4°C.
  • buffer A 75 mM sodium phosphate dibasic, 25 mM sodium phosphate monobasic, 150 mM NaCl, 10 mM sodium metabi sulfite, and 0.6% (v/v) protease inhibitor cocktail, pH 7.4
  • the extract was then centrifuged at 13,000 x g
  • the column was washed with 50 mM sodium phosphate pH 7.4, 300 m : NaCl, 40 mM imidazole buffer and the bound proteins were eluted with 250 mM of imidazole.
  • the eluate from the nickel column was concentrated using an AMICON® 30K ultra centrifugal filter (Millipore, Billerica, MA). Total soluble protein concentration was estimated by the Bradford method (Bradford, 1976) using Coomassie blue G-250 dye (Thermo Scientific) and bovine serum albumin (Pierce, Rockford, IL) as a standard. Protein fractions from the purification procedure were separated by SDS-PAGE and the ⁇ tGNL was detected by Western blot and silver staining using Pierce® Silver Stain Kit (Thermo Scientific).
  • the lactonase activity was assayed in vitro based on the decrease in absorbance (405 nm) of the ?-nitrophenol pH indicator that resulted from the enzymatic opening of the lactone ring when D-glucono-6-lactone was used as substrate in the presence of the AtGNL as previously described (Ishikawa et a!., 2008). Enzyme preparations were made fresh for individual experiments at room temperature.
  • the optimum concentration for enzyme activity was 30 ⁇ ig per reaction.
  • One mL of the reaction typically contained: 10 mM PIPES pH 6.5, 5 mM D-glucono-6-lactone, 75 ⁇ MnCl 2 , 2.5 mM / nitrophenol, and an aliquot of the purified enzyme. An equal amount of boiled enzyme was used as control for these experiments.
  • the substrates used in this experiment were: D-glucono-6-lactone (D-GuIL), L-galactono-r-lactone (L-GalL), L- galactonic acid (L-Gal A), L-gulono-y-lactone (L-GulL), and L-gulonie acid (L-Gul A).
  • D-GuIL D-glucono-6-lactone
  • L-GalL L-galactono-r-lactone
  • L-Gal A L- galactonic acid
  • L-GulL L-gulono-y-lactone
  • L-Gul A L-gulonie acid
  • the cameras in the system are as follows: VIS camera, piA2400-17gc CCD (Easier, Ahrensburg, Germany) with resolution of 2454 x 2056 pixels; FLUO camera, scA1600-14gc CCD (Easier, Ahrensburg, Germany) with resolution 1624 x 1234 pixels; and NIR camera, Goideye GIGE P-008 SWIR (Allied Vision Technologies, Stadtroda, Germany) with resolution 320 x 256 pixels and with spectral sensitivity between 900 and 1700 run.
  • the relative area of the plants displaying normal green color versus the area with detectable yellow color (chlorosis) were calculated.
  • the analysis of the NIR images was similar to the color classification of VIS camera, using the acquired gray-scale images, where high water content corresponds to darker tones while low water corresponds to lighter gray tones.
  • the software used this information to calculate the relative area with low, medium, and high water content.
  • the fluorescence camera acquires red-scale images and in this case the red tones were divided into four equidistant bins, and the software calculated the relative area with zero, low, medium, and high fluorescence. Quantitative data obtained from the images were analyzed.
  • Arabidopsis thaliana var. Columbia was transformed by the floral dip method (Clough and Bent, 1998) with Agrobacterium tumefaciens GV3101 carrying the construct of interest (p ⁇ tGNL:pCAMBIA1305.1).
  • a different set of plants was also transformed with bacteria carrying the empty vector control (pCAMBIAl 305.1 ).
  • TO seeds were selected with hygromycin and the antibiotic resistant seedlings were transferred to soil and grown to maturity under the above mentioned conditions.
  • the presence of the transgene of interest was established via PGR using gene specific primers, and genomic cDNA as a template.
  • Seeds of the PGR positive plants were sterilized and transferred to a petri dish containing MS media with 20 mg/L hygromycin. Plated seeds were vernalized for 3 days at 4°C and then transferred to an environmentally controlled chamber. Hygromycin resistant seedlings were transferred to soil and grown until maturity.
