Preliminary Functional Analysis Of AtPME31 In ABA-regulated Seed Germination In Arabidopsis

Abscisic acid(ABA)is an important plant hormone that accumulates in developing embryo and regulates seed germination.ABA functions through complex signaling networks,some components of which have been identified,The core components include ABA receptors PYR/PYL/RCAR,protein phosphatases PP2Cs,protein kinases SnRK2s,and various transcription factors,such as AP2,VP1/ABI3 and bZIP transcription factor.Recent studies have shown that pectin methylesterases is also involved in ABA inhibition of seed germination,PMEs is an important enzyme in plant cell wall metabolism.During the synthesis of pectin,the galacturonic acid carboxyl group is methylated by methyltransferase,and is secreted into the cell wall by methylation of pectin methylesterase,which plays an important role in the seeds germination.In this study,we investigated the roles of AtPME31 in the ABA-regulated germination of Arabidopsis seeds using their deletion and overexpression mutants.The results are as follows:To detect the tissue expression specificity of AtPME31,a transgenic plant driven by the promoter of AtPME31 with GUS as the reporter gene were constructed;The tissue specific expression analysis indicated that the expression level of AtPME31 in seeds was significantly higher than that in other parts.In order to further study whether AtPME31 is involved in ABA regulation of seed germination,the expression pattern of AtPME31 in seed germination was firstly determined.Secondly,the expression of AtPME31 after ABA treatment was detected during seed germination.The results showed that the expression of AtPME31 was significantly decreased during seed germination,and the relative expression of AtPME31 was significantly up-regulated after ABA treatment.This indicated that ABA could induce the expression of AtPME31,suggesting that AtPME31 may play an important role in the process of ABA-regulated seed germination.The germination rate and cotyledon-greening percentage were tested.In the absence of ABA,the AtPME31 mutants showed no obvious difference with wild-type plants;when germinated and grown on MS medium containing 0.5 ?M ABA,the AtPME31 mutants were germinated more slowly and displayed significantly reduced cotyledon-greening percentages compared with wild-type plants.We observed the growth of seedlings on MS medium and ABA treatment and found that the seedlings growth was no significant difference between wild-type and AtPME31 mutant.We constructed AtPME31 overexpressing lines and detected the expression level of AtPME31 by qRT-PCR,and selected two lines(OE-PME31#3 and OE-PME31#4)with high expression level.In the presence of ABA,the AtPME31-overexpressing lines showed ABA insensitive phenotypes compared with wild-type plants during seed germination and seedling growth.It was found that compared with the wild type,the activity of pme31-1 and pme31-2 mutant in mature seeds was significantly decreased.To preliminarily analyze the mechanism of AtPME31 in ABA regulation of seed germination,we analyzed the expression pattern of AtPME31 in abi4-1 and abi5-1 mutants of ABA-dependent germination related genes.It was found that the expression of AtPME31 in abi4-1 and OE-ABI4 mutants was not significantly different from wild type,while the expression of AtPME31 in abi5-1 mutants was significantly down-regulated compared with wild type.To determine the genetic relationship between AtPME31 and ABI5,we hybridized abi5-1 with pme31-1,it was found that the sensitivity of pme31-1 abi5-1 to ABA was between pme31-1 and abi5-1 after ABA treatment.These results indicated that AtPME31 was involved in the ABA-regulated seed germination process and may be regulated by ABI5.In addition,AtPME31 is epistatic to ABI5.