Functional Characterization Of GmPYLs As ABA Receptors In Soybean And DTF1Gene Related To DNA Methylation In Arabidopsis

This dissertation includes two studies:(1) a study on a family of abscisic acid (ABA)receptors in soybean (Glycine max);(2) an investigation of DTF1that is associated withRdDM pathway in Arabidopsis thaliana. The main results are as following:(1) As plant hormone, ABA plays important roles in regulating plant growth,development, and proper responses to environmental stresses. Recently some members ofArabidopsis Pyrabactin Resistance (PYR)/PYR1-Like (PYL)/Regulatory Component ofABA Receptor (RCAR)(PYR/PYL/RCAR) family, which are able to bind ABA, havebeen identified as ABA receptors. Currently most of studies focused on a single or a fewABA receptor(s) in model species, such as Arabidopsis and rice (Oryza Sativa). However,systemic investigation of ABA receptors in soybean is still lacking. Here we reportedcloning and characterization of21ABA receptors (GmPYR/PYL/RCARs, hereafterreferred as GmPYLs) in soybean. Our results showed that all GmPYLs are localizedwithin cytoplasm and nucleus in Nicotiana benthamiana. Yeast two-hybrid assaysrevealed that GmPYLs interact with two soybean protein phosphotase2C (GmPP2Cs)and AtABI1(an Arabidopsis PP2C) in an ABA-dependent or-independent manner.Furthermore, ABA dependency of GmPYLs-GmPP2Cs or-AtABI1interactions is notalways correlated with isoleucine and lysine in CL2region of GmPYLs, indicatingselection between GmRYLs and GmPP2Cs. Additionally, overexpressing GmPYL1partially impairs ABA insensitivity of Arabidopsis triple mutant pyr1/pyl1/pyl4seedlings.The present results show that soybean GmPYLs can physically interact with twoGmPP2Cs and AtABI1in yeast, and GmPYL1is a functional ABA receptor inArabidopsis, suggesting conservation of ABA receptors between soybean andArabidopsis.(2) RNA-directed DNA methylation (RdDM) is a very important de novo DNAmethylation pathway in many plant species. RdDM pathway mediates the transcriptionalsilencing of many endogenous genomic loci, most of which are transposon regions. Weused a forward genetics screen approach to identify DTF1(DNA-binding transcriptionfactor1) as a new component for RdDM pathway in Arabidopsis. The dtf1mutations canrelease some transcriptional silencing of RdDM target locis and reduce the accumulation of24-nt small interfering RNAs (siRNAs) from some of the targets. In the dtf1mutantplants, the release of transcriptional silenced gene at solo-LTR is accompanied bydecreased accumulation of24-nt siRNAs, but not by reduced DNA methylation. Theseresults suggest DTF1is an atypical component of RdDM pathway and has both DNAmethylation-dependent and-independent roles in transcriptional gene silencing. Thestudy suggests that besides DNA methylation, siRNAs may cause some otheruncharacterized epigenetic modifications that lead to transcriptional gene silencing.