National Key Laboratory of Plant Molecular Genetics, Chinese Academy of Sciences Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China.
Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences, Shanghai 201602, China.
Proc Natl Acad Sci U S A. 2024 Jan 16;121(3):e2319335121. doi: 10.1073/pnas.2319335121. Epub 2024 Jan 10.
The phytohormone cytokinin has various roles in plant development, including meristem maintenance, vascular differentiation, leaf senescence, and regeneration. Prior investigations have revealed that cytokinin acts via a phosphorelay similar to the two-component system by which bacteria sense and respond to external stimuli. The eventual targets of this phosphorelay are type-B ARABIDOPSIS RESPONSE REGULATORS (B-ARRs), containing the conserved N-terminal receiver domain (RD), middle DNA binding domain (DBD), and C-terminal transactivation domain. While it has been established for two decades that the phosphoryl transfer from a specific histidyl residue in ARABIDOPSIS HIS PHOSPHOTRANSFER PROTEINS (AHPs) to an aspartyl residue in the RD of B-ARRs results in a rapid transcriptional response to cytokinin, the underlying molecular basis remains unclear. In this work, we determine the crystal structures of the RD-DBD of ARR1 (ARR1) as well as the ARR1-DNA complex from . Analyses of the ARR1-DNA complex have revealed the structural basis for sequence-specific recognition of the GAT trinucleotide by ARR1. In particular, comparing the ARR1 and ARR1-DNA structures reveals that unphosphorylated ARR1 exists in a closed conformation with extensive contacts between the RD and DBD. In vitro and vivo functional assays have further suggested that phosphorylation of the RD weakens its interaction with DBD, subsequently permits the DNA binding capacity of DBD, and promotes the transcriptional activity of ARR1. Our findings thus provide mechanistic insights into phosphorelay activation of gene transcription in response to cytokinin.
植物激素细胞分裂素在植物发育中具有多种作用，包括分生组织维持、血管分化、叶片衰老和再生。先前的研究表明，细胞分裂素通过类似于双组分系统的磷酸传递发挥作用，细菌通过该系统感知和响应外部刺激。该磷酸传递的最终靶标是 B-拟南芥反应调节剂 (B-ARRs)，包含保守的 N 端受体结构域 (RD)、中间 DNA 结合结构域 (DBD) 和 C 端转录激活结构域。虽然已经确定了两个十年来，来自 ARABIDOPSIS HIS PHOSPHOTRANSFER PROTEINS (AHPs) 中特定组氨酸残基的磷酸转移到 B-ARRs 的 RD 中的天冬氨酸残基导致对细胞分裂素的快速转录反应，但潜在的分子基础仍不清楚。在这项工作中，我们确定了 ARR1（ARR1）的 RD-DBD 以及. 的 ARR1-DNA 复合物的晶体结构。ARR1-DNA 复合物的分析揭示了 ARR1 对 GAT 三核苷酸进行序列特异性识别的结构基础。特别是，比较 ARR1 和 ARR1-DNA 结构表明，未磷酸化的 ARR1 以与 RD 和 DBD 之间存在广泛接触的封闭构象存在。体外和体内功能测定进一步表明，RD 的磷酸化会削弱其与 DBD 的相互作用，随后允许 DBD 结合 DNA 的能力，并促进 ARR1 的转录活性。因此，我们的研究结果为细胞分裂素响应基因转录的磷酸传递激活提供了机制见解。
