The Molecular Mechanism Of Auxin Response Factor TaARF15-A1 In Regulating Wheat Senescence

Leaf senescence,as the final stage of leaf development,involves the accumulation of photosynthetic products and nutrient remobilization.Therefore,identifying senescence-associated genes and studying their roles in regulating leaf senescence will help improve crop yield and quality.Auxin plays an important role in mediating leaf senescence.However,how auxin response factors(ARFs),which are crucial components of the auxin signaling pathway,participate in the regulation of senescence in wheat is unclear.In this study,based on the previous laboratory research,TaARF15-A1 with auxin response characteristic was selected,and the genetic effects of TaARF15-A1 were analyzed by transgenic and gene editing technology;Meanwhile,to dissect the underlying molecular mechanism,transcriptome sequencing,protein-protein and protein-DNA interaction assays,and other technical means were performed;Finally,the allelic variations of TaARF15-A1 in natural populations were explored and the molecular marker of TaARF15-A1 was developed.The main results are as follows:1.Isolation and characterization of TaARF15-A1TaARF15-A1 is 6876 bp in length,consisting of 14 exons and 13 introns,and is a nucleus-localized transcriptional activator.TaARF15-A1 is a constitutively expressed gene and is highly expressed in stems at various developmental stages.2.TaARF15-A1 negatively regulates wheat senescenceThe genetic effects of TaARF15-A1 overexpression and RNA interference lines and CRISPR/Cas9-based arf15 mutants confirmed that TaARF15-A1 overexpression plants showed delayed wheat senescence,whereas TaARF15 knockdown lines and knockout mutants showed accelerated senescence.Meanwhile,the statistical analysis of agronomic traits of arf15 lines showed that the thousand kernel weight(TKW)and plant height(PH)of arf15 lines were significantly lower.3.Mechanism of TaARF15-A1 mediating wheat senescenceTranscriptome analysis of flag leaves 15 days post-anthesis(DPA)in wild type(WT)and three independent overexpression lines suggests that TaARF15-A1 delays leaf senescence by negatively regulating senescence-promoting processes and positively modulating senescence-delaying genes.Of the differentially expressed genes(DEGs),TaARF15-A1 could directly bind to the promoter regions of TaNAM-1;TaMYC2 physically interacted with TaARF15-A1,and positively modulated wheat senescence.What’s more,TaMYC2 directly bound to the promoters of TaNAM-1 homoeologs and up-regulated its expression.TaARF15-A1 could interfere with the regulation of TaNAM-1 by TaMYC2 through competitive bindings to the similar promoter region of TaNAM-1 and protein-protein interaction,and then indirectly repressed the transcription of TaNAM-1 to negatively modulate wheat leaf senescence.4.Polymorphism analysis and marker development of TaARF15-A1A total of 20 mutation sites were identified by detecting natural variations of TaARF15-A1 in 32 representative cultivars with high genetic diversity,and all of these variations formed two haplotypes,which were named TaARF15-A1-Hap I and TaARF15-A1-Hap Ⅱ.A In Del marker was developed based on the variations of TaARF15-A1 haplotypes,and the association analysis of traits / markers showed that the TaARF15-A1 haplotype was significantly associated with mature days(MD)and plant height(PH)in multiple environments,which appeared to be closely associated with the variations in the TaARF15-A1 promoter region.And TaARF15-A1-Hap I underwent selection during Chinese and global wheat breeding.