Mechanism Research Of BPC1/BPC2-GALS1 In Response To Salt Stress In Arabidopsis

Salinity is one of the major problems in modern agriculture which severely limits crop yields.Salinity not only seriously affects the normal agricultural production,but also hinders the development of agricultural economy.Therefore,it is very important to unravel the mechanism of plants in response to salt stress.As the thick wall of the plant cell,cell wall provides the mechanical supports to the plant cell in the normal growth,development and response to various stresses.It is mainly composed of cellulose,hemicellulose,pectin and lignin.Cellulose plays an important role in plant response to salt stress.As another important component of plant cell wall,pectin has a complex composition and structure,and little is known about its role in resisting external stress.Rhamnogalacturonan I(Rhamnogalacturonan I,RGI)is a kind of pectin polysaccharide with a large content in the cell wall.Its molecular weight is very large,with a side chain structure of different lengths.β-1,4-galactan is an important side chain polysaccharide in RGI.Our previous study has found that salt stress obviously induced the expression of GALS1 to accumulate the β-1,4-galactan,thus caused the salt sensitivity.However,the molecular mechanism of regulation of the GALS1 expression is still unclear.Our previous study has found that two transcription factors BPC1 and BPC2 might bind to the GALS1 promoter using the yeast one-hybrid experiment.Therefore,this paper analyzes the relationship between BPC1/BPC2 and GALS1,and studies the mechanism of BPC1/BPC2-GALS1 in response to salt stress in Arabidopsis.The main results are as follows:To confirm the interactions between the GALS1 promoter and the BPC1/BPC2 transcription factors,the Y1 H assay was used.The assay showed that BPC1/BPC2 could bind to the promoter of GALS1 in yeast,but the another BPC class I member,BPC3,could not directly bind to the GALS1 promoter in yeast.Next,EMSA and ChIP assay were performed.The results clearly suggest that BPC1/BPC2 directly binds to GALS1 promoter in vitro and in vivo.To explore whether BPC1/BPC2 are involved in salt tolerance,the BPC1 and BPC2 expression in response to salt stress were analyzed.The results showed that salt stress significantly reduced the BPC1 and BPC2 expression.Next,two T-DNA insertion lines(bpc1-1 and bpc2)were used to perform salt sensitivity assays.The results showed that there are no obvious differences in primary root length between wild type and bpc1-1 or bpc2 mutants under the salt stress,which imply that there exists a functional redundancy of BPC1 or BPC2 gene in this process.Thus,we used the bpc1-1 bpc2 double mutant to perform salt sensitivity test and we found that the primary root length of bpc1-1 bpc2 double was shorter than that in the wild type.To further confirm the function of BPC1/BPC2,we obtained the BPC1 and BPC2 overexpressor lines to perform salt sensitivity assay.Under NaCl treatment,compared to wild type,BPC1 overexpressors and BPC2 overexpressors showed a longer primary root length.These results showed that BPC1/BPC2 positively regulate the salt tolerance.Furthermore,we analyzed the GALS1 expression and found that the GALS1 expression was obviously increased in bpc1-1 bpc2 double mutant,while GALS1 expression was repressed in BPC1 and BPC2 overexpressors with or without salt stress.We also detected the β-1,4-galactan level in these genotypes.The results showed that the β-1,4-galactan level is increased in bpc1-1 bpc2 double mutant,while the β-1,4-galactan level was significantly suppressed in BPC1 and BPC2 overexpressors.Taken together,these results suggest that BPC1/BPC2 positively affects the salt tolerance by repressing the GALS1 expression to modulate the β-1,4-galactan accumulation.To confirm the genetic relationship between BPC1/BPC2 and GALS1,the bpc1-1 bpc2gals1-1 triple mutant was obtained.Next,we investigated the salt sensitivity of wild type,gals1-1,bpc1-1 bpc2 and bpc1-1 bpc2 gals1-1 mutants.The results showed that the primary root growth of bpc1-1 bpc2 gals1-1 triple mutant was comparable to that of gals1-1 single mutant under NaCl treatment.Furthermore,the β-1,4-galactan level in bpc1-1 bpc2 gals1-1triple mutant was also as low as that of the gals1-1 single mutant,even though the bpc1-1bpc2 double mutant showed a high β-1,4-galactan level.These results indicate that GALS1 is genetically epistatic to BPC1/BPC2.Based on these results,we propose a model about BPC1/BPC2-GALS1 in salt tolerance.Under normal conditions,BPC1/BPC2 inhibits the expression of GALS1 by binding to the GALS1 promoter to maintain a low β-1,4-galactan level with no inhibition on plant growth.Under NaCl treatment,the transcription level of BPC1/BPC2 is rapidly reduced,their suppression on GALS1 transcription is relieved,then the synthesis of β-1,4-galactan is increased,finally the plants growth is severely inhibited.