Arabidopsis miR171-Targeted Scarecrow-Like Proteins Bind to GT cis-Elements and Mediate Gibberellin-Regulated Chlorophyll Biosynthesis under Light Conditions

摘要

An extraordinarily precise regulation of chlorophyll biosynthesis is essential for plant growth and development. However, our knowledge on the complex regulatory mechanisms of chlorophyll biosynthesis is very limited. Previous studies have demonstrated that miR171-targeted scarecrow-like proteins (SCL6/22/27) negatively regulate chlorophyll biosynthesis via an unknown mechanism. Here we showed that SCLs inhibit the expression of the key gene encoding protochlorophyllide oxidoreductase (POR) in light-grown plants, but have no significant effect on protochlorophyllide biosynthesis in etiolated seedlings. Histochemical analysis of β-glucuronidase (GUS) activity in transgenic plants expressing pSCL27::rSCL27-GUS revealed that SCL27-GUS accumulates at high levels and suppresses chlorophyll biosynthesis at the leaf basal proliferation region during leaf development. Transient gene expression assays showed that the promoter activity of PORC is indeed regulated by SCL27. Consistently, chromatin immunoprecipitation and quantitative PCR assays showed that SCL27 binds to the promoter region of PORC in vivo . An electrophoretic mobility shift assay revealed that SCL27 is directly interacted with G(A/G)(A/T)AA(A/T)GT cis -elements of the PORC promoter. Furthermore, genetic analysis showed that gibberellin (GA)-regulated chlorophyll biosynthesis is mediated, at least in part, by SCLs. We demonstrated that SCL27 interacts with DELLA proteins in vitro and in vivo by yeast-two-hybrid and coimmunoprecipitation analysis and found that their interaction reduces the binding activity of SCL27 to the PORC promoter. Additionally, we showed that SCL27 activates MIR171 gene expression, forming a feedback regulatory loop. Taken together, our data suggest that the miR171-SCL module is critical for mediating GA-DELLA signaling in the coordinate regulation of chlorophyll biosynthesis and leaf growth in light. Author Summary Chlorophyll biosynthesis is essential for plant growth and development. To date, the regulatory mechanisms of chlorophyll biosynthesis have been well understood only in dark conditions. Previous reports showed that miR171-targeted SCL6/22/27 proteins were involved in chlorophyll biosynthesis. However, the molecular mechanism of SCL action remains unclear. In this study, we found that SCLs negatively regulated chlorophyll biosynthesis though suppressing the expression of the key gene PROTOCHLOROPHYLLIDE OXIDOREDUCTASE ( POR ). SCL27 is highly expressed at the basal cell proliferation region of young leaves, suggesting an important role of SCLs in inhibiting chloroplast development before cell expansion. In addition, GT- cis elements were required for SCL27 directly binding to the PORC promoter. Furthermore, we showed that SCLs mediated GA-regulated chlorophyll biosynthesis through direct interaction with DELLA proteins. The interaction between SCLs and DELLAs reduced the DNA binding activity of SCL27. Our uncovered GA-DELLA-SCL module and its DNA binding targets provide new insights into molecular mechanisms by which chlorophyll biosynthesis and cell proliferation are coordinately regulated during leaf development in response to developmental and environmental cues.