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A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa.

Dalal J, Lopez H, Vasani NB, Hu Z, Swift JE, Yalamanchili R, Dvora M, Lin X, Xie D, Qu R, Sederoff HW - Biotechnol Biofuels (2015)

Bottom Line: Hydrogenation-derived renewable diesel from camelina is environmentally superior to that from canola due to lower agricultural inputs, and the seed meal is FDA approved for animal consumption.The photorespiratory bypass is an effective approach to increase photosynthetic productivity in camelina.By reducing photorespiratory losses and increasing photosynthetic CO2 fixation rates, transgenic plants show dramatic increases in seed yield.

View Article: PubMed Central - PubMed

Affiliation: Department of Crop Science, North Carolina State University, Campus Box 7287, Raleigh, NC 27695-7287 USA.

ABSTRACT

Background: Camelina sativa is an oilseed crop with great potential for biofuel production on marginal land. The seed oil from camelina has been converted to jet fuel and improved fuel efficiency in commercial and military test flights. Hydrogenation-derived renewable diesel from camelina is environmentally superior to that from canola due to lower agricultural inputs, and the seed meal is FDA approved for animal consumption. However, relatively low yield makes its farming less profitable. Our study is aimed at increasing camelina seed yield by reducing carbon loss from photorespiration via a photorespiratory bypass. Genes encoding three enzymes of the Escherichia coli glycolate catabolic pathway were introduced: glycolate dehydrogenase (GDH), glyoxylate carboxyligase (GCL) and tartronic semialdehyde reductase (TSR). These enzymes compete for the photorespiratory substrate, glycolate, convert it to glycerate within the chloroplasts, and reduce photorespiration. As a by-product of the reaction, CO2 is released in the chloroplast, which increases photosynthesis. Camelina plants were transformed with either partial bypass (GDH), or full bypass (GDH, GCL and TSR) genes. Transgenic plants were evaluated for physiological and metabolic traits.

Results: Expressing the photorespiratory bypass genes in camelina reduced photorespiration and increased photosynthesis in both partial and full bypass expressing lines. Expression of partial bypass increased seed yield by 50-57 %, while expression of full bypass increased seed yield by 57-73 %, with no loss in seed quality. The transgenic plants also showed increased vegetative biomass and faster development; they flowered, set seed and reached seed maturity about 1 week earlier than WT. At the transcriptional level, transgenic plants showed differential expression in categories such as respiration, amino acid biosynthesis and fatty acid metabolism. The increased growth of the bypass transgenics compared to WT was only observed in ambient or low CO2 conditions, but not in elevated CO2 conditions.

Conclusions: The photorespiratory bypass is an effective approach to increase photosynthetic productivity in camelina. By reducing photorespiratory losses and increasing photosynthetic CO2 fixation rates, transgenic plants show dramatic increases in seed yield. Because photorespiration causes losses in productivity of most C3 plants, the bypass approach may have significant impact on increasing agricultural productivity for C3 crops.

No MeSH data available.