  • Explants seedlings, leaves, flowers, and fruits were cut from plants 4, 8, 12, and 30 days after germination. Next, the explants were incubated in fresh and cold phosphate buffer pH 7.0 with 4% formaldehyde at room temperature for 30 min.
  • the explants were washed several times with cold phosphate buffer for 1 h, then vacuum infiltrated with X- Gluc substrate solution containing: 1 mg 5-bromo-4-chloro-3-indolyl ⁇ -D-glucuronide in 100 ⁇ of methanol, 1 n L 2X phosphate buffer, 20 iL 0.1 M potassium ferrocyanide, 20 ⁇ , 0.1 M potassium ferricyanide, 10 iL 10% (w/v) solution of Triton X-100, and 850 ⁇ , of water. Tissues were incubated in darkness at room temperature overnight until a distinct blue staining appeared. Finally, explants were incubated in 70% ethanol until the chlorophyll was removed. Photographs were taken with AxioCam MRc camera connected to a Stemi 2000-C stereo microscope (Zeiss).
  • FIG. 9 shows the list of buffers tested for protein purification.
  • FIG. 10 shows the AtGNL protein extracted with different buffers. Western blot of total protein extracted from N. benthamiana leaves with different buffers as described in FIG. 7.
  • lane 1 crude extract with buffer- l
  • lane 2 crude extract with buffer-2
  • lane 3 crude extract with buffer-3
  • lane 4 crude extract with buffer-4
  • lane 5 crude extract with buffer-5
  • lane 6 sample extracted in buffer- 5 and resuspended in buffer-4
  • lane 7 crude extract in buffer-5
  • lane 8 sample extracted in buffer-5 and resuspended in buffer-3
  • lane 9 sample extracted in buffer-5 and resuspended in buffer-6
  • lane 10 crude extract in buffer-6.
  • the optimal buffer to recover more recombinant protein was buffer 6.
  • FIG. 11 shows the ⁇ tGNL protein eluted with different imidazole concentrations.
  • Western blot of AtGNL protein with different concentration of imidazole M: marker
  • B6 crude extract in buffer 6
  • FT flow through W: wash buffer.
  • W wash buffer.
  • 40 mM and 250 mM were best for washing and eluting conditions, respectively.
  • FIG. 12 shows the Purification of the ⁇ 4tGNL:pBIB-kan-6XHIS expressed in N. benthamiana leaves.
  • M marker
  • lane 1 crude extract
  • lane 2 flow through
  • lane 3 wash
  • lane 4 enzyme
  • lane 5 concentrated enzyme.
  • FIG. 12 illustrates the result of the purification of At&NL from N. benthamiana tissue using nickel affinity chromatography. Western blot results showed the presence of AtGNL in the crude extract and flow through or wash indicating protein had a partial binding to the cation column. The silver-stained gel indicates that the protein preparation contained mostly the protein of interest with a few minor contaminants.
  • Gluconolactonase (GNL, EC 3.1.1.17) catalyzes the hydrolysis of D-glucono-a-lactone (D-GulL) to D-gluconic acid (Ogawa et a!., 2002),
  • the Iactonase activity was assayed in vitro based on the decrease in absorbance (405 nm) of the ?-nitrophenol pH indicator that resulted from the enzymatic opening of the lactone ring when D-glucono-5-lactone (D-GuIL) was used as substrate in the presence of the ⁇ GNL as previously described (Hucho and Wallenfels, 1972),
  • the enzymatic activity was assayed at 25°C with 10 mM PIPES pH 6.5, 5 mM D-GuIL, 75 ⁇ MnCh, 2,5 mM p-nitrophenol, and 30 ⁇ g
  • FIG. 14 shows the Effects of temperature and pH on the activity of the AtGNL enzyme.
  • A pH effect on GNL activity.
  • B Temperature effect on GNL activity. Measurements were made in duplicate. Values are means ⁇ SD. Ogawa et al, (2002) reported that the GNL. enzyme from niger had higher activity at 30°C, while the activity of the GNL from P. aeruginosa is optimal at 24°C (Tarighi et al, 2008), which are similar to the AtGNL.
