Investigation Of Mechanism For ZmSRO1c-mediated Hypocotyl Elongation And Leaf Senescence

Similar to RCD One(SRO)is a small protein family unique to plants,which play important roles in development regulation or responses to abiotic stress.The family members can be divided into two subgroups.Currently,the function and mechanism of the first subgroup members RCD1 and SRO 1 to abiotic stresses was widely studied,while their functions in regulating photomorphogenesis and senescence is not well known.Moreover,there is almost no report on the study of the functions and mechanisms of the second subgroup members involved in the regulation of development.Based on previous studies,we further studied the function and mechanism of the second subgroup member ZmSRO1c of maize and the second subgroup members of Arabidopsis SRO family in regulating hypocotyl development and senescence of leaves.1.ZmSRO1c and the second subgroup members of Arabidopsis SRO family promote hypocotyl elongationZmSRO1c transgenic maize lines exhibited longer coleoptile compared with WT,and ZmSRO1c promoted hypocotyl elongation when heterologous overexpressed(OE)in Arabidopsis,which suggest that ZmSRO1c is a negative regulator of photomorphogenesis.Electron microscopy illustrate that the hypocotyl cells of ZmSRO1c OE lines were longer than that of Col-0,indicating that ZmSRO1c promote hypocotyl elongation mainly due to enhancing cell elongation.In order to further verify the function of the second subgroup of SRO family in photomorphogenesis,we selected three members of the second subgroup,AtSRO2,AtSRO3 and AtSRO4,which are closely related with ZmSRO1c,and found that the overexpression of the three genes promoted hypocotyl elongation under white light.The hypocotyls of AtSRO3 and AtSRO4 overexpression lines were also significantly elongated under blue and far red light,indicating that ZmSRO1c and Arabidopsis SRO homologous genes negatively regulate photomorphogenesis.2.ZmSROlc promote hypocotyl elongation through interacting with the N-terminal of HY5 via its RST domainIt was confirmed that ZmSRO1c interacts with HY5,a key regulator of photomorphogenesis,in the nucleus by Y2H,BiFC and Co-IP,which negatively regulates photomorphogenesis and promotes hypocotyl elongation.Further analyzes found that the C-terminal RST domain of ZmSRO1c and the N-terminus of HY5 are participate in the interaction of ZmSRO1c with HY5.In addition,some members of the second subgroup of the SRO family in Arabidopsis can also interact with HY5.However,no direct interaction was found between PIFs,another key regulators of photomorphogenesis,and ZmSRO1c or the second subgroup members of Arabidopsis SROs.3.ZmSRO1c can relieve the repression of HY5 on downstream target genes to promote hypocotyl elongationWe found that the expression levels of some of the HY5 and PIFs target genes associated with hypocotyl development were significantly up-regulated in ZmSRO1c overexpression lines,indicating that ZmSRO1c might regulate the expression of HY5 and PIF target genes through interacting with HY5.Further,the dual luciferase reporter gene assay showed that HY5 inhibited the expression of the target gene AtIAA19,but ZmSRO1c released the inhibitory effect on AtIAA19 through interacting with HY5,thus enhancing the expression of AtIAA19 and promoting hypocotyl elongation.4 ZmSRO1c inhibits chlorophyll synthesis and promotes chlorophyll degradation,leading to leaf senescencePrevious experiments showed that ZmSRO1c heterologous expression in Arabidopsis resulted in lower chlorophyll content and premature senescence of leaves.Here,we found that the chlorophyll content of ZmSRO1c transgenic maize lines were lower than WT,and they showed a certain degree of premature senescence compared with WT.Moreover,the overexpression of AtSRO2,AtSRvO3,AtSRO4 in Arabidopsis also led to premature senescence of leaves,indicating that the second subgroup of SROs involved in the regulation of plant senescence.We found that ZmSRO1c can inhibit the expression of chlorophyll synthesis-related gene and promote the expression of chlorophyll degradation-related genes,resulting in decreased chlorophyll content.qRT-PCR analysis revealed that the expression levels of some senescence-associated genes targeted by HY5 and PIFs were significantly up-regulated in ZmSRO1c overexpression lines,suggesting that ZmSRO1c may affect the expression of these senescence-associated genes by interacting with HY5 and leading to premature senescence of the leaves.In summary,we conclude that the second subgroup of the SRO family may promote hypocotyl elongation and leaf senescence by interacting with HY5 and regulating the expression of downstream hypocotyl elongation and leaf premature senescence-related target genes.