The Function Analysis Of AtPSL4 In Regulation Of ABA Signalling In Arabidopsis Thaliana

The abscisic acid?ABA?is involved in the regulation of many growth and development process of plant as one of the most important plant hormone,such as the seed mature,seed germination,seedling growth,stoma movement and flowering.In addition,ABA also plays a very important role in response to many environmental stress named as "stress hormone".Now the ABA signal transduction pathway accepted by most people consist of three core components:ABA receptors PYR/PYL/RCAR proteins,negative regulation factor protein phosphatase 2C?PP2C?,and positive regulation factor SNF1 related protein phosphatase 2?SnRK2?.They form a dual negative regulation system to control the ABA signal transduction.We focus on studying the function of AtPSL4 using Arabidopsis thaliana materials by treating AtPSL4 overexpression lines and mutant with NaCl,mannitol,ABA.The main results are as follows:?1?We identified the underlying candidate genes that response to salt stress by searching the GENEVESTIGATOR and Arabidopsis eFP Browser database combined with the result of iTRAQ data of our lab.Finally we choose the AtPSL4 for further research.AtPSL4 gene encodes a homologous protein of yeast glucosidase ?? subunit.There are 647 amino acids in the peptide which corresponding CDS consists of 1944nt.PSL4 protein is conserved in evolution and lacks paralogous genes by comparison with other species.A signal peptide and PRKCSH-like domain exist in the N end,and a endoplasmic reticulum retention signal and PRKCSH₁ domain exist in the C end of PSL4 protein.?2?The germination rates of AtPSL4 overexpression lines are faster than WT when treated with NaCl and mannitol.And the greening rates are also higher in AtPSL4 overexpression lines.The results indicate that AtPSL4 positively regulates germination and the growth of seedlings.?3?The expression of AtPSL4 is tissue-specific to a certain degree by high throughput data and qRT-PCR,for example,the expression of AtPSL4 is maximum in root,however it is hard to detect the expression in flower.In addition,ABA induces the expression of AtPSL4,which is more distinct than NaCl or mannitol.?4?PSL4 locates in endoplasmic reticulum by the construction of PSL4-EGFP fusion protein and performing instantaneous infection in the leaves of tobacco.The sequence deletion experiment indicates that the sigal peptide in N end of PSL4 plays an essential role in the location of itself.?5?The germination rates of AtPSL4 overexpression lines is faster than WT when treated with paclobutrazole which functions as the inhibitor of GA synthesis.But there are not significant differences in the expression of genes of GA synthesis or GA signal pathway,which indicates that AtPSL4 is not involved in the GA signal pathway.?6?The germination rates of AtPSL4 overexpression lines is faster than WT when treated with ABA and the germination difference becomes more significant with the ABA concentration enhancement.The expression of marker genes in ABA signal pathway is distinct lower in AtPSL4 overexpression lines,such as ABI3,ABI4,ABI5.But there are not significant differences in the expression of PYL/PYRs,PP2Cs,SnRK2s.The results indicate that AtPSL4 is indeed involved in ABA signal pathway,and may function in the downstream of SnRKs and the upstream of ABI5.?7?PSL4 protein does not interact with CaM2.1,CaM4.1,CaM6 or CaM7 by yeast two hybrid.And the expression of CaMs is nearly same in AtPSL4 overexpression lines and WT by qRT-PCR.The results indicate that AtPSL4 may not be involved in the ABA signal pathway mediated by CaMs.?8?PSL4 protein interact with SnRK2.2,SnRK2.3,SnRK2.6 by performing yeast two hybrid experiment and BiFC experiment.But the interaction between PSL4 protein and 14 ABA receptors was not detected in yeast two hybrid experiment.The results indicate that AtPSL4 may function in the downstream of SnRKs.