SlbHLH068 interacts with FER to regulate the iron-deficiency response in tomato
SlbHLH068 interacts with FER to regulate the iron-deficiency response in tomato
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SlbHLH068 与 FER 相互作用调节番茄缺铁反应
DOI：
10.1093/aob/mcv058
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发表时间：
2015-07-01
期刊：
影响因子：
4.2
通讯作者：
Wu, Huilan
中科院分区：
文献类型：
--
作者：
Du, Juan;Huang, Zongan;Wu, Huilan
背景与目的铁是所有生物体必需的微量营养素，其在植物体内的吸收、转运、分配和利用受到复杂的调控。FER是从番茄(Solanum Lycopsicum)中分离得到的第一个参与高等植物铁稳态的转录因子。T3238fer突变体中的FER缺陷显著下调铁摄取基因的表达，如铁络合物还原酶1(LeFRO1)和铁调节转运蛋白1(LeIRT1)；然而，FER调节下游基因的分子机制尚不清楚。因此，本工作的目的是寻找与FER相互作用调节番茄缺铁反应的基因。方法利用BLASTP程序，根据番茄基因组中的AtbHLH39氨基酸序列，鉴定了拟南芥碱性螺旋-环-螺旋(BHLH)蛋白Ib亚群的同源基因SLbHLH068。通过酵母双杂交和双分子荧光互补(BIFC)检测SlbHLH068与FER的相互作用。此外，还利用病毒诱导的基因沉默技术(VIGS)获得了SLbHLH068表达下调的番茄植株。用Northern印迹杂交和多重RT-PCR分析基因的表达。结果SLbHLH068在根、叶和茎中高度表达，对缺铁有响应。通过酵母双杂交和BIFC实验证明SlbHLH068与FER之间存在相互作用。酵母实验中，FER与SLbHLH068形成的异源二聚体直接与LeFRO1启动子结合，激活了其报告基因的表达。VIGS下调SlbHLH068的表达，导致LeFRO1和LeIRT1的表达减少，叶片和根中铁的积累减少。结论SLbHLH068作为一个可能的转录因子，通过与FER的相互作用参与了番茄体内的铁平衡。
Background and Aims Iron is an essential micronutrient for all organisms and its uptake, translocation, distribution and utilization are regulated in a complex manner in plants. FER, isolated from tomato (Solanum lycopersicum), was the first transcription factor involved in the iron homeostasis of higher plants to be identified. A FER defect in the T3238fer mutant drastically downregulates the expression of iron uptake genes, such as ferricchelate reductase 1 (LeFRO1) and iron-regulated transporter 1 (LeIRT1); however, the molecular mechanism by which FER regulates genes downstream remains unknown. The aim of this work was therefore to identify the gene that interacts with FER to regulate the iron-deficiency response in tomato.Methods The homologue of the Arabidopsis Ib subgroup of the basic helix-loop-helix (bHLH) proteins, SlbHLH068, was identified by using the program BLASTP against the AtbHLH39 amino acid sequence in the tomato genome. The interaction between SlbHLH068 and FER was detected using yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. In addition, virus-induced gene silencing (VIGS) was used to generate tomato plants in which SlbHLH068 expression was downregulated. The expression of genes was analysed using northern blot hybridization and multiple RT-PCR analysis. Seedlings of wild-type and mutant plants were grown under conditions of different nutrient deficiency.Key Results SlbHLH068 is highly upregulated in roots, leaves and stems in response to iron deficiency. An interaction between SlbHLH068 and FER was demonstrated using yeast two-hybrid and BiFC assays. The heterodimer formed by FER with SlbHLH068 directly bound to the promoter of LeFRO1 and activated the expression of its reporter gene in the yeast assay. The downregulation of SlbHLH068 expression by VIGS resulted in a reduction of LeFRO1 and LeIRT1 expression and iron accumulation in leaves and roots.Conclusions The results indicate that SlbHLH068, as a putative transcription factor, is involved in iron homeostasis in tomato via an interaction with FER.