| Transcription factors mediate plant developmental and growth processes.They perform vital roles in transmitting stimulatory or inhibitory signals depending on their associations with cis-regulatory elements of the promoters of target genes.Abscisic acid(ABA)is an important phytohormone regulating plant growth,development and stress response through the ABA core signaling pathway.It has an essential role in multiple physiological processes of plants through transcriptional reprogramming together with numerous transcription factors and related regulatory networks.Drought stress greatly affects plant growth and harm crop productivity heavily as one of the most common form of environmental stresses.It regulates the expression of associated transcription factors in ABA-dependent or ABA-independent manners which mediates the response to drought stress.Accordingly,as sessile organisms,transcriptional regulation of plants play an important role in balancing the crosstalk between the physiological processes and stress responses and integrating internal and external signals.R2R3-MYB subgroup 4 proteins,AtMYB32 and its homologs AtMYB4 and AtMYB7,act as transcriptional regulators of the plant phenylpropanoid pathway and physiological processes of plants.AtMYB4 and AtMYB7 proteins also play crucial roles in response to abiotic stress and related signaling pathway.At present,the potential role of AtMYB32 in plant responses to abiotic stress and ABA signaling pathway is not completely clear.In this paper,a series of biochemical and genetic phenotypic analysis experiments have been carried out to solve this scientific problem,and the following research results have been obtained:(1)AtMYB32 positively regulates ABA-induced inhibition of seed germination and cotyledon greening.It was found that the transcript levels of AtMYB32 and the levels of transgenic Myc-AtMYB32 protein are induced by ABA during seed germination and seedling development.Under different concentrations of ABA treatment,the germination rate and cotyledon greening rate of the AtMYB32-overexpressing plants were significantly lower and the mutant atmyb32-1 were higher compared with that of the wild-types.These findings suggest that AtMYB32 functions as a positive regulator of ABA-mediated inhibition of seed germination and early seedling development.(2)AtMYB32 positively regulates the inhibition of seed germination and cotyledon greening in an ABA-dependent manner under osmotic stress.The percentages of seed germination and cotyledon greening were markedly lower for the AtMYB32 ox lines compared with the Col-4 wild-type plants,while the atmyb32-1 mutant was resistant to the osmotic stress.AtMYB32 transcription was induced in response to Na Cl during early seedling development.It was found that the salt induced transcription of AtMYB32 was significantly attenuated in the ABA-deficient mutant and the ABA signaling mutants,indicating that ABA is at least in part required for AtMYB32 expression in plants subjected to salt stress during early seedling development.(3)AtMYB32 functions as a positive regulator of the ABA-mediated inhibition of seed germination and early seedling development by directly promoting the expression of positive regulators of ABA singnalling ABI3,ABI4 and ABI5.Compared to those in the wild type,the expression levels of ABI3,ABI4 and ABI5 in AtMYB32 ox were significantly higher during seed imbibition both under normal conditions and following 24 h of ABA treatment.In contrast,the expression levels of ABI3,ABI4 and ABI5 were lower in the atmyb32-1 mutant than in the wild type.The chromatin immune-precipitation(ChIP)and electrophoretic mobility shift assays(EMSA)revealed that AtMYB32 could bind to the promoters of ABI3,ABI4 and ABI5 both in vitro and in vivo.Dual-luciferase reporter assays(LUC)further confirmed that AtMYB32 could activate ABI4 expression.By crossing the plants in the genetic backgrounds of AtMYB32ox8 and abi4-1,we found that ABI4 is epistatic to AtMYB32 during ABA-mediated seed germination and postgermination stages.Based on these findings,we concluded that AtMYB32 functions as a positive regulator of the ABA-mediated inhibition of seed germination and early seedling development by directly promoting ABI3,ABI4 and ABI5 expression.(4)AtMYB32 serves as a negative regulator of plant drought tolerance.AtMYB32 expression is repressed by drought stress at both the transcriptional and posttranscriptional levels,and atmyb32-1 mutant exhibited enhanced drought tolerance.Therefore,these results suggest that AtMYB32 plays a negative role in the plant drought stress response.(5)AtMYB32 plays a negative role in ABA-independent regulation of drought tolerance.Consistent with the enhanced drought tolerance phenotype,expression of the drought-induced marker gene CBF4 was upregulated in the atmyb32-1 mutant compared to the wild-type following drought treatment.However,the transcript levels of ABA catabolism-related genes and ABA signaling genes did not differ between the atmyb32-1 mutant and wild type under drought stress.Consistent with these results,the ABA content did not change in the atmyb32-1 mutant after drought treatment.Moreover,our results from EMSA and ChIP assays confirmed that AtMYB32 can bind to the promoter of CBF4 both in vitro and in vivo.We therefore conclude that AtMYB32 acts as a negative regulator of plant drought tolerance,likely by directly repressing CBF4 expression.In conclusion,this paper reveals novel functions for the R2R3-MYB transcription factor AtMYB32,i.e.,regulation of the ABA response and drought tolerance.AtMYB32 plays a positive role in the ABA-induced inhibition during seed germination and early seedling growth by directly promoting ABI3,ABI4,and ABI5 expression,but plays a negative role in ABA-independent regulation of drought tolerance by directly inhibiting CBF4 expression.Thus,manipulating the expression of AtMYB32 may be an effective means of controlling seed germination and plant drought tolerance for agricultural purposes. |
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