Functional Analysis Of Cell Wall Genes GALS1,XTH30 And PME31 In Response To Salt Stress In Arabidopsis

Salt stress is a major abiotic stress that severely reduces plant growth and limits agricultural productivity.Plants have evolved various strategies to adapt to the saline environments.Plants sense the stress and transmit the signal,and then regulate the downstream genes to activate the adaptive responses to salt stress.Cell wall is the thick wall of the periphery of the plant cell,which determines the cell growth and strengthens the plant body.However,the detailed cell wall metabolism in response to stress conditions remains largely unknown.It has been shown that cellulose is heavily involved in salt stress response.However,the role of the other polysaccharides,like hemicellulose and pectin,is still unclear.In this paper,we started with analyzing cell wall polysaccharide composition of Arabidopsis leaves exposed to salt stress,and further explored the role of cell wall polysaccharides response to salt stress.The results are as follows:To study whether cell wall is involved in response to salt stress,we firstly analyzed the sugar composition of Arabidopsis exposed to salt stress to determine which cell wall components are involved in salt stress response.The results showed that salt stress can significantly increase the galactose content,which is the major monosaccharide component of ?-1,4-galactan in cell wall.NaCl treatment can increase the GALS1 expression.Loss-of-function in GALS1 in Arabidopsis showed more resistant to salt stress than the wild type.Complementation of GALS1 in gals1-1 mutant restored the salt resistant phenotype of gals1-1.Conversely,overexpression of GALS1 in Arabidopsis showed salt hypersensitivity.The presence of exogenous D-gal in growth medium could increase P-1,4-galactan content in cell wall,and aggravated the salt hypersensitivity.These results showed that the accumulation of ?-1,4-galactan could cause salt hypersensitivity.To further explore the mechanism of GALS1 in salt stress response.Two chemical inhibitors isoxaben(a cellulose synthesis inhibitor)and oryzalin(a microtubule inhibitor)were used,and the results showed that overexpression of GALS1 enhanced the sensitivity of dark-induced hypocotyl growth in response to both isoxaben and oryzalin.The salt stress induced cellulose reduction level is heavily affected in GALS1 overexpressors.Together,these data suggested that salt stress could increase the GALS1 expression to synthesize more ?-1,4-galactan,then affected cellulose synthesis and cortical microtubules depolymerization,thus caused the salt hypersensitivity.Meanwhile,we fused various fragments of GALS1 promoter with LUC report gene,and obtained several transgenic Arabidopsis lines.We found that the fragment of Pro/GALS1(Pro/1-600 of GALS1)retained the GALS1 promoter activity during the salt stress treatment by detecting the LUC activity and qRT-PCR assay.We used the fragments of Pro/GALS1(Pro/93-600 of GALS])to screen the cDNA library.Total 14 positive colonies were confirmed and sequenced.CBF2 can interact with the GALS1 promoter fragment(Pro/93-206 of GALS1)in yeast.Overexpression of CBF2 in Arabidopsis significantly decreased the GALS1 expression,therefore enhanced the salt tolerance.However,there is no salt phenotype between the wild type and cbf2 mutant,suggesting the functional redundancy of CBF2.Further investigations found that CBF1 and CBF3 could also directly bind to the promoter of GALS1.Taken together,we provide a mechanism for involvement of ?-1,4-galactan and cellulose in salt stress.Salt stress increases the GALS1 expression to synthesize more ?-1,4-galactan,then affects the cellulose synthesis and cortical microtubules depolymerization,thus causes the salt hypersensitivity.In this process,CBF1,CBF2 and CBF3 inhibit the GALS1 expression by directly binding to the GALS1 promoter to alleviate the salt symptoms caused by ?-1,4-galactan.XTH30,encoding xyloglucan endohydrolase(XEH)and xyloglucan endotransglucosylase(XET)activities,are involved in cell wall extension by cutting and rejoining xyloglucan chains.To study whether xyloglucan is involved in response to salt stress,we used T-DNA insertional mutants of XTH30 to explore the function of xyloglucan during salt stress.Here,we showed that loss-of-function mutants in XTH30 were more resistant to salt stress than the wild type.Conversely,overexpression of XTH30 in Arabidopsis showed salt hypersensitivity.We found XTH30 did not affect the xyloglucan content in response to salt stress.The accumulation of XyG oligosaccharide,XLFG,can be induced by salt stress in dark-induced hypocotyl.However,this induction was partly inhibited in xth30 mutants.xth30 mutants were less hypersensitive to isoxaben and oryzalin.The cellulose content reduced by salt stress is slightly affected in xth30 mutants.Taken together,these results indicated that XTH30 could alter the XLFG abundance to affect the xyloglucan structure,thus modulate the cellulose synthesis,and finally negatively regulate salt tolerance.To determine whether pectin methylesterases(PMEs)are involved in response to salt stress,T-DNA insertional mutants of PME31 were used.Salt stress significantly increased PME31 expression.The expression level of PME31 was high in dry seeds.Knock-down mutants in PME31 conferred hypersensitive phenotypes to salt stress in seed germination and post-germination growth.Real-time PCR analysis revealed that the transcript levels of several stress genes(DREB2A,RD29A and RD29B)are lower in pme31-2 mutant than that in the wild type in response to salt stress.These results suggested that PME31 could positively modulate salt stress tolerance.