Functional Analysis Of Glyoxylate Reductase Isozymes GR1 And GR2 In Rice

Glyoxylate is an intermediate in the photorespiratory glycolate metabolism.Toxicity may occur if it accumulates in plant cells.In plants,non-toxic levels of glyoxylate are mainly realized by metabolizing it via photorespiration,but part of glyoxylate can still be reduced back to glycolate by glyoxylate reductase(GR).It has been known that there exist two isozymes of GR,one is localized in the cytosol and the other in the chloroplast.In this study,by using approaches of gene knockout and overexpression,in combination with the heterologous expression,the biological functions of the GR isozymes were investigated.The main results are described as follows:1.Temporal and spatial-specific expressions of OsGR1 and OsGR2 in riceThe expression patterns of time-space for Os GR1 and OsGR2 were analyzed by quantitative PCR and the specific primers were designed based on the gene sequence.The results showed that the expression level of OsGR2 is extremely low and amounts to only6%of the reference gene Actin.The abundance of OsGR1 is 20-folds higher than that of OsGR2.Both genes are mainly expressed in leaves,stems and small shells?,and OsGR1 is also highly expressed in roots.At different times during a day and at different growth stage,The activity of NADPH-OsGR is 2 times that of NADH-Os GR.Both enzymes showed the same diurnal rhythm as photosynthesis,and a noon-nap phenomenon exists for GR activity changes during a day and plants at the vegetative stage showed relatively high enzyme activities.2.Construction of the OsGR knockout and overexpression transgenic plantsTwoknockoutandoneconstitutiveoverexpressionvectors(pYLCRISPR/Cas9-OsGR1,pYLCRISPR/Cas9-OsGR2andpOE-OsGR1)were constructed,which were then transformed into rice by Agobacterium-mediated infection,and finally the transgenic plants were generated with OsGR1 or OsGR2 knocked out and OsGR1 overexpressed.Molecular,western blot and activity assays showed that OsGR1 and OsGR2 were totally knocked out at the DNA level,and down-regulated by 70~90%at the mRNA level in the Cas9 transgenic lines;no enzyme protein was produced and the enzymatic activity was reduced by 30~40%in gr1 plants,and by only 10%in the gr2 plants.It was also verified that both genes were highly up-regulated at the mRNA,protein,activity levels.3.The relationship between OsGR and oxalate accumulation in riceNo phenotypic differences were noticed for all knockout lines and overexpression plants.The oxalate content was determined in different positional leaves of plants and at different growth stage.The results turned out that oxalate content was increased by about26~33%in all the gr mutants,and dropped by 16%in the overexpression lines GR2OE4-6 and GR1OE168-2-2-5.4.Heterogeneous expressions of OsGR1 and OsGR2 and their enzymatic propertiesThe heterogeneous expression vectors of pET30a-OsGR1 and pET28a-OsGR2 were constructed,then induciblely expressed in E.coli.It was found that abundant soluble proteins of both OsGR1 and OsGR2 could be produced in E.coli with high enzymatic catalytic activities.OsGR protein was purified by the Ni column affinity chromatography with.Kinetic analyses on both enzymes OsGR1 and OsGR2 showed that OsGR1 and OsGR2,when using NADPH as cofactor,K_m for glyoxlate was 0.03 mM and 0.072 mM,respectively,and if using glyoxylate as substrate,K_m for NADPH was 0.267 mM and 0.144mM,respectively.which was about 1.5 and 3 folds lower than for NADH(0.42 and 0.403mM).OsGR1 and OsGR2 both have higher affinity for than for NADH,but OsGR1preferred glyoxylate as substrate to OsGR2.V_m of OsGR1 was only 1/3 of OsGR2 for the glyoxylate-NADPH reaction(68 U/mg protein,262 U/mg protein).Oxalate has no inhibitive effect on the NADPH-glyoxylate reaction catalyzed by both Os GR1 and OsGR2,but highly inhibited the NADH-glyoxylate reaction,with K_i(oxalate)3 mM and 8 mM,respectively.To sum up,this study demonstrates OsGR1 and OsGR2 may play roles in detoxifying glyoxylate and regulating photorespiration,an in addition,the result further confirmed that glyoxylate is an efficient precursor for oxalate biosynthesis and both GR isozymes are important for oxalate regulation in plants.