Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jul;116(1):23-34.
doi: 10.1093/aob/mcv058. Epub 2015 Jun 12.

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

Juan Du  1 , Zongan Huang  1 , Biao Wang  1 , Hua Sun  1 , Chunlin Chen  1 , Hong-Qing Ling  2 , Huilan Wu  2
Affiliations

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

Juan Du et al. Ann Bot. 2015 Jul.

Abstract

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 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.

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.

Keywords: FER; SlbHLH068; Solanum lycopersicum; Tomato; ferric-chelate reductase 1; iron deficiency; iron homeostasis; iron-regulated transporter 1; transcription factor.

PubMed Disclaimer

Figures

F<sc>ig</sc>. 1.
Fig. 1.
Gene structure and sequence comparison of AtbHLH38, AtbHLH39, AtbHLH100, AtbHLH101 and SlbHLH068 (XP_004249750). (A) Alignment of the amino acid sequences of SlbHLH068 and four Arabidopsis homologues. Multiple sequence alignment was performed using the ClustalW program in the Lasergene software. Identical residues are shown in black. Underlined amino acid sequences are conserved bHLH domains predicted by the CDD program (http://www.ncbi.nlm.nih.gov/Structure/cdd/ March, 2015). (B) Gene structure comparison of SlbHLH068 and its four homologues in Arabidopsis. Gene structure was predicted by aligning coding sequence and genome DNA sequence of each gene. Grey boxes represent upstream/downstream untranslated region, black boxes denote exons and thin lines indicate introns.
F<sc>ig</sc>. 2.
Fig. 2.
Expression profiling of SlbHLH068 in wild-type tomato (Lycopersicon esculentum ‘Moneymaker’). (A) Northern blot analysis of SlbHLH068 expression in roots (R), leaves (L), stems (S), flowers (Fl) and fruits (Fr) under differing iron supplies. Roots, leaves and flowers were collected from seedlings treated hydroponically for 6 d with sufficient (+) or deficient (−) iron. Flowers and young fruits were harvested from seedlings grown in a field with a sufficient iron supply. (B) Time-course experiment of SlbHLH068 expression in roots. The + and – signs indicate plants grown under conditions with 100 µm and 0·1 µm Fe(III)-EDTA, respectively. (C) Response of SlbHLH068 in leaves (L) and roots (R) to different elemental supply. – and H indicate deficient and high elemental levels, respectively. CK (complete nutrient condition), N (nitrogen), P (phosphorus), K (potassium), Fe (iron), Cu (copper), Zn (zinc), Mn (manganese). RNA stained with ethidium bromide was used as a control for equal loading.
F<sc>ig</sc>. 3.
Fig. 3.
Interaction analysis of SlbHLH068 and FER by a yeast two-hybrid assay. (A) Yeast cells grown on SD plates without Trp, Leu and His. (B) Filter lift assay of lacZ expression for yeast cells grown on SD plates without Trp and Leu. The numbers 1–7 indicate yeast cells transformed with the following plasmid combinations: 1, pAD-FER/pBD-bHLH068; 2, pAD-FER/pBD; 3, pAD/pBD-bHLH068; 4, pAD/pBD-FER; 5, pAD-bHLH068/pBD; 6, pAD-FER/pBD-FER; 7, pAD-bHLH068/pBD-bHLH068.
F<sc>ig</sc>. 4.
Fig. 4.
Transcriptional activation assay of the GUS gene driven by the LeFRO1 promoter in yeast. (A) Filter lift assay of GUS activity in yeast cells grown on SD plates without Trp and Leu. (B) Quantitative analysis of GUS activity in yeast cells cultured in liquid SD medium without Trp and Leu. Values are the mean of three replicates ±s.d. Different lowercase letters indicate significance as determined using ANOVA and Tukey’s post-hoc multiple comparison test at P < 0.05. The numbers 1–6 indicate plasmid combinations as follows: 1, pAD/pBD-PLeFRO1::GUS; 2, pAD/pBD-FER-PLeFRO1::GUS; 3, pAD-SlbHLH068/pBD-PLeFRO1::GUS; 4, pAD-SlbHLH068/pBD-FER-PLeFRO1::GUS; 5, pAD-AtbHLH40/pBD-PLeFRO1::GUS; 6, pAD-AtbHLH40/pBD-FER-PLeFRO1::GUS.
F<sc>ig</sc>. 5.
Fig. 5.
BiFC assay of the interaction between FER and SlbHLH068 in Arabidopsis protoplast cells. Arabidopsis mesophyll protoplasts were transformed with different plasmid or plasmid pairs as follows. pFER-cCFP/pbHLH068-nYFP: co-transformation of FER and SlbHLH068 integrated in the N terminus of fluorescent protein; pcCFP-FER/pnYFP-bHLH068: co-transformation of FER and SlbHLH068 integrated in the C terminus of fluorescent protein; pFER-cCFP/pFER-nYFP: co-transformation of cCFP- and nYFP-fused FER proteins; pbHLH068-cCFP/pbHLH068-nYFP: co-transformation of cCFP- and nYFP-fused SlbHLH068 proteins; pA7-YFP: a positive control containing full-length fluorescent protein; pX-cCFP/pX-nYFP: empty plasmid pair as a negative control. Images were captured 16–20 h after transient expression using an Olympus confocal microscope. The protoplasts transformed with pFER-cCFP/pbHLH068-nYFP, pcCFP-FER/pnYFP-bHLH068 and pFER-cCFP/pFER-nYFP displayed fluorescent signals in the nucleus, whereas no fluorescent signal was observed in the nucleus of protoplasts transformed with other plasmid combinations. Scale bar = 20.0 µm.
