Effect Of Maize C₄ Pepc,ppdk,nadp-me On Physiological Characterization Of Arabidopsis Under High Light Stress

This study assessed the effects of maize?Zea mays?phosphoenolpyruvate carboxylase?PEPC,coding gene pepc?,pyruvate phosphate dikinase?PPDK,coding gene ppdk?,and NADP-malic enzyme?NADP-ME,coding gene nadp-me?,and their combinations on physiological characteristics of Arabidopsis under high light stress,and investigate the ideal combination of three genes to improve photosynthetic performance and its mechanism.This study provides a theoretical guidance for the genetic improvement of biosmass and stress resistance of wheat,C₃ crops by using C₄ photosynthetic enzyme genes.In this study,Zmpepc?PC?,Zmppdk?PK?,Zmnadp-me?ME?,Zmpepc+Zmppdk?PCK?,Zmppdk+Zmnadp-me?PKM?,Zmpepc+Zmnadp-me?PCM?,Zmpepc+Zmppdk+ Zmnadp-me?PCKM?transgenic Arabidopsis and wild type?WT?were conducted as experimental materials.By analysis and compare systematically for the differences of photosynthetic performance,physiological parameters and chloroplast proteome of transgenic Arabidopsis and WT using various methods such as chlorophyll fluorescence and i TRAQ under normal light and high light treatment,the main results and conclusion are listed as follows:1.The recombinant expression vector containing Zmnadp-me gene were transformed into the PCK transgenic Arabidopsis stable lines mediated by Agrobacterium tumefaciens.The results of PCR,Southern blot,q RT-PCR,Western blot analysis showed that the three photosynthetic enzymes genes of maize were integrated into the Aribidopsis genome with a single copy,and the genes were correctly in transcription and translation level.The results indicated that the PCKM transgenic Arabidopsis lines were successfully created.2.The results of photosynthetic rate,stomatal conductance,transpiration rate and intercellular CO₂ concentration of seven transgenic Arabidopsis genotypes and WT showed that the photosynthetic rate of four genotypes,including PC,PCK,PCM and PCKM transgenic Arabidopsis,increased significantly,and the increase was caused by non-stomatal limitation.The photosynthetic rate of PC,PCK,PCM and PCKM transgenic Arabidopsis were 11.4%,16.5%,11.9% and 23.2% higher than WT under normal light treatment,respectively;and were 17.3%,32.5%,17.9% and 46.6% higher than WT under high light treatment,respectively.The increase of photosynthetic rate of PC,PCK,PCM,and PCKM transgenic Arabidopsis,that compared with WT under high light treatment,which that bigger than the increase under normal light treatment.The trend of significant difference of photosynthetic rate of these four genotypes was as follows,PCKM>PCK>PC,PCM,while photosynthetic rate of PK,ME and PKM transgenic Arabidopsis were not different from those of WT.These results indicated that Zmpepc gene was the prerequisite factor for improving photosynthetic rate,Zmppdk had a positive synergistic effect on Zmpepc for improving photosynthetic rate,and the combination of three genes had the most significant synergistic effect.3.The results of four photosynthetic enzymes activities,including PEPC,PPDK,NADP-ME and Ru BPCase,and carboxylation efficiency,biomass of seven transgenic Arabidopsis showed that the overexpression of four photosynthetic enzymes activities in PC,PCK,PCM and PCKM transgenic Arabidopsis,improved significantly the carboxylation efficiency,and then increased significantly the photosynthetic rate,and correspondingly increased biomass of the four genotypes.The carboxylation efficiency of PC,PCK,PCM and PCKM transgenic Arabidopsis were 9.1%,13%,9.9% and 19.6% higher than WT under normal light treatment,respectively;and were 10.1%,16.6%,10.8% and 25.9% higher than WT under high light treatment,respectively.The carboxylation efficiency was no difference among PK,ME,PKM transgenic Arabidopsis that compared with WT.The biomass of PC,PCK,PCM and PCKM transgenic Arabidopsis were 6.5%,11.1%,7.6% and 17.1% higher than WT under normal light treatment,respectively;and were 9.8%,16.4%,9.7% and 23.8% higher than WT under high light treatment,respectively.The biomass was no difference among PK,ME,PKM transgenic Arabidopsis that compared with WT.4.The results of chlorophyll fluorescence,PSI activity,soluble protein,reactive oxygen species?ROS?,ROS scavenging enzymes and malondialdehyde of seven transgenic Arabidopsis under high light stress showed that four genotypes including PC,PCK,PCM,PCKM transgenic Arabidopsis had better tolerance to high light stress.The results indicated that PC,PCK,PCM,PCKM transgenic Arabidopsis had increased photosystem activity?K-phase,J-phase and PSI activity?,electron transfer efficiency?ETo/TRo?,photochemistry efficiency?TRo/ABS?;These genotypes had increased SOD,APX activity and soluble proteins content,reduced ROS accumulation.These genotypes had better tolerance to high light stress through effectively reducing the damage degree of photosynthetic apparatus,and maintaining the number of active reaction centers?RC/CSm?.5.The results of chloroplast proteomics analysis of PCKM transgenic Arabidopsis and WT under normal and high light treatment,which indicated that increased the dark reaction efficiency,and improved the light reaction characteristics by the expression of PCKM in Arabidopsis.The introduction of these three genes up-regulated the protein abundances related to light capture,conversion and CO₂ assimilation in the photosynthetic apparatus of transgenic Arabidopsis,which correspondingly improved the light absorption,the activity and stability of PSII and PSI,the electron transfer efficiency,the production of ATP and NADPH,and the efficiency of carbon assimilation,especially under high light treatment.A total of 8904 peptides,2405 proteins were identified based on i TRAQ technology,and 1961 proteins localized to chloroplasts were obtained based on the database from the University of Nebraska Medical Center.Forty-one,559 and 562 differentially expressed proteins?DEPs?were identified by PCKM responsive,high light responsive and their common responsive,respectively.The enriched KEGG analysis showed that DEPs were mainly involved in metabolic pathways,photosynthesis,secondary metabolic biosynthesis,carbon fixation of photosynthetic organisms,carbon metabolism,photosynthesis-antenna protein and other related pathways.Further analysis of the pathways of photosynthesis-antenna protein?ath00196?,photosynthesis?ath00195?and carbon fixation of photosynthetic organisms?ath00710?revealed that the DEPs of PCKM responsive involved up-regulation of two antenna proteins,nine photo-reactive proteins and seven carbon assimilation proteins;the DEPs of common PCKM and high light treatment responsive included DEPs of PCKM responsive,and additional 8,16 and 5 DEPs corresponding metabolic pathways,which that most of the DEPs were up-regulated.