Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa, 921-8836, Japan.
Crop Biotech Institute and Graduate School of Biotechnology, Kyung Hee University, Yongin, 17104, Korea.
Plant Mol Biol. 2019 Nov;101(4-5):471-486. doi: 10.1007/s11103-019-00917-8. Epub 2019 Sep 24.
Subgroup IVc basic helix-loop-helix transcription factors OsbHLH058 and OsbHLH059 positively regulate major iron deficiency responses in rice in a similar but distinct manner, putatively under partial control by OsHRZs. Under low iron availability, plants transcriptionally induce the expression of genes involved in iron uptake and translocation. OsHRZ1 and OsHRZ2 ubiquitin ligases negatively regulate this iron deficiency response in rice. The basic helix-loop-helix (bHLH) transcription factor OsbHLH060 interacts with OsHRZ1, and positively regulates iron deficiency-inducible genes. However, the functions of three other subgroup IVc bHLH transcription factors in rice, OsbHLH057, OsbHLH058, and OsbHLH059, have not yet been characterized. In the present study, we investigated the functions of OsbHLH058 and OsbHLH059 related to iron deficiency response. OsbHLH058 expression was repressed under iron deficiency, whereas the expression of OsbHLH057 and OsbHLH060 was moderately induced. Yeast two-hybrid analysis indicated that OsbHLH058 interacts with OsHRZ1 and OsHRZ2 more strongly than OsbHLH060, whereas OsbHLH059 showed no interaction. An in vitro ubiquitination assay detected no OsbHLH058 and OsbHLH060 ubiquitination by OsHRZ1 and OsHRZ2. Transgenic rice lines overexpressing OsbHLH058 showed tolerance for iron deficiency and higher iron concentration in seeds. These lines also showed enhanced expression of many iron deficiency-inducible genes involved in iron uptake and translocation under iron-sufficient conditions. Conversely, OsbHLH058 knockdown lines showed susceptibility to iron deficiency and reduced expression of many iron deficiency-inducible genes. OsbHLH059 knockdown lines were also susceptible to iron deficiency, and formed characteristic brownish regions in iron-deficient new leaves. OsbHLH059 knockdown lines also showed reduced expression of many iron deficiency-inducible genes. These results indicate that OsbHLH058 and OsbHLH059 positively regulate major iron deficiency responses in a similar but distinct manner, and that this function may be partially controlled by OsHRZs.
亚组 IVc 碱性螺旋-环-螺旋转录因子 OsbHLH058 和 OsbHLH059 以相似但又不同的方式正向调控水稻对缺铁的主要响应，推测受 OsHRZs 的部分控制。在铁供应不足的情况下，植物会转录诱导参与铁吸收和转运的基因的表达。OsHRZ1 和 OsHRZ2 泛素连接酶负调控水稻的这种缺铁响应。碱性螺旋-环-螺旋（bHLH）转录因子 OsbHLH060 与 OsHRZ1 相互作用，正向调控缺铁诱导基因。然而，水稻中另外三个亚组 IVc bHLH 转录因子 OsbHLH057、OsbHLH058 和 OsbHLH059 的功能尚未得到表征。在本研究中，我们研究了 OsbHLH058 和 OsbHLH059 与缺铁响应相关的功能。OsbHLH058 的表达在缺铁时受到抑制，而 OsbHLH057 和 OsbHLH060 的表达则适度诱导。酵母双杂交分析表明，OsbHLH058 与 OsHRZ1 和 OsHRZ2 的相互作用强于 OsbHLH060，而 OsbHLH059 则没有相互作用。体外泛素化试验检测到 OsHRZ1 和 OsHRZ2 对 OsbHLH058 和 OsbHLH060 没有泛素化作用。过表达 OsbHLH058 的转基因水稻品系对缺铁有耐受性，种子中铁浓度较高。这些品系在铁充足条件下，还表现出许多与铁吸收和转运相关的缺铁诱导基因的表达增强。相反，OsbHLH058 敲低系对缺铁敏感，许多缺铁诱导基因的表达减少。OsbHLH059 敲低系也对缺铁敏感，在缺铁的新叶中形成特征性的棕色区域。OsbHLH059 敲低系还表现出许多缺铁诱导基因的表达减少。这些结果表明，OsbHLH058 和 OsbHLH059 以相似但又不同的方式正向调控水稻对缺铁的主要响应，并且这种功能可能部分受 OsHRZs 的控制。
