Effects Of OsGLO5 And OsCATA Genes On Respiratory Metabolism In Rice

Photorespiration is important carbon metabolic pathway, and important energy metabolic pathways in plants. Photorespiration could reduce CO₂ fixing of Calvin cycle by depletion of Calvin cycle metabolites, decline nitrogen reassimilation and accumulation of photorespiratory metabolites, photorespiratory pathway has been shown to be attributed to inhibition of the repair of photodamaged PSII owing to suppression of the de novo synthesis of the D| protein at the translation step, photosynthetic CO₂ fixation could be limited by photorespiration, it’s causes an imbalance between the amount of the light energy absorbed and the capacity of the plant for its utilization, inhibition of photorespiratory pathway and photosystem can be attributed directly to the consequences of excessive light, such as ROS generation. The H2O₂ produced by photorespiratory pathway in the peroxisomal, not simply related to H2O₂ toxicity, but are rather mediated by redox signaling pathways, lead to programmed cell death. This study is construct interference vector of OsGLO5 and transform into Oryza sativa; analyze the impact of photorespiration, Calvin cycle, photosystem and antioxidant system by OsGLO5, OsCATA and changing the ratio of CO₂ and O₂. The results are as follows:1. A 404bp OsGLO5 gene sequence, was amplified from Oryza sativa for construct interference vector, sequencing shows 100% homology to database, it have six exons 2,3,4,5, 6 and 7 in OsGLO5, only a fragment in 2 and 7 exons. OsGLO5 interference vector contain UBi-1 promoter have been construct, which has a GUS and hygromycin by 35S promoter controled. It’s transformed into rice with or without OsGLO5 interference fragment by Agrobacterium-mediated method.2. Identification of transgenic plants by GUS staining, extract DNA, quantitative PCR, Western blot and GLO（CAT） activity. It’s obtained one lines for blank vector and two lines for OsGLO5i vector for further research by GUS staining and PCR identification. Compared with Oryza Sativa, the expression degrees of OsGLO5 decreased to 20% and 17% by quantitative PCR; the protein content of OsGLO decreased to 61% and 67% by western blot, the glycolate oxidase activity decreased to 54% and 48% in OsGLO5i transgenic plant lines. Meanwhile the expression degrees of OsGLO1, OsGLO₂ and OsGLO3 also significantly down-regulate in some lines, the expression degrees of OsGLO4 also declined. Transgenic rice has shorter plant height and shorter leaf length. The expression degrees of OsCATA gene decreased to 28% and 15% by quantitative PCR; the protein content of OsCATA difficult to be detected by western blot, the catalase activity decreased to 7% and 6% in OsCATAi transgenic plant lines. Meanwhile the expression degrees of OsCATC have significantly increased, up to 750% and 816%, the expression degrees of OsCATB also have significant change. Transgenic rice has shorter plant height, shorter leaf length, white spots in leaf and flowering delayed 5-10 days.3. Light saturation point and CO₂ saturation point all declined in OsGLO5i transgenic plant lines, and net photosynthetic rate decreased slightly at 400 μmol·mol-1 CO₂ concentration and 800 μmol·m-2·s-1 light intensity. Quantitative PCR results showed that:the expression degrees of photorespiration pathway genes OsHPR and OsGDC decreased, rubisco initial activity and total activity also have significant differences, the expression degrees of genes in Calvin cycle fructose bisphosphate aldolase, phosphate ribulokinase, ribose-5-phosphate isomerase et al., all shows significant downward. The chlorophyll fluorescence parameters, photosystems-related genes, antioxidant enzyme activity and the expression of antioxidant enzymes gene et al., shows no significant differences in OsGLO5i transgenic plant lines with Oryza Sativa and empty vector transgenic plant lines, ASC content, DHA content, GSH content and GSSG content which shows significant increase. Field data indicate that panicle length, seed width and 100 seed weight were decreased significantly in OsGLO5i transgenic plant lines with Oryza Sativa and empty vector transgenic plant lines, yield per plant also decreased in some lines, seed length and panicle number did not change significantly.4. Light saturation point and CO₂ saturation point all declined in OsCATAi transgenic plant lines, and net photosynthetic rate decreased significantly at 400 μmol·mol-1 1 CO₂ and 800 μmol·m-2·s-1 illuminations. Quantitative PCR results showed that:the expression degrees of photorespiration pathway genes