Related in: MedlinePlus

Expression of photorespiratory bypass genes in camelina. a Constructs were generated for introducing photorespiratory bypass genes into Camelina. Each coding sequence was preceded by a constitutive promoter and fused to a chloroplast transit peptide sequence. The DEF2 construct contains GlcD, GlcE and GlcF sequences cloned into a modified pCAMBIA2300-mCherry vector where NPTII from pCAMBIA2300 has been replaced by the mCherry gene. The TG1 construct contains the GCL and TSR sequences cloned into the pEG100 vector. Plants were either transformed with the DEF2 construct alone, TG1 construct alone, or co-transformed with DEF2 and TG1. b Plants passing selection were tested for gene insertion using PCR of gDNA. Primers (Additional file 1: Table S1) were used to amplify the endogenous reference gene SVP1(+) and transgenes GlcD, GlcE, GlcF, TSR and GCL. c The expression of transgene mRNAs was tested by semi-quantitative RT-PCR using the same primers as above. d The proteins GlcD, GlcE and GlcF combine to form the glycolate dehydrogenase (GDH) enzyme complex. The activity of GDH was tested using isolated chloroplasts from transgenic and WT plants. e The activity of enzymes TSR and GCL was evaluated in a coupled NADH depletion assay. Using sodium glyoxylate as substrate, NAD generation in chloroplast extracts was compared
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Fig2: Expression of photorespiratory bypass genes in camelina. a Constructs were generated for introducing photorespiratory bypass genes into Camelina. Each coding sequence was preceded by a constitutive promoter and fused to a chloroplast transit peptide sequence. The DEF2 construct contains GlcD, GlcE and GlcF sequences cloned into a modified pCAMBIA2300-mCherry vector where NPTII from pCAMBIA2300 has been replaced by the mCherry gene. The TG1 construct contains the GCL and TSR sequences cloned into the pEG100 vector. Plants were either transformed with the DEF2 construct alone, TG1 construct alone, or co-transformed with DEF2 and TG1. b Plants passing selection were tested for gene insertion using PCR of gDNA. Primers (Additional file 1: Table S1) were used to amplify the endogenous reference gene SVP1(+) and transgenes GlcD, GlcE, GlcF, TSR and GCL. c The expression of transgene mRNAs was tested by semi-quantitative RT-PCR using the same primers as above. d The proteins GlcD, GlcE and GlcF combine to form the glycolate dehydrogenase (GDH) enzyme complex. The activity of GDH was tested using isolated chloroplasts from transgenic and WT plants. e The activity of enzymes TSR and GCL was evaluated in a coupled NADH depletion assay. Using sodium glyoxylate as substrate, NAD generation in chloroplast extracts was compared

Mentions: For the catabolic conversion of glycolate to glycerate and CO2 in chloroplasts (photorespiratory bypass, Fig. 1), genes encoding three bacterial enzymes—GDH, GCL and TSR were introduced into C. sativa nuclear genome and the proteins were targeted to chloroplasts. The bacterial sequences were codon-optimized for expressing in plants (GenBank: KP967458-KP967462). The first step of the photorespiratory bypass is the oxidation of glycolate to glyoxylate. This step is catalyzed by the enzyme glycolate dehydrogenase (GDH), containing three subunits, GlcD, GlcE and GlcF (construct DEF2, Fig. 2a). The second step involves ligation of two glyoxylate molecules to form a 3-carbon tartronic semialdehyde (TSA) and the release of a CO2 molecule. This conversion is catalyzed by glyoxylate carboxyligase (GCL). The third step involves conversion of TSA to glycerate by tartronic semialdehyde reductase (TSR). The gene constructs of GCL and TSR are included in vector TG1 (Fig. 2a). Plants were either transformed with DEF2 (DEF2 lines; partial bypass), TG1 (TG1 lines) or co-transformed with DEF2 and TG1 constructs together (DEF2+TG1 lines; full bypass). The integration and expression of the transgenes in plants was verified by PCR using gDNA (Fig. 2b) and cDNA templates (Fig. 2c). Using PCR, we were able to isolate homozygous lines for all the three combinations—DEF2, TG1 and DEF2+TG1. In T3 homozygous plants, the protein expression of the transgenes was quantified by ELISA with antibodies specific to each protein. Green leaves of 3-week-old plants were used to quantify bypass proteins. On average, in DEF2 transgenics, GlcD expression was detected to be 0.76 µg/g fresh weight (FW), GlcE was 0.61 µg/g FW, and GlcF was 0.89 µg/g FW. On average, in TG1 plants, the expression of GCL was 5.7 µg/g FW and TSR was 11.8 µg/g FW (Additional file 1: Table S3). The catalytic activity of the bypass enzymes was evaluated by isolating the chloroplasts from transgenic and WT plants, and using chloroplast extracts to perform enzymatic assays. The activity of GDH was determined by measuring glyoxylate generation after feeding chloroplast extracts with glycolate [31]. Chloroplasts from DEF2 and DEF2+TG1 transgenics showed 65–120 % higher GDH activity than WT chloroplasts (Fig. 2d). The activities of enzymes GCL and TSR were evaluated in a coupled NADH depletion assay [32]. Chloroplasts from TG1 and DEF2+TG1 transgenics showed 50–300 % higher GCL+TSR activity than WT chloroplasts (Fig. 2e).Fig. 2


A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa.

