The Analysis Of The Physiological Functions Of Glycolate Oxidase GLO1 And GLO4 In Rice

Glycolate oxdase?GLO?is a key enzyme in photorespiration metabolism,which is closely related to plant stress response and other processes.There are four GLO genes in rice genome,GLO1 and GLO4 are the photorespiratory genes.In this study,GLO1 and GLO4knockout mutants were generated by crispr-cas9 system respectively.Then the GLO1 and GLO4 mutant were used to study the physiological functions of GLO1 and GLO4.The main results obtained are as follows:1.Generation of GLO knockout mutantsThe T0-generation mutant plants were obtained by crispr-cas9 system,and the DNA was extracted from the T1 or T2 generation plants.Specific primers were designed according to the GLO1 and GLO4 mutant target sequences,and the GLO1 and GLO4 target fragments in the mutant plants were obtained by PCR.The PCR products were sequenced and analysedby online tool DSDecodeM to confirm the mutation.Afterthat,the GLO1 and GLO4 mutant homozygous plants were screened by HPT amplification and 50 mg/L hygromycin medium.The obtained plants were mutant homozygous plants without HPT gene?crispr-cas9 vector sequence?.2.GLO mutant phenotype analysisBoth GLO1 and GLO4 knockout mutants showed dwarfish and their tiller number decreased.Further analysis of GLO enzyme activity showed that the GLO activity of GLO1mutants reduced by 50%,while the GLO activity of GLO4 mutants reduced by 30%.Subsequent Western blot results showed that there were two GLO protein bands in WT?GLO1 and GLO4?,while only one corresponding GLO protein band was found in GLO1and GLO4 mutants respectively.These results indicated that the down-regulation of GLO activity in GLO1 and GLO4 mutants was caused by their specific GLO deletion,and this result further confirmed that GLO1 and GLO4 were the dominant photorespiration GLO genes in rice leaves.3.Analysis of related physiological and biochemical indexes of GLO mutantsThe CAT activity of GLO mutant plants decreased,and the DAB-H₂O₂ staining showed that the content of H₂O₂ in GLO1 and GLO4 mutant plants decreased.We hypothesized that the decreased H₂O₂ content might function as feedback regulation of CAT expression.In addition,the auxin biosynthesis could be affected by the peroxisomal H₂O₂ homeostasis,the expression of some auxin biosysnthesis related gene was up-regulated in GLO1 and GLO4mutant?such as ASA2,LBD18,LBD29,IAA29 and IAA1 etc.?.Furthermore,the auxin content was also increased in GLO1 and GLO4 mutants.These results suggestedthat there was cross-talking between GLO mediated photorespiration H₂O₂ metabolism and auxin signaling.