F<sc>ig</sc>. 6.
Fig. 6.
Iron-deficiency response in SlbHLH068-silenced tomato plants. (A) CH42-silenced tomato with pale green leaves. The plant on the left is infiltrated with the empty vector. The plant on the right is infiltrated with pTRV1/pTRV2-CH42. (B) Tomato infiltrated with an empty vector (upper) or pTRV1/pTRV2-bHLH068 (lower) after 6 d of iron-deficiency treatment. (C) Multiplex RT-PCR analysis of SlbHLH068 and LeFRO1 expression in leaves of control and SlbHLH068-silenced plants. 1, plant infiltrated with empty vector; 2 and 3, plants infiltrated with TRV1/TRV2-bHLH068. Values are the mean of five replicates ±s.d. (D) Multiplex RT-PCR analysis of SlbHLH068, FER, LeFRO1 and LeIRT1 expression in the roots of plants infiltrated with an empty vector or TRV1/TRV2-bHLH068. (E) Northern blot analysis of LeFRO1 and LeIRT1 expression in the roots of plants infiltrated with an empty vector or TRV1/TRV2-bHLH068. RNA stained with ethidium bromide was used as a control for equal loading. (F,G) Iron content in leaves and roots of control and silenced plants. Values are the mean ± s.d. in three control plants, and 13 out of 20 plants treated with pTRV1/pTRV2-bHLH068 showed reduced expression of bHLH068. Asterisks indicate significant difference at **P < 0.01 and *P < 0.05, respectively.
F<sc>ig</sc>. 7.
Fig. 7.
Northern blot analysis of SlbHLH068 in roots and leaves of the tomato mutants. chln, fer, chln/fer and the corresponding wild-types BB (Bonner Beste) and BF (T3238FER). Young wild-type and mutant seedlings were grown on half-strength Hoagland’s solution until emergence of the fourth true leaf. The roots were completely washed with deionized water, and plants were transferred into culture pots containing Hoagland’s solution with 100 µm (+) or 0·1 µm (−) Fe(III)–EDTA for 6 d. Total RNA was extracted from roots and leaves, and the expression intensity of SlbHLH068 was determined by northern blotting. RNA stained with ethidium bromide was used as a control for equal loading.
F<sc>ig</sc>. 8.
Fig. 8.
Regulatory model of transcription factors FER and SlbHLH068 in tomato. The iron-deficient signal initiates expression of the transcription activator genes FER and SlbHLH068 in root cells. The heterodimers formed by FER and SlbHLH068 directly bind to the LeFRO1 promoter and activate its transcription. LeIRT1 and LeNRAMP1 may be regulated in the same way. FER is able to form homodimers, but their biological function remains to be investigated. SlbHLH066 and SlbHLH067, closely grouped with Ib subgroup of bHLH proteins in Arabidopsis by phylogenetic analysis, were determined as iron-deficiency responsive proteins (Sun et al., 2015). They may play redundant roles with SlbHLH068 in tomato, as AtbHLH38/AtbHLH39/AtbHLH100/AtbHLH101 in Arabidopsis. Further experimental evidence is needed to prove this hypothesis.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Becker R, Grün M, Scholz G. 1992. Nicotianamine and the distribution of iron into the apoplasm and symplasm of tomato (Lycopersicon esculentum Mill.): I. Determination of the apoplasmic and symplasmic iron pools in roots and leaves of the cultivar Bonner Beste and its nicotianamine-less mutant chloronerva. Planta 187: 48–52. - PubMed
    1. Bereczky Z, Wang HY, Schubert V, Ganal M, Bauer P. 2003. Differential regulation of nramp and irt metal transporter genes in wild-type and iron uptake mutants of tomato. The Journal of Biological Chemistry 278: 24697–24704. - PubMed
    1. Brumbarova T, Bauer P. 2005. Iron-mediated control of the basic helix–loop–helix protein FER, a regulator of iron uptake in tomato. Plant Physiology 137: 1018–1026. - PMC - PubMed
    1. Chen JC, Jiang CZ, Gookin TE, Hunter DA, Clark DG, Reid MS. 2004. Chalcone synthase as a reporter in virus-induced gene silencing studies of flower senescence. Plant Molecular Biology 55: 521–530. - PubMed
    1. Chen QR, Vansant G, Oades K, et al. 2007. Diagnosis of the small round blue cell tumors using multiplex polymerase chain reaction. Journal of Molecular Diagnostics 9: 80–88. - PMC - PubMed

Publication types

MeSH terms