Dalal J, Lopez H, Vasani NB, Hu Z, Swift JE, Yalamanchili R, Dvora M, Lin X, Xie D, Qu R, Sederoff HW - Biotechnol Biofuels (2015)

Expression of photorespiratory bypass genes in camelina. a Constructs were generated for introducing photorespiratory bypass genes into Camelina. Each coding sequence was preceded by a constitutive promoter and fused to a chloroplast transit peptide sequence. The DEF2 construct contains GlcD, GlcE and GlcF sequences cloned into a modified pCAMBIA2300-mCherry vector where NPTII from pCAMBIA2300 has been replaced by the mCherry gene. The TG1 construct contains the GCL and TSR sequences cloned into the pEG100 vector. Plants were either transformed with the DEF2 construct alone, TG1 construct alone, or co-transformed with DEF2 and TG1. b Plants passing selection were tested for gene insertion using PCR of gDNA. Primers (Additional file 1: Table S1) were used to amplify the endogenous reference gene SVP1(+) and transgenes GlcD, GlcE, GlcF, TSR and GCL. c The expression of transgene mRNAs was tested by semi-quantitative RT-PCR using the same primers as above. d The proteins GlcD, GlcE and GlcF combine to form the glycolate dehydrogenase (GDH) enzyme complex. The activity of GDH was tested using isolated chloroplasts from transgenic and WT plants. e The activity of enzymes TSR and GCL was evaluated in a coupled NADH depletion assay. Using sodium glyoxylate as substrate, NAD generation in chloroplast extracts was compared
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Fig2: Expression of photorespiratory bypass genes in camelina. a Constructs were generated for introducing photorespiratory bypass genes into Camelina. Each coding sequence was preceded by a constitutive promoter and fused to a chloroplast transit peptide sequence. The DEF2 construct contains GlcD, GlcE and GlcF sequences cloned into a modified pCAMBIA2300-mCherry vector where NPTII from pCAMBIA2300 has been replaced by the mCherry gene. The TG1 construct contains the GCL and TSR sequences cloned into the pEG100 vector. Plants were either transformed with the DEF2 construct alone, TG1 construct alone, or co-transformed with DEF2 and TG1. b Plants passing selection were tested for gene insertion using PCR of gDNA. Primers (Additional file 1: Table S1) were used to amplify the endogenous reference gene SVP1(+) and transgenes GlcD, GlcE, GlcF, TSR and GCL. c The expression of transgene mRNAs was tested by semi-quantitative RT-PCR using the same primers as above. d The proteins GlcD, GlcE and GlcF combine to form the glycolate dehydrogenase (GDH) enzyme complex. The activity of GDH was tested using isolated chloroplasts from transgenic and WT plants. e The activity of enzymes TSR and GCL was evaluated in a coupled NADH depletion assay. Using sodium glyoxylate as substrate, NAD generation in chloroplast extracts was compared
Mentions: For the catabolic conversion of glycolate to glycerate and CO2 in chloroplasts (photorespiratory bypass, Fig. 1), genes encoding three bacterial enzymes—GDH, GCL and TSR were introduced into C. sativa nuclear genome and the proteins were targeted to chloroplasts. The bacterial sequences were codon-optimized for expressing in plants (GenBank: KP967458-KP967462). The first step of the photorespiratory bypass is the oxidation of glycolate to glyoxylate. This step is catalyzed by the enzyme glycolate dehydrogenase (GDH), containing three subunits, GlcD, GlcE and GlcF (construct DEF2, Fig. 2a). The second step involves ligation of two glyoxylate molecules to form a 3-carbon tartronic semialdehyde (TSA) and the release of a CO2 molecule. This conversion is catalyzed by glyoxylate carboxyligase (GCL). The third step involves conversion of TSA to glycerate by tartronic semialdehyde reductase (TSR). The gene constructs of GCL and TSR are included in vector TG1 (Fig. 2a). Plants were either transformed with DEF2 (DEF2 lines; partial bypass), TG1 (TG1 lines) or co-transformed with DEF2 and TG1 constructs together (DEF2+TG1 lines; full bypass). The integration and expression of the transgenes in plants was verified by PCR using gDNA (Fig. 2b) and cDNA templates (Fig. 2c). Using PCR, we were able to isolate homozygous lines for all the three combinations—DEF2, TG1 and DEF2+TG1. In T3 homozygous plants, the protein expression of the transgenes was quantified by ELISA with antibodies specific to each protein. Green leaves of 3-week-old plants were used to quantify bypass proteins. On average, in DEF2 transgenics, GlcD expression was detected to be 0.76 µg/g fresh weight (FW), GlcE was 0.61 µg/g FW, and GlcF was 0.89 µg/g FW. On average, in TG1 plants, the expression of GCL was 5.7 µg/g FW and TSR was 11.8 µg/g FW (Additional file 1: Table S3). The catalytic activity of the bypass enzymes was evaluated by isolating the chloroplasts from transgenic and WT plants, and using chloroplast extracts to perform enzymatic assays. The activity of GDH was determined by measuring glyoxylate generation after feeding chloroplast extracts with glycolate [31]. Chloroplasts from DEF2 and DEF2+TG1 transgenics showed 65–120 % higher GDH activity than WT chloroplasts (Fig. 2d). The activities of enzymes GCL and TSR were evaluated in a coupled NADH depletion assay [32]. Chloroplasts from TG1 and DEF2+TG1 transgenics showed 50–300 % higher GCL+TSR activity than WT chloroplasts (Fig. 2e).Fig. 2

