Auxin Response Factor(ARF) Genome-Wide Identification,Characterization And Functional Analysis Of CcARF5 And CcARF28 Under Al Stress In Cajanus Cajan

Auxin response factors(ARFs)are essential for regulating plant growth and adapting to unfavorable environments by modulating the auxin signaling pathway.Aluminium(Al)toxicity severely limits the growth of wood bean in acidic soils.However,the identification of the ARF family in pigeon pea and its functional role in Al stress remain unclear.Therefore,this study provided the first detailed bioinformatics information on the ARF family members of pigeon pea,and constructed CcARF5 and CcARF28 overexpression vectors.Transgenic pigeon pea plants were obtained by transient transformation system.The functions of the CcARF5 and CcARF28 genes in pigeon pea under Al stress were revealed by phenotypic,physiological and genetic level studies in transgenic plants.These results provide new insights into the potential functions of ARF family members,and will help to elucidate the role of ARF genes in the accumulation of flavonoids and lignin under Al stress,and will better understand the molecular mechanisms of Al resistance in pigeon pea.The main results are as follows:1.In this study,30 members of the ARF gene family were identified from the pigeon pea genome.Further analysis showed that 30 CcARFs were sporadically dispersed across the 11 chromosomes of pigeon pea.Phylogenetic analysis classified all CcARFs into four groups,with different function in different biological processes.CcARFs contained BDD,ARF,and CTD domains,whereas 9 CcARFs lacked CTD domains.A total of 15 cis-elements of the CcARFs promoter associated with abiotic stresses were found.2.Tissue-specific and Al stress analysis of nine CcARF genes showed that most genes from the four subfamilies were low in roots compared to stems and leaves,but were significantly up-regulated under Al treatment.The elevated levels of genistein and apigenin under Al stress demonstrated their Al-induced synthesis in pigeon pea roots.Further correlation analysis confirmed a strong correlation between CcARFs and flavonoids accumulation.3.To further validate the function of ARF genes in Al stress,CcARF5(CcARF5-OE)and CcARF28(CcARF28-OE)were transiently overexpressed in pigeon pea.Transgenic CcARF5-OE and CcARF28-OE lines of pigeon pea showed reduced root elongation compared to the null plants(Empty)under Al stress.This phenotype was mainly due to its increased Al content,elevated oxidative damage and reduced antioxidant enzymes active.CcARF5 and CcARF28 reduced the accumulation of lignin in roots under Al stress,which in turn affected the accumulation of Al in roots.In summary,CcARF5 and CcARF28 weaken resistance to Al stress by transcriptionally repressing the expression of key genes in the phenylpropane synthesis pathway and reducing the accumulation of lignin and flavonoids in the roots.