Regulation Of AtDOF4.7-involved In Floral Organ Abscission By Ethylene-dependent And -independent Pathways

Organ abscission, which means plant body discards some unwanted organs (e. g., leaves, flowers and fruits), is an important plant developmental process. In Arabidopsis, organ abscission regulated by plant hormone ethylene and INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) ligand signals that are studied widely. Our lab previously reported that AtDOF4.7, a DOF transcription factor family gene, might involve in floral organ abscission. Overexpression of AtDOF4.7 directly suppresses the expression of POLYGALACTURONASE ABSCISSION ZONE A. THALIANA (PGAZAT), the key cell wall-loosening gene, resulting in a deficiency of floral organ abscission. However, we still have no idea about how ethylene-dependent and -independent abscission pathways regulate AtDOF4.7.In this study, I investigated floral organ abscission pathways upstream of AtDOF4.7 and regulation network downstream of AtDOF4.7. First of all, The timing of GUS expression in AZ cells of siliques was advanced when PromoterAtDOF4.7∷GUS lines were treated with ethylene; secondly, expression pattern of PromoterAtDOFf4.7∷GUS was investigated in ethylene-insensitive mutants ein2-1 and etrl-1 through genetic process, the GUS expression was delayed in AZ cells of siliques in PromoterAtDOF4.7∷GUS/ein2-1 (or PromoterAtDOF4.7∷GUS/etr1-1). And Real-time PCR analysis proved that the timing of AtDOF4.7 expression was delayed in ein2-1 mutant. These data suggest that ethylene temporally regulates AtDOF4.7 expression. Meanwhile, ethylene-independent floral organ abscission pathway also can regulate AtDOF4.7 expression.GUS expression of PromoterAtDOF4.7∷GUS was also investigated in ethylene-independent abscission deficiency mutants ida-2, and the expression pattern is similar with that in WT. However, Real-time PCR analysis showed that relative expression level of AtDOF4.7 was higher in ida-2 mutant, compared with in WT. These data suggest that IDA negatively regulates AtDOF4.7 at transcriptional level. In order to confirm the relationship between AtDOF4.7 and IDA, overexpression of AtDOF4.7 (named S107) was crossed into 35S:IDA that prematurely abscises its organs. However, floral organ abscission was not rescued in S107/35S:IDA lines. These results show that AtDOF4.7 is downstream of IDA.To investigate and explain the regulation of AtDOF4.7 by ethylene-independent floral organ abscission pathway, detection of relationship between AtDOF4.7 and key abscission-related MAPK cascade was performed. BiFC and yeast two-hybrid assays demonstrate that the interaction with AtDOF4.7 and MPK3/MPK6. Activated MPK3/MPK6 by MKK5DD strongly phosphorylated AtDOF4.7 MBP-tagged fusion protein in vitro. Further in vivo experiment was performed to study biological function of phosphorylated AtDOF4.7, suggesting that the level of AtDOF4.7 protein may be regulated by MAPK phosphorylation.In order to study the function mechanism of AtDOF4.7, detection and screening of AtDOF4.7 regulating downstream target genes with RNA-seq method. There were 5 down-regulated cell wall-loosening enzyme genes(AT2G43620, AT2G43610, AT4G02330, AT4G16260 and AT4G30290) in 232 down-regulated differentially expressed genes, Real-time PCR analysis proved these genes were actually down regulated in S107 siliques. These data reveal that AtDOF4.7 negatively regulates many cell wall-loosening enzymes to efficiently regulate abscission.In summary, our results suggest that ethylene-dependent and -independent floral organ abscission pathways co-regulate AtDOF4.7. Ethylene influences the timing of AtDOF4.7 expression, and IDA regulates expression of AtDOF.47 at transcriptional and post-transcriptional level to control degree of organ abscission undrlying ethylene-independent abscission pathway. AtDOF4.7 negatively regulates downstream of five cell wall-hydrolyzing enzyme genes.