The ARF2-PIF5 Functional Module Executes Transcriptional Reprogramming In ABS3-Mediated Senescence Pathway In Arabidopsis

Senescence,or aging,is the final chapter of life,and an intricate and active process that is regulated at multiple levels.Proper regulation of plant senescence is of great importance for plant reproduction success,adaptation to the changing environment,and crop production.It is well-established that one of the hallmarks of plant senescence is the global transcriptional reprogramming coordinated by a plethora of transcription factors(TFs).However,molecular mechanisms underlying the interactions between different TFs in modulating senescence remain obscure.Previously,we found that the Arabidopsis ABS3 subfamily MULTIDRUG AND TOXIC COMPOUND EXTRUSION(MATE)transporter genes regulate senescence and senescence-associated transcriptional changes.This study mainly focuses on the transcriptional regulation in ABS3-mediated senescence pathway.The main findings are as follows:(1)Genetic screens for mutants suppressing the accelerated senescence phenotype of the Arabidopsis abs3-1D mutant,an activation-tagging gain-of-function mutant of ABS3,identified AUXIN RESPONSE FACTOR 2(ARF2)and PHYTOCHROME-INTERACTING FACTOR 5(PIF5)as key TFs responsible for the transcriptional regulation in the ABS3-mediated senescence pathway.In addition,the overexpression of ARF2 or PIF5 can promote senescence under C-deprivation and natural growth conditions.(2)The activation of ABS3 expression in abs3-1D leads to a larger-scale transcription reprogramming upon carbon deprivation.The impact of abs3-1D on gene expression is dependent on transcription factors ARF2 and PIF5.A significant overlap was observed between ARF2-regulated and PIF5-regulated genes in the abs3-1D background.These results demonstrate that ARF2 and PIF5 execute transcriptional reprogramming in the ABS3-mediated senescence pathway(3)PIF5 and PIF4 share redundant roles in the ABS3-mediated senescence pathway and interact directly with ARF2.Although the progression of senescence in pif4-2 abs3-1D was overall similar to that of abs3-1D,pif4-2 pif5-10 abs3-1D triple mutants showed further delayed senescence compared with pif5-10 abs3-1D under C-deprivation.These observations suggest that PIF5 and PIF4 share redundant functions in the ABS3-mediated senescence pathway.Analyses using yeast two-hybrid,Bi FC,GST pull-down,and Co-IP experiments,indicated that both PIF5 and PIF4 physically interact with ARF2.(4)ARF2 and PIF5 regulate senescence interdependently and independently.When PIF5 is mutated,the ability of ARF2 overexpression to promote senescence was attenuated in pif5-10 or pif4-2 pif5-10 background.Similarly,the loss of arf2-20 delayed the senescence of PIF5-GFP OEs under C-deprivation and natural senescence conditions.These data suggest an interdependency of ARF2 and PIF5 in promoting senescence.On the other hand,arf2-20 pif5-10 double mutants displayed further delayed senescence and stronger suppression of the accelerated senescence phenotype of abs3-1D,compared with the respective single mutants,suggesting that ARF2 and PIF5 also regulate senescence through pathways that are independent of one another.(5)ORE1 and SGR1 are common target genes of ARF2 and PIF5 in the ABS3-mediated senescence pathway.ARF2 and PIF5 specifically regulate the expression of target genes in an interdependent manner,but their mutual regulation is different.The binding of PIF5 to the promoters of senescence target genes is dependent on ARF2.In the case of ARF2,although ARF2 can bind to the promoters of ORE1 and SGR1 in a PIF5/4-independent manner,it is dependent on PIF5/4 to activate the expression of ORE1 and SGR1.The loss-of-function mutant of ORE1 or SGR1 can suppress the premature senescence of abs3-1D under Cdeprivation.Furthermore,the overexpression of ORE1 and SGR1 accelerated senescence of mateq,which has a strong stay-green phenotype.These data demonstrate that the induction of ORE1 and SGR1 is a direct output of the ARF2-PIF5 functional module involved in the ABS3-mediated senescence pathway.(6)The promotion of senescence by ARF2 and PIF5 requires the ABS3 subfamily MATEs.The senescence-promoting effect of pARF2:ARF2-GFP or p PIF5:PIF5-GFP overexpression is dampened in the mateq background.(7)ARF2 directly binds to the promoter of ABS3 and represses the transcription of ABS3,and the transcriptional repression of ABS3 by ARF2 is independent of PIF5/4.These findings suggest the existence of a negative feedback regulatory loop of ARF2 to ABS3 in which an increased expression of ABS3 promotes senescence through ARF2-PIF5-mediated transcriptional regulation,whereas the transcription of ABS3 itself is directly repressed by ARF2.In conclusion,this study reveals the molecular mechanism of ARF2-PIF5/4 functional modules in regulating plant senescence,further elucidates the ABS3-mediated senescence pathway,and shines more light on the genetic regulatory network of plant senescence.