Bottom Line: Hydrogenation-derived renewable diesel from camelina is environmentally superior to that from canola due to lower agricultural inputs, and the seed meal is FDA approved for animal consumption.The photorespiratory bypass is an effective approach to increase photosynthetic productivity in camelina.By reducing photorespiratory losses and increasing photosynthetic CO2 fixation rates, transgenic plants show dramatic increases in seed yield.

View Article: PubMed Central - PubMed

Affiliation: Department of Crop Science, North Carolina State University, Campus Box 7287, Raleigh, NC 27695-7287 USA.

ABSTRACT

Background: Camelina sativa is an oilseed crop with great potential for biofuel production on marginal land. The seed oil from camelina has been converted to jet fuel and improved fuel efficiency in commercial and military test flights. Hydrogenation-derived renewable diesel from camelina is environmentally superior to that from canola due to lower agricultural inputs, and the seed meal is FDA approved for animal consumption. However, relatively low yield makes its farming less profitable. Our study is aimed at increasing camelina seed yield by reducing carbon loss from photorespiration via a photorespiratory bypass. Genes encoding three enzymes of the Escherichia coli glycolate catabolic pathway were introduced: glycolate dehydrogenase (GDH), glyoxylate carboxyligase (GCL) and tartronic semialdehyde reductase (TSR). These enzymes compete for the photorespiratory substrate, glycolate, convert it to glycerate within the chloroplasts, and reduce photorespiration. As a by-product of the reaction, CO2 is released in the chloroplast, which increases photosynthesis. Camelina plants were transformed with either partial bypass (GDH), or full bypass (GDH, GCL and TSR) genes. Transgenic plants were evaluated for physiological and metabolic traits.

Results: Expressing the photorespiratory bypass genes in camelina reduced photorespiration and increased photosynthesis in both partial and full bypass expressing lines. Expression of partial bypass increased seed yield by 50-57 %, while expression of full bypass increased seed yield by 57-73 %, with no loss in seed quality. The transgenic plants also showed increased vegetative biomass and faster development; they flowered, set seed and reached seed maturity about 1 week earlier than WT. At the transcriptional level, transgenic plants showed differential expression in categories such as respiration, amino acid biosynthesis and fatty acid metabolism. The increased growth of the bypass transgenics compared to WT was only observed in ambient or low CO2 conditions, but not in elevated CO2 conditions.

Conclusions: The photorespiratory bypass is an effective approach to increase photosynthetic productivity in camelina. By reducing photorespiratory losses and increasing photosynthetic CO2 fixation rates, transgenic plants show dramatic increases in seed yield. Because photorespiration causes losses in productivity of most C3 plants, the bypass approach may have significant impact on increasing agricultural productivity for C3 crops.

No MeSH data available.


Related in: MedlinePlus