Raffinose accumulation is positively correlated with plant chilling stress tolerance; however, the understanding of the function and regulation of raffinose metabolism under chilling stress remains in its infancy. RAFFINOSE SYNTHASE (RAFS) is the key enzyme for raffinose biosynthesis. In this study, we report that two independent maize (Zea mays) zmrafs mutant lines, in which raffinose was completely abolished, were more sensitive to chilling stress and their net photosynthetic product (total soluble sugars and starch) accumulation was significantly decreased compared with controls after chilling stress. A similar characterization of the maize dehydration responsive element (DRE)-binding protein 1A mutant (zmdreb1a) showed that ZmRAFS expression and raffinose content were significantly decreased compared with its control under chilling stress. Overexpression of maize ZmDREB1A in maize leaf protoplasts increased ZmDREB1A amounts, which consequently upregulated the expression of maize ZmRAFS and the Renilla LUCIFERASE (Rluc), which was controlled by the ZmRAFS promoter. Deletion of the single dehydration-responsive element (DRE) in the ZmRAFS promoter abolished ZmDREB1A's influence on Rluc expression, while addition of three copies of the DRE in the ZmRAFS promoter dramatically increased Rluc expression when ZmDREB1A was simultaneously overexpressed. Electrophoretic mobility shift assays and chromatin immunoprecipitation-quantitative PCR demonstrated that ZmDREB1A directly binds to the DRE motif in the promoter of ZmRAFS both in vitro and in vivo. These data demonstrate that ZmRAFS, which was directly regulated by ZmDREB1A, enhances both raffinose biosynthesis and plant chilling stress tolerance.

中文翻译：

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ZmDREB1A调节RAFFINOSE合酶，控制玉米中的棉子糖积累和植物耐冷胁迫。

棉子糖的积累与植物抗冷胁迫的能力呈正相关。然而，对低温胁迫下棉子糖代谢功能和调节的了解仍处于起步阶段。棉子糖合成酶（RAFS）是棉子糖生物合成的关键酶。在这项研究中，我们报告了棉子糖被完全废除的两个独立的玉米（Zea mays）zmrafs突变株系对低温胁迫更加敏感，其净光合产物（总可溶性糖和淀粉）的积累与对照相比显着降低。放松压力后。玉米脱水反应元件（DRE）结合蛋白1A突变体（zmdreb1a）的相似特征显示，与低温胁迫下的对照相比，ZmRAFS表达和棉子糖含量显着降低。玉米叶原生质体中玉米ZmDREB1A的过表达增加了ZmDREB1A的量，因此上调了ZmRAFS启动子控制的玉米ZmRAFS和海肾LUCIFERASE（Rluc）的表达。ZmRAFS启动子中单个脱水反应元件（DRE）的删除消除了ZmDREB1A对Rluc表达的影响，而当ZmDREB1A同时过表达时，在ZmRAFS启动子中添加三份DRE则显着提高了Rluc表达。电泳迁移率变动分析和染色质免疫沉淀定量PCR证明，ZmDREB1A在体内和体外均直接与ZmRAFS启动子中的DRE基序结合。这些数据表明，受ZmDREB1A直接调节的ZmRAFS，