Molecular Mechanisms Of Negative Feedback Regulation Of ABA Signaling

Abscisic acid(ABA),a classical plant hormone,plays an important role in the whole life cycle of plants,especially in plants' responses to stress conditions.Over the past two decades,the molecular mechanisms of ABA signal transduction have been studied in depth,and ABA receptors,i.e.PYR/PYL/RCARs proteins,and the co-receptors,i.e.group A PP2Cs,have been identified.Group A PP2Cs negatively regulate ABA signaling by inhibiting the activity of SnRK2 protein kinases.It has been repeatedly observed in previous studies that the expression of some group A PP2C genes is induced by ABA,but the molecular mechanisms and biological significance remain largely unknown.In this study,by using specific antibodies against ABI1 and ABI2,I found that ABA can significantly induce ABI1 and ABI2 protein accumulation.Further analysis indicated that this induction was mainly due to increased gene expression.Moreover,the expression of all nine group A PP2C genes in Arabidopsis was significantly induced by ABA,and the homologous genes in maize also displayed ABA-induced expression,indicating that ABA-induced expression of group A PP2C genes is conserved in monocot maize and dicot Arabidopsis.In order to study the molecular mechanisms underlying ABA control of group A PP2C gene expression,the transgenic plants that contain a series of 5' deletions of the ABI1 and ABI2 promoters fused with a GUS reporter were generated.Then,these transgenic plants were subjected to histochemical staining,and the promoter fragments of ABI1 and ABI2 responding to ABA were identified.Yeast one-hybrid assays showed that the pivotal bZIP family transcription factors of the ABA signaling pathway,i.e.ABF1-ABF4,could directly bind to these cis-elements.It was further confirmed using ChIP assays that ABFs could bind to the promoters of group A PP2C genes in vivo,and this binding was induced by ABA treatments.Accordingly,ABA-induced expression of group A PP2C genes and the ABI1 and ABI2 protein levels were dramatically reduced in the abfs mutants compared with those in the wild-type seedlings,indicating that ABFs indeed mediate ABA induction of group A PP2C gene expression and the ABI1 and ABI2 protein accumulation.It was previously shown that ABFs could be phosphorylated and activated by SnRK2s protein kinases,which serves as a major molecular mechanism of ABA regulation towards ABF proteins.In this study,we generated ABFs antibodies and found that ABA can significantly induce ABFs protein levels.Further studies showed that this induction was also due to ABA-induced expression of ABF genes,and ABFs themselves are involved in this process as well.These data indicate that in addition to the well-characterized ABA-induced phosphorylation by SnRK2 protein kinases,ABA also significantly induces the gene expression and protein levels of ABFs.By these mechanisms,ABA signal could be rapidly amplified by ABFs.Light is one of the most important environmental signals regulating plant growth and development.However,little is known regarding how light regulates ABA signaling.I compared ABA-induced ABFs protein accumulation in light and darkness,and found that light could greatly enhance ABA induction of ABF proteins.Accordingly,ABA induction of group A PP2C gene expression and protein levels were also stimulated by light.Further,yeast two-hybrid assays showed that the photoreceptors,i.e.phyA and phyB,can directly interact with ABFs.These data suggest that light may regulate the ABA signaling pathway by regulating ABF proteins.It was observed long ago that under water deficit conditions,the root:shoot biomass ratio increases,allowing the roots to grow at the expense of the leaves.Further research confirmed that,ABA plays an important role in the regulation of the root:shoot ratio during water stress.In this study,histochemical staining of ABI1p:GUS and ABI2p:GUS lines indicated that ABA significantly induced the expression of and genes in roots and stomata.These observations have prompted us to speculate that ABA-induced expression of group A PP2C genes may serve as a desensitization mechanism acting in the roots,thus allowing roots to continue to grow under water stress conditions.Collectively,my study demonstrates that ABFs mediate rapid ABA induction of group A PP2C genes,thus playing a role in the negative feedback regulation of ABA signaling.