Functional Analyses Of OsPLGG1 And OsPLGG2 In Rice

Photorespiration is the second highest metabolic flux only behind photosynthesis in C₃ plants,which may consume 25-30%of photosynthate.Three organelles plus cytosol are involved in photorespiration,it has been well studied in terms of the enzymes which catalyze the core photorespiratory pathway,in contrast,identification of those transporters responsible for the trans-membrane transportation of photorespiratory metabolites is far lagging behind.So far,only dicarboxylate transport Di T1 and Di T2,glycolate/glycerate transporter At PLGG1 and glycolate transporter At BASS6 were identified in Arabidopsis.There exist two PLGG homologs in the rice genome,but it has not yet been investigated whether these two genes can function for the trans-membrane transportation of glycolate/glycerate and how functionally different they are in rice,as an important staple crop plant.In this study,the function of OsPLGG1 and OsPLGG2 is comparatively investigated,and the results are summarized as follows:1.Expression patterns of OsPLGG1 and OsPLGG2 in riceBioinformatic analysis identified that there are two PLGG homologs in the rice genome,here named as OsPLGG1(Os01g0511600)and OsPLGG2(Os10g0578800),their encoded proteins share homology of 77.7%,68.7%,respectively,with At PLGG1,and has 79.9%similarity between the two homologs themselves.Both of OsPLGG1 and OsPLGG2 belong to Lrg B protein family and fall into the same branch in the evolutionary tree,implicating that the two homologs may have similar functions.Transcriptional analyses showed that OsPLGG1 and OsPLGG2 are ultimately expressed in rice leaves and sheath,and their expression can be affected by light,SA,ABA,both of which are in parallel regulated.2.Subcellular localization of OsPLGG1 and OsPLGG2Bioinformatic analysis predict that both OsPLGG1 and OsPLGG2 harbor the chloroplastic peptide with transmembrane structure and domain.Rice and tobacco transient expression analysis verified that OsPLGG1 and OsPLGG2 are localized in the inner and outer membrane of chloroplasts,respectively.This implicates that these both proteins may be involved in the trans-membrane transportation of chloroplasts.3.Generation of the mutants and transgenic rice lines with OsPLGG1 and OsPLGG2 suppressed and over-expressedWe first successfully generated the rice mutants and transgenic lines with OsPLGG1 and OsPLGG2 suppressed and over-expressed,all of which are experimentally verified by quantitative RT-PCR.The results showed that all the materials had achieved the expected gene regulation effect.In the OsPLGG1 RNAi transgenic plants,the m RNA level of OsPLGG1 decreased by42%to 51%,and the m RNA level of OsPLGG2 also decreased by 12%to 41%;in the RNAi transgenic plants of OsPLGG2,the m RNA level of OsPLGG2 decreased by 25%-57%,and the m RNA level of OsPLGG1 also decreased by 37%-66%;in the T-DNA inserted mutant of OsPLGG2,the expression of OsPLGG2 was almost undetectable,at this time,the expression of OsPLGG1 was up-regulated by 2-3 times;in the heterozygous plants knocked out of OsPLGG1 by Cas9,the m RNA level of OsPLGG1 decreased by 88%to 93%,while the m RNA level of OsPLGG2 increased by 2.2-3.9 times;in the homozygous plants where Cas9knocked out OsPLGG2,the expression of OsPLGG2 was barely detectable,at which point the expression of OsPLGG1 was also upregulated by 1.5-1.6 times.In OsPLGG1 overexpression plants,the m RNA level of OsPLGG1 was increased by250-450 times,and the expression of OsPLGG2 was also increased by 2.4-3.6 times simultaneously;in OsPLGG2 overexpression plants,the m RNA level of OsPLGG2 was increased by 13.8-22.7 times,and the gene expression of OsPLGG1 was also increased by2.5-3 times.4.Phenotypes of the rice mutants and transgenic linesAs previously reported,Arabidopsis with At PLGG1 suppressed or up-regulated showed yellowish and dwarf phenotypes.In contrast,our transgenic lines with the two genes suppressed or up-regulated looked even greener than WT,and the content of total chlorophyll was increased,with chlorophyll an enhanced more significantly.The growth and development of RNAi plants showed negative regulation phenotype.The plant height,effective tillering,leaf length,leaf width,length of panicles and setting rates of the OsPLGG1RNAi plants reduced by 19%-34%,25%-31%,30%-40%,16%-36%,20%-32%,19%-41%,respectively.In the RNAi plant of OsPLGG2,the plant height,effective tillering,leaf length,leaf width,length of panicles and setting rates were reduced by 14%to 15%,24%to 34%,24%to 30%,10%to 12%,18%to 15%,and 17%to 23%,respectively.The growth and development of T-DNA inserted mutant plants also presented a negative regulation phenotype,with plant height,effective tillering,leaf length,leaf width,length of panicles and setting rates decreased by 25%to 29%,31%to 48%,18%to 30%,11%to 27%,18%to 19%,and 35%to 38%,respectively.Under ambient CO₂ conditions(400 ppm)the CRISPR-Cas9 OsPLGG1 mutant developed yellowish on the leaf lamina and dwarf phenotypes,and the plant height was 41.4%and 43.9%shorter than the wild type.Whereas the phenotype of CRISPR-Cas9 OsPLGG2mutant plants is consistent with that of T-DNA inserted mutants,showed similar leaf color to WT,but dwarf growth occurred.Under normal air condition,photosynthetic rates and light saturating points were reduced in the lines with both genes down-regulated,the reduction was more significant as compared with reduction for the OsPLGG2 knockout mutants.Photosynthesis could be rescued to some extent by high CO₂(3000 ppm),though it was still lower than that of WT.Surprisingly,no matter OsPLGG1 or OsPLGG2 was overexpressed,transgenic plants showed similar phenotypic traits with its downregulation.In OsPLGG1 overexpressed plants,plant height,effective tillering,leaf length,leaf width,length of panicles and setting rates decreased by 8%to 10%,18%to 24%,13%to 19%,4%to 7%,7%to 11%,and 14%to 69%,respectively.In OsPLGG2 overexpression plants,plant height,effective tillering,leaf length,leaf width,length of panicles and setting rates were reduced by 10%-14%,26%-54%,16%-18%,6%-8%,8%-13%,35%-84%,respectively.The results above indicate that appropriate expression of OsPLGG1 and OsPLGG2 is necessary for normal growth and development of rice.5.Functional analysis of OsPLGG1 and OsPLGG2GC/MS analysis found that both glycolate and glycerate were increased in the transgenic lines with both genes down-regulated and the mutant with OsPLGG2 knocked out,and the increase was more significant in the former transgenic lines than that in the plgg2 mutant.Further yeast heterologous expression analysis showed that glycolate was reduced while glycerate was increased in the yeast cells with either of the genes expressed or both genes co-expressed,and the change was much more significant when the two genes were co-expressed,implicating occurrence of a synergistic effect.These data suggest that OsPLGG1 and OsPLGG2 can transport glycolate/glycerate,both of which work in a synergistic manner.In summary,OsPLGG1 and OsPLGG2 play important roles in plant growth and development,as well as in photosynthetic regulation;The two genes are ultimately expressed in rice leaves,and the encoded proteins are located in the inner and outer membrane of chloroplasts,respectively;both OsPLGG1 and OsPLGG2 can transport glycolate/glycerate cross chloroplastic membrane,and moreover,both proteins work in a synergistic manner.