Salt Stress Proteomic Analysis Of Salt-tolerant R. Chinensis

Salt stress is the main abiotic stress to limit plant growth and productivity in the world.Sequential GISH-FISH,DIA proteomics and salt-tolerant identification methods were used to investigate the chromosome composition of salt-tolerant Tritipyrum Y1805,to explore its key proteins and metabolic pathways in response to salt stress and recovery conditions,and to evaluate the salt tolerance of the F₂ progeny from Y1805×wheat in this study.This work would lay a foundation for genetics and breeding of salt-tolerant wheat.The main results were as follows.(1)The Y1805 chromosomes were analyzed by sequential GISH-FISH.The block genomic DNA of Chinese Spring wheat digested with boiled water,and the genomic DNA probe(green)of Thinopyrum elongatum were used for GISH analysis in Y1805 chromosomes.The results showed that Y1805 was an octoploid Tritipyrum containing 7 pairs of E-genome chromosomes from Thinopyrum elongatum.FISH analysis of Y1805 chromosome was carried out by the repeat probes p As1-1,p As1-3,p As1-4,p As1-6,AFA-3 and AFA-4(red)and p Sc119.2-1 and(GAA)₁₀(green).The results showed that red signals were mainly distributed in the D-genome chromosomes,green signals were mainly concentrated in the B-genome chromosomes,and obvious red signals appeared in the E-genome chromosomes,but with few green signals.There were less red signals on the A-and B-genome chromosomes in wheat Sa20 and W2,while green signals were mainly distributed on the B-genome chromosomes.(2)The plant length and dry weight at the T2(1 h after recovery)and T3(24 h of recovery)stages were significantly greater than those at the T1 stage(5 h under salt stress)in Y1805.There was no significant difference between their treatment and control at the three stages.So Y1805 could maintain normal growth under salt stress.At the T3 stage,the plant length and dry weight of wheat Chinese Spring(CS)were significantly less than those of the controls.The plant length of Y1805 and CS decreased by 1.99%and 6.37%at the T3 stage,respectively,and their dry weight decreased by 11.83%and 15.69%,respectively.Therefore,the effects of salt stress on the length and dry weight of Y1805 was less than those of CS,and its ability to restore growth after recovery was stronger,indicating the strong salt tolerance.(3)Under salt stress,44 and 69 differentially expressed proteins(DEPs)were identified in Y1805 and CS,respectively.Among these DEPs,Y1805 had 23 specific DEPs(11 up-regulated and 12 down-regulated),which played key roles in salt tolerance.Transcriptome-proteome association analysis showed that 13 DEPs had the same expression pattern,while one DEP had the opposite expression pattern at the transcriptome and proteome levels under salt stress.15 genes closely related to salt stress were selected from Y1805 for q RT-PCR analysis,which confirmed the accuracy of proteomic data.Compared with CS,“antioxidant activity”and“molecular function regulator”were Y1805 specific pathways under salt stress.In addition,some Y1805 specific DEPs related to the response to salt stress was up-regulated,such as peroxidase 5,ATP dependent DNA helicase,trehalose phosphatase 3,ABA induced protein PHV A1,1-aminocyclopropane-1-carboxylate oxidase 4.Y1805 has strong salt tolerance,which might be attributed to active oxygen scavenging,osmoregulation,plant hormones(abscisic acid and ethylene)and transient growth arrest.(4)During the recovery process,102 DEPs(66 up-regulated and 36 down-regulated)were identified in Y1805.In CS,525 DEPs(307 up-regulated and 218down regulated)were detected.There were 73 specific DEPs(48 up-regulated and 25down regulated)in Y1805,which might contribute to rapid recovery of Y1805 after removing salt stress.Transcriptome-proteome associated analysis displayed that 25DEPs had the same expression pattern,while 2 DEPs had the opposite expression pattern at the transcriptome and proteome levels.Compared with CS,“virus”and“virus component”were the specific pathways in Y1805.In addition,UDP glucose 6-dehydrogenase 2,xylanase inhibitor,glycerin-3-phosphate dehydrogenase[NAD(+)]2,DEAD box ATP dependent RNA helicase 14,CLP protease regulatory subunit CLPX1,cytoplasmic glutamine synthetase,peptide proline CIS trans isomer CYP59,DEx H box ATP dependent RNA helicase DEx H10 could contribute to rapid growth recovery of Y1805 by strengthening cell wall,enhancing respiration,ATP and protein hydrolysis,ammonium detoxification,transcriptional regulation,promoting nucleic acid synthesis,and timely handling incomplete splicing and pseudotranscription.(5)The salt tolerance of three parental materials Y1805,SA20 and W2 were identified.It was found that Y1805 showed the strongest salt tolerance,and W2 was the weakest.The salt damage ratio and yellowing ratio of three F₂ populations 1903(Y1805×SA20),1905(W2×Y1805)and 1906(Y1805×W2)were comprehensively analyzed.1903 population had the strongest comprehensive salt tolerance.In this population,1903-68,1903-106,1903-185,1903-84 and 1903-134 had strong salt tolerance,while 1903-209,1903-71,1903-204,1903-103 and 1903-101 were poor in salt tolerance.In 1905 population,1905-29,1905-79,1905-23,1905-28 and 1905-78had strong salt tolerance,while 1905-69,1905-57,1905-83,1905-48 and 1905-75were poor in salt tolerance.In 1906 population,1906-128,1906-121,1906-181,1906-120 and 1906-21 had strong salt tolerance,while 1906-14,1906-35,1906-67,1906-18 and 1906-29 were poor in salt tolerance.Y1805 is an octoploid Thinopyrum(AABBDDEE)with 7 pairs of E chromosomes.The key proteins and metabolic pathways of Y1805 in response to salt stress and recovery were found,and salt tolerance of the F₂ progeny from Y1805×wheat was preliminarily understood.These results would provide the reference for understanding the molecular mechanism of salt tolerance and cloning key genes in Tritipyrum.