Molecular Mechanism Of The Arabidopsis ANAC069 Negatively Regulate Abiotic Stress Response

NAC proteins are plant-specific transcription factors involved in abiotic stress.However,their roles in stress response are still not fully understood.Here,we studied regulatory networks in response to abiotic stress that Arabidopsis ANAC069 protein participated,and the mechanisms of abiotic stress sensitivity mediated by ANAC069.The main results are shown as following:(1)GUS analysis suggested that ANAC069 expression is tissue and developmental stage-specific and it can be induced by osmotic stress and ABA.(2)Germination rates root lengths,fresh weights,and survival rates assays showed that compared with wild type(WT)plants,overexpression of ANAC069 in Arabidopsis displayed increased sensitivity to ABA,salt,and osmotic stress.Conversely,ANAC069 knockout plants showed enhanced tolerance to salt and osmotic stress,but no overt ABA sensitivity difference.(3)ROS levels analysis showed that the steady state levels of both H2O2 and O2-were highest in overexpression lines,and were lowest in mutants under salt,mannitol treatments;Under ABA treatments conditions,the ROS levels of overexpression lines were also highest,while there was no overt difference between mutants and WT plants.(4)Physiological index assays showed that the expression levels of ANAC069 negatively correlated with proline amounts,and activity of SOD,POD and GST.Real time RT-PCR further suggested that the expression levels of ANAC069 negatively correlated with the expression levels of most of SOD,POD,GST,and P5CS genes.(5)The promoter sequence of ANAC069 contains a lot of motifs related to stress response,hormone response such as ABRE,EBOX,WBOX and DOF et.by cis-elements motif prediction tool PLANTCARE and PLACE.Y1H assays and transient cotransformation assays showed a DOF protein(ATDOF5.8)binds to DOF motifs present in ANAC069 promoter to activate its expression.The chromatin immunoprecipitation assays further suggested ATDOF5.8 protein binds to ANAC069 promoter in Arabidopsis.Moreover,real-time RT-PCR showed both of ATDOF5.8 and ANAC069 share similar expression patterns,suggesting that these two genes might be involved in the same regulatory network in response to salt,osmotic and ABA.(6)Y1H assays and transient cotransformation assays also showed ANAC069 can specifically bind to NACRS that contains core sequence of "CACG".The expression profiles of overexpression ANAC069 plants and ANAC069 knockout plants under salt stress conditions were compared using Affymetrix Arabidopsis gene chips.A total of 567 differentially expression genes with p-value<0.05 and fold Change>2 were obtained,among which 341 were upregulated and 226 genes were downregulated in overexpression ANAC069 plants relative to ANAC069 knockout plants.MEME analysis showed that most of these differentially expressed genes contain a conserved[GCA][CA]C[AG]CG[TG]sequence in there promoters.Y1H assays confirmed ANAC069 protein can binding to this conserved sequence.Transient cotransformation assays further showed salt stress enhanced the ability of ANAC069 binding to the[GCA][C A]C[AG]CG[T G]sequence.This result indicates that[GCA][CA]C[AG]CG[TG]is the primary sequence motif recognized by ANAC069 to regulate gene expression during salt-promoted stress response.Our data suggested a working model for ANAC069-mediated stress regulatory network:Abiotic stress signals trigger ATDOF5.8 which in turn activates ANAC069.The activated ANAC069 binds to[GCA][CA]C[AG]CG[TG]motif to regulate its target genes,leading to physiological changes involved in stress tolerance,including decreased ROS scavenging capability,POD,SOD and GST activity,altered proline biosynthesis,and increased water loss rate,which enhance plants sensitivity to abiotic stress.