The transcription factor CsbHLH18 of sweet orange (Citrus sinensis) functions in modulation of cold tolerance and reactive oxygen species homeostasis by regulating the antioxidant gene

The basic helix-loop-helix (bHLH) transcription factors (TFs) comprise one of the largest gene families in plants and participate in various physiological processes, but physiological role and regulatory function of the majority of bHLHs remain poorly understood. Here, a total of 56 putative CsbHLH genes were identified in sweet orange (Citrus sinensis) based on a genome-wide analysis. The CsbHLHs, except four members, were distributed throughout nine chromosomes and divided into 19 subgroups. Most of the CsbHLH genes were responsive to cold stress, with the greatest upregulation being observed in CsbHLH18. CsbHLH18 is localized in the nuclei and has transcriptional activation activity. Overexpression of CsbHLH18 conferred enhanced cold tolerance in transgenic tobacco. The transgenic plants accumulated significantly less reactive oxygen species (ROS), concurrent with increased activities and transcript levels of antioxidant enzymes (抗氧化酶). By contrast, knockdown of bHLH18 by RNA interference in trifoliate orange (枸桔) promoted cold susceptibility, accompanied by (伴随着) down-regulation of antioxidant genes and accumulation of more ROS. Protein-DNA interaction assay demonstrates that CsbHLH18 directly and specifically binds to and activate the promoter of CsPOD. Taken together, these findings indicate that CsbHLH18 plays a positive role in cold tolerance through, at least partly, modulation of ROS homeostasis by directly regulating the antioxidant gene.

bHLH转录因子构成了植物中最为庞大的基因家族之一，在植物多个生理过程中扮演重要作用，但bHLHs大多数基因的生理作用及功能调节仍然不清楚。本文总共鉴定了甜橙中56假定的CsbHLH基因。这些CsbHLH基因除了4个，其他的分布在9个染色体，并划分为19个亚群。大多数的CsbHLH基因响应于冷胁迫，其中又以CsbHLH18基因响应最为强烈。CsbHLH18蛋白定位于核，并且具有转录激活活性。在烟草中过表达CsbHLH18基因会增强烟草的抗寒冷能力。过表达植株中同样积累更少的活性氧物质，这与增强的抗氧化酶活性和转录水平等结果一致。相反，通过RNA干扰敲除枸桔中的bHLH18基因促进植株对寒冷的敏感性，并伴随着抗氧化基因的下调表达，并且积累更多的活性氧物质。蛋白-DNA的互作试验揭示了CsbHLH18直接并特异结合和激活CsPOD基因的启动子。综上，本文的发现揭示了CsbHLH18基因通过，或者说部分通过直接调控抗氧化基因调节植物体内的活性氧物质内稳态进而在植物的抗寒性中扮演正向的作用。

通讯：刘继红 (http://chfs.hzau.edu.cn/info/1028/1137.htm)

个人简介：1999年，华中农业大学，博士。

doi: https://doi.org/10.1093/jxb/ery065