iTRAQ-Based Proteomics Investigation of Critical Response Proteins in Embryo and Coleoptile During Rice Anaerobic Germination

Direct-seeding of rice has become popular in recent years due to its low cost and convenience,however,hypoxic condition limits seedling establishment.In this study,weedy rice WR04-6 with high germination ability under anaerobic conditions was used as a gene donor,and we successfully improved the seedling establishment rate of rice cultivar Qishanzhan(QSZ)based on selection of a new rice line R42 from the recombinant inbred line population.R42 inherited high anaerobic germination(AG)ability,and was used for isobaric tags for relative and absolute quantitation(iTRAQ)-based comparative proteomic studies with QSZ to further explore the molecular mechanism of AG.A total of 719 differentially abundant proteins(DAPs)were shared by R42 and QSZ responded to AG,and thus defined as common response DAPs.A total of 300 DAPs that responded to AG were only identified from R42,which were defined as tolerance-specific DAPs.The common response and tolerance-specific DAPs had similar biochemical reaction processes and metabolic pathways in response to anoxic stress,however,they involved different proteins.The tolerance-specific DAPs were involved in amino acid metabolism,starch and sucrose metabolism,tricarboxylic acid cycle pathway,ethylene synthesis pathway,cell wall-associated proteins and activity of active oxygen scavenging enzyme.The in silico protein-protein interactions for the top 60 DAPs indicated that tolerance-specific DAPs had relatively independent protein interaction networks in response to an anoxic environment compared with common response DAPs.The results of physiological indicators showed thatα-amylase and superoxide dismutase activities of R42 were significantly increased under anoxic conditions compared with aerobic conditions.Multiple lines of evidence from western blot,physiological analysis and quantitatDirect seeding of rice has become popular in recent years due to its low cost and convenience,however,hypoxic conditions can limit seedling establishment.In the present study,weedy rice WR04-6 with high germination ability in anaerobic conditions was used as the gene donor and successfully improved the seedling establishment rate of rice cultivar Qishanzhan(QSZ)based on selection of a new rice line R42 from the recombinant inbred line(RIL)population.R42 inherited the had high anaerobic germination(AG)ability,which was used for the isobaric tags for relative and absolute quantitation(iTRAQ)based comparative proteomic studies with QSZ to further explore the molecular mechanism of AG.A total of 719 differentially abundant proteins(DAPs)shared by R42 and QSZ responded to AG and were thus defined as common response DAPs.A total of 300 DAPs that responded to AG were only identified from R42,which were defined as tolerance-specific DAPs.The common response and tolerance-specific DAPs had similar biochemical reaction processes and metabolic pathways in response to anoxic stress,however they involved different proteins.The 300 tolerance-specific DAPs were involved in amino acid metabolism,starch and sucrose metabolism,TCA cycle pathways,ethylene synthesis pathway,cell wall-associated proteins and activity of active oxygen scavenging enzyme.The in silico protein-protein interactions for the top 60 DAPs indicated that tolerance-specific DAPs had relatively independent protein interaction networks in response to an anoxic environment compared with common response DAPs.The results of physiological indicators showed thatα-amylase and SOD activities of R42 were significantly increased under anoxic conditions compared with aerobic conditions.Multiple lines of evidence from western blot,physiological analyses and real-time PCR jointly supported the reliability of proteomics data.In summary,our findings deepened the understanding of the molecular mechanism for the rice response to AG.ive real-time PCR jointly supported the reliability of proteomics data.In summary,our findings deepened the understanding of the molecular mechanism for the rice response to AG.

Effects of AG1 and AG2 QTLs on Nonstructural Carbohydrate and Seed Management Options for Rice Seedling Growth and Establishment under Flooding Stress

Rice(Oryza sativa)plants acquired excess photosynthates in the form of nonstructural carbohydrates(NSCs)in their stems and grain.Despite keen interest in rice NSC,the dynamics of NSC accumulation,translocation and re-accumulation have not yet been well investigated.AG1 and AG2 QTLs associated with flooding tolerance through catalyzing starch into soluble sugar in germinating seeds.Here we conducted three experiments,greenhouse and field to lay the groundwork for large-scale diversity studies on grain NSC and some agronomic traits under direct-seeded rice(DSR)system,using elite lines incorporating AG1,AG2 and AG1-AG2 QTLs into the popular varieties PSB Rc82 and Ciherang-Sub1 along with the donors Kho Hlan On(AG1)and Ma-Zhan Red(AG2).In germinating seedlings,soluble sugars increased,while starch concentration decreased gradually especially in the tolerant checks and AG1-AG2 introgression lines under flooded soil.Soluble sugar accumulation in stem started to increase from the vegetative stage and peaked at the panicle initiation stage then gradually decreased towards the maturity stage.But Sub1-AG lines had higher sugar and starch concentrations at different growth stages than other genotypes in wet season 2016 and dry season 2017.Plant survival rate was positively correlated with the stem NSC at the early vegetative stage(21 days after sowing),and stem NSC was positively associated with plant height at different growth stages.Among the tested seeding rate,the most suitable seeding rate,4 g/m2 with shallow burial depth(0.5 cm),resulted in better seedling establishment,relatively higher seedling vigor index and higher leaf area index under flooding in DSR system.Introgression of AG1-AG2 QTLs had no any negative impact on nonstructural carbohydrate,germination rate,and growth and biomass production.