SlbHLH068 interacts with FER to regulate the iron-deficiency response in tomato

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 ferric-chelate 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.

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.

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.

The results indicate that SlbHLH068, as a putative transcription factor, is involved in iron homeostasis in tomato via an interaction with FER.