  • FIG. 15 shows the Effects of cofactor and substrate on the activity of the AtGNL enzyme.
  • A Cofactors effect on GNL activity.
  • B D-GuIL substrate concentration effect on GNL activity. Measurements were made in duplicate. Values are means ⁇ SD. The 3 mM: of D-glucono-5-lactone was the most effective substrate concentration for this assay.
  • FIG. 16 shows the Enzyme kinetics of the recombinant yi?lg56500 enzyme.
  • A Michaelis-Menten.
  • B Double reciprocal Lineweaver-Burke. Measurements were made in duplicate. Values are means ⁇ SD.
  • FIG. 17 summarizes the comparison between the kinetic parameters of the AtGNL with the one of known GNLs. Based on these results the G. oxidans GNL is the most similar to the Arabidopsis GNL.
  • Seeds expressing the ⁇ tGNL-6XHIS:pBIB-kan (.4 GNL) and empty pB IB-Kan (control) were screened in the Lorence Laboratory (unpublished).
  • One hundred and thirty primary transformants that were PGR positive were screened to identify high AsA expressers.
  • three lines per group were selected for further analysis: over-expressers (WT + AtGNL), restored 1 (SALK_026172 + AtGNL), and restored 2 (SALK_011623 + AtGNL).
  • FIG. 18 shows the total foliar As A level of AtGNL lines under normal light conditions.
  • A Over-expressers and wild type (WT).
  • B Restored lines and knockout control (S 026 ! 72).
  • C Restored lines and knockout control (S_011623).
  • FIG. 19 shows the Phenotype of AtGNL lines grown under normal conditions.
  • A Representative images of ⁇ /GNL lines acquired with the visible camera (aka RGB).
  • FIG. 20 shows the experimental set up for studying the effect of light on the phenotype of AtGNL lines. Light intensity was measured four times per day (9:00 am, 12:00 pm. 3 :00 pm, and 6:00 pm) to cover the sunlight period. The light intensity for these three treatment was defined as: low light (35 - 1 10 mol/m 2 /s), normal light ( 10 - 350 i umol/m /s), and high light (350 - 700 ⁇ 1/ ⁇ 2 /3).
  • FIG. 21 shows the Total foliar AsA levels of AtGNL lines under low, normal and high light conditions.
  • WT Over- expressers and wild type
  • B Restored lines and knockout control
  • C Restored lines and knockout control
  • S026172 had a lower significant difference compared with wild type control at high light treatment.
  • WT wild type
  • EV empty vector
  • OE over-expresser
  • KO knockout
  • R restored.
  • the table in FIG. 22 indicates lines are significantly different and that there is a significant interaction between the lines and light treatments.
  • This knockout line has a lower AsA level in the leaves, lower biomass, and projected leaf area, and also higher fluorescence compared with rest of the lines. Overall these results show that the AiGNL enzyme is essential to support normal AsA content in leaves and normal growth and development in Arabidopsis.
  • FIG. 25 shows the Effect of darkness on the expression of genes in the AsA metabolic network.
  • Microarray data deposited at Genevesti gator was mined. Genes that are down regulated in darkness are shown in red, while genes that are up-regulated are shown in green. Yellow color indicates genes isoforms that are upregulated under dark conditions.
  • the D-mannose/L-galactose, L-gulose and D- glucuronate pathway are repressed under low light conditions while the wyo-inositol pathway keeps working. Suza and Lorence, unpublished.
  • Photosynthetic efficiency is the fraction of light (photons) that plants obtain from the sun to convert into chemical energy during photosynthesis. Under normal light conditions there was no penalty in the photosynthetic efficiency of plants lacking AtGNL expression.
  • LEF linear electron flow
  • NPQt non-photochemical quenching
  • the linear electron flow rate (LEF) has a direct correlation to photosynthetic efficiency.
  • LEF facilitates the movement of H + ions across the thvlakoid membrane to create an electrochemical gradient that is used by ATP-synthase to produce energy (ATP).
  • FIG. 28 shows that under low light conditions both knockouts had lower LEF values than the controls, while KO-1 was the line with the worst performance under normal light conditions.