OsHPR and OsGDC decreased, rubisco initial activity and total activity also have significant differences, the expression degrees of genes in Calvin cycle fructose bisphosphate aldolase, phosphate ribulokinase, ribose-5-phosphate isomerase et al., all shows significant downward. The chlorophyll fluorescence parameters Fv/Fm, qN, qP and Yield were significant declined, the expression of photosystems-related genes OsPsb H, OsPsb R and OsLHC also were declined in OsCATAi transgenic plant lines with Oryza Sativa and empty vector transgenic plant lines, the activity of antioxidant enzyme SOD, APX, GPX and GR were significantly increased, and the expression of related gene OsSOD, OsAPX, OsGPX and OsGR also were increased, the content of anti-oxidants ASC, DHA, GSH and GSSG were increased, H2O₂ content also showed a significant increase, but there has been no change to increase exponentially, MDA concentration have been declined.5. The digital gene expression of OsGLO5i transgenic rice and Oryza Sativa in CO₂ and O₂ treatment shows that there are 451 up-regulation gene and 496 down-regulation gene in Oryza Sativa at high CO₂ treat compare with normal air; 282 up-regulation gene and 397 down-regulation gene at low O₂ treat compare with normal air; and 445 up-regulation gene and 564 down-regulation gene at high O₂ treat compare with normal air. there are 763 up-regulation gene and 191 down-regulation gene in OsGLO5i transgenic rice at normal air compare with Oryza Saliva; The upregulated and downregulated genes in CO₂ and O₂ treatment compare with Oryza Saliva at the same condition is:1272:289（high CO₂ treatment）, 1337:885（low O₂ treatment）,1505:727（high O₂ treatment）.6. Rubisco initial activity and total activity, glycolate oxidase activity, net photosynthetic rate and gross photosynthetic rate all have been decreased in Oryza Saliva L. spp. Japonica at high CO₂ treat compare with normal air, the expression of gene in Calvin cycle and photorespiration pathway rubisco large subunit, ribose-5-phosphate isomerase, OsGLO5, OsHPR, OsGDC el al., also have been declined; chlorophyll fluorescence parameters qP, Yield declined too; the activity of SOD and APX increased, but the activity of CAT is declined, the content of GSH and GSSG also have been declined. There are same changes on physiological and molecular at low O₂ treat with high CO₂ treat, rubisco initial activity and total activity declined, glycolate oxidase activity declined, net photosynthetic rate and gross photosynthetic rate declined, but the value is high than high CO₂ treat. Chlorophyll fluorescence parameters qP, Yield and activity of antioxidant enzyme shows same change with high CO₂ treat. There are little changes on physiological and molecular at high O₂ treat with normal air, rubisco initial activity and total activity, glycolate oxidase activity, net photosynthetic rate and gross photosynthetic rate, chlorophyll fluorescence parameters qP, Yield, activity of antioxidant enzyme, content of antioxidants et al., all is higher than high CO₂ treat, but don’t have different with normal air.7. Glycolate oxidase activity and gross photosynthetic rate are significant declined at different treatment in OsGLO5i transgenic rice compare with Oryza Saliva; rubisco initial activity and total activity declined, net photosynthetic rate, chlorophyll fluorescence parameters qN, qP and Yield all declined at high O₂ treat compare with Oryza Saliva, and the expression of genes in related metabolic pathways is declined too; there are no consistency significant change on the activity of antioxidant enzyme and the content the anti-oxidants at different treatment. It’s don’t show much different in OsGLO5i transgenic rice compare with Oryza Sativa L. spp. Japonica at high CO₂ and low O₂ treat.The result of field in OsGLO5i transgenic rice and OsCATAi transgenic rice and different CO₂ and O₂ concentration treatment shows that:Photorespiration have feedback to the Calvin cycle, when the rate of photorespiration exceeds a certain range, the CO₂ fixed by Calvin cycle will decrease, and even within a certain range, it does not exhibit when photorespiration decline, the CO₂ fixed by Calvin cycle increase; Photorespiration has a protective effect to photosystem, this protection may be related to Calvin cycle, that is caused when photorespiration falls and the Calvin cycle decline, the photosystem will be hurt; the impact of photorespiration against oxidation system may not be directly by H2O₂ produced in peroxisomes, but through the reactive oxygen species generate by photosystem, affect the antioxidant system.