  • Arabidopsis plants grown under field conditions were compared with plants grown indoors. Indoor-grown plants had larger leaves, modified leaf shapes and longer petioles and less NPQt, while field-grown plants had a high capacity to perform state transitions (Mishra et al, 2012). If photosynthesis is inefficient, excess light energy is dissipated as heat to avoid damaging the photosynthetic apparatus. When plants are under abiotic stress, such as low light intensity, the photosynthetic efficiency and the NPQt are opposite. The KO-1 line had high NPQt, indicating inefficient photosynthesis at both low and normal light conditions.
  • the table indicates the treatment light has a significant effect value 0 001
  • the over-expressers, restored lines, wild type, and empty vector lines had higher values of photosynthetic efficiency and LEF compared with KO lines under normal and low light conditions, while the NPQt values were opposite with the KO-1 having the highest value.
  • AtGNL empty vector and wild type plants were treated with the X-Gluc substrate.
  • FIG. 32 GUS activity was evident in plants expressing the AtGNL promoter in ail developmental stages from cotyledons to roots, although much less staining was observed in 4-day-old seedlings compared with the controls. The oldest seedlings stained most intensely, especially at the leaf tips and margins.
  • FIG. 32 shows the Temporal and spatial expression of AtGNL using the GUS-PLUS reporter gene. The ⁇ tGNL is expressed in the whole plant and at all developmental stages, indicating that the GNL enzyme is important in the physiological development of the plant from beginning to maturity.
  • a phylogenetic tree for /-Mg56500 was generated.
  • /Mg5650Q (.4 G L) was compared with known GNLs and with putative GNLs for many other organisms.
  • the protein sequences were aligned using the MEGA6 software (Tamura et al., 2013). Only the sequences that had between 90 and 100% of identity with the AtGNL protein of interest were included in this analysis.
  • a BLASTP search was done against the Arabidopsis protein database (www.arabidopsis.org) using the A.
  • This enzyme has been characterized in A. niger (Ogawa et al, 2002), E. gracillis (Ishikawa et al, 2008), P. aeruginosa (Tarighi et al., 2008), R. norvegicus (Kondo et al, 2006), Z mobilis (Pedruzzi et al, 2007), and now also in Arabidopsis (this work).
  • the BLASTP result revealed the presence of 37 candidates in different organisms with 90-100% identity to the AtGNL query, FIG. 33 shows the Phylogenetic analysis of known and putative GNLs.
  • AtGNL AtGNL groups with proteins from plant species including plant crops of agricultural importance including Cucumis sativus (cucumber), Cucumis melo (melon), Citrus sinensis (orange), Vitis vinifera (grapes), Theohroma cacao (cacao), Glycine max (soybean), and Fragaria vesca (strawberry), trees such as Populus trichocarpa (poplar) and Pnmns mume (Chinese plum), and energy crops such as Jatropha curcas.
  • the constitutive expression of the gene of interest leads to higher seed yield in plants, such as Arabidopsis. Such higher seed yield is shown in FIG. 35.
  • FIG. 36 shows the GNL DNA sequence.
  • FIG. 37 shows the G L amino acid sequence.
  • the GNL over- expressed in the plant may include at least 70% of the sequence shown in FIG. 36 or FIG. 37.
  • the , GNL enzyme had highest activity at temperatures between 25°C to 35°C, while the bacteria P. aeruginosa has optimal temperature activity at 24°C.
  • the optimum temperature of the AtGNL is consistent with the preferred growth temperature of Arabidopsis.
  • the enzyme characterized in this work is very specific with the D-GuIL substrate.
  • the recombinant GNLs from E. gracilis and R. norvegicus are promiscuous as they displayed activity with additional substrates (Kondo et al., 2006; Ishiwaka et al, 2008b).
  • GNL enzymes require a divalent cofactor for activity.
  • the AtGNL displayed similar activity when incubated with MnCl 2 , MgCl 2 , or ZnC .
  • other GNLs such as the E. gracilis isoform prefer ZnCh and activity dropped significantly with other cofactors (Ishikawa et al., 2008).
  • the ability of AtGNL to work with MnCh is consistent with an enzyme that is active in the chloroplasts, as Mn is abundant in that organelle (Alberts et al, 2002). Based on optimum pH, optimum T, and kinetic parameters of the known GNLs the one that is the most similar to the one here characterized is the G. oxydans GNL.
  • the present invention provides a method of controlling the role of this enzyme in AsA biosynthesis in planta.
  • this enzyme leads to over-expressers with up to 3-fold increase in foliar AsA content.
  • Two T-DNA insertion knockouts in this gene SALK lines
  • SALK lines Two T-DNA insertion knockouts in this gene (SALK lines) had reduced AsA content compared to the WT control.
  • the functional gene was inserted into the knockout background this led to plants with restored AsA content.
  • Overall this data indicates that /iiGNL is functional in planta.
  • ⁇ tG L is a chloroplastic enzyme (FIG. 7)
  • FOG. 7 chloroplastic enzyme
  • transgenic A thaliana lines expressing the GUS-PLUS reporter gene under the control of the ⁇ rG L promoter (p ⁇ tGNL).
  • p ⁇ tGNL ⁇ rG L promoter
  • AtGNL is an important enzyme to sustain sufficient AsA content and to maintain plant growth and efficient photosynthesis.
  • a phylogenetic tree of known and putative GNLs was developed (FIG. 33). At least 37 candidates with 90 - 100% sequence identity to the ⁇ 4 GNL at the amino acid level exist. This analysis indicates the presence of GNLs in a wide array of plants including crops of agricultural importance, mammals, bacteria, and fungi. Interestingly it appears to be a GNL in Sellaginella moellendorffii, an ancient vascular plant that is widely used as a model to study the evolution of plants as a whole (Banks et al., 2011).

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Abstract

L'ascorbate protège les tissus contre des dommages provoqués par des espèces réactives de l'oxygène (ROS) produites par le métabolisme normal ou générées par une contrainte. La voie de transformation de l'inositol en molécules AsA implique quatre enzymes : myo-inositol oxygénase, glucuronate réductase, gluconolactonase (GNL), et L-gulono-1,4-lactone oxydase (GulLO). Dix-huit GNL putatifs ont été identifiées chez l'Arabidopsis, dont l'une, l'AtGNL est intéressante car elle possède un peptide d'adressage au chloroplaste. Les invalidations de ce gène entraînaient une teneur en AsA foliaire réduit et une inhibition de la croissance par rapport aux témoins. Le gène fonctionnel a restauré le phénotype des invalidations et ces plantes avaient une teneur en AsA supérieure, une capacité photosynthétique améliorée et un rendement des semences supérieur.
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WO2011050286A1 (fr) * 2009-10-23 2011-04-28 Arkansas State University Méthodes et compositions permettant d'augmenter la production d'un polypeptide
US20110244512A1 (en) * 2010-03-31 2011-10-06 E. I. Du Pont De Nemours And Company Pentose phosphate pathway upregulation to increase production of non-native products of interest in transgenic microorganisms

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CA2602843A1 (fr) * 2005-03-28 2006-10-05 Virginia Tech Intellectual Properties, Inc. Plantes transgeniques tolerantes au stress surexprimant des genes de synthese de la paroi cellulaire de l'acide ascorbique

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US20020012979A1 (en) * 1998-06-08 2002-01-31 Alan Berry Vitamin c production in microorganisms and plants
EP1026257A1 (fr) * 1999-01-18 2000-08-09 F. Hoffmann-La Roche Ag Production d'acide L-ascorbique et d'acide D-érythorbique
WO2011050286A1 (fr) * 2009-10-23 2011-04-28 Arkansas State University Méthodes et compositions permettant d'augmenter la production d'un polypeptide
US20110244512A1 (en) * 2010-03-31 2011-10-06 E. I. Du Pont De Nemours And Company Pentose phosphate pathway upregulation to increase production of non-native products of interest in transgenic microorganisms

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US12180494B2 (en) 2016-12-01 2024-12-31 Arkansas State University—Jonesboro Method of improving chloroplast function and increasing seed yield

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