bHLH transcription factor bHLH115 regulates iron homeostasis in Arabidopsis thaliana

Abstract

Iron (Fe) deficiency is a limiting factor for the normal growth and development of plants, and many species have evolved sophisticated systems for adaptation to Fe-deficient environments. It is still unclear how plants sense Fe status and coordinate the expression of genes responsive to Fe deficiency. In this study, we show that the bHLH transcription factor bHLH115 is a positive regulator of the Fe-deficiency response. Loss-of-function of bHLH115 causes strong Fe-deficiency symptoms and alleviates expression of genes responsive to Fe deficiency, whereas its overexpression causes the opposite effect. Chromatin immunoprecipitation assays confirmed that bHLH115 binds to the promoters of the Fe-deficiency-responsive genes bHLH38/39/100/101 and POPEYE (PYE), which suggests redundant molecular functions with bHLH34, bHLH104, and bHLH105. This is further supported by the fact that the bhlh115-1 mutant was complemented by overexpression of any of bHLH34, bHLH104, bHLH105, and bHLH115. Further investigations determined that bHLH115 could interact with itself and with bHLH34, bHLH104, and bHLH105. Their differential tissue-specific expression patterns and the severe Fe deficiency symptoms of multiple mutants supported their non-redundant biological functions. Genetic analysis revealed that bHLH115 is negatively regulated by BRUTUS (BTS), an E3 ligase that can interact with bHLH115. Thus, bHLH115 plays key roles in the maintenance of Fe homeostasis in Arabidopsis thaliana.

Keywords: Arabidopsis; BTS; Fe deficiency; PYE.; bHLH115; iron.

Fig. 1.

Characterization of bhlh115 mutants. (A) Location of T-DNA in the bHLH115 gene. Thick bars and lines indicate the exons and introns, respectively. Grey bars indicate the 5′ and 3′ untranslated regions. Triangles indicate the location of the T-DNA. (B) Confirmation of the bhlh115-1 and bhlh115-2 mutants. The full-length cDNA of bHLH115 was amplified by RT-PCR. (C)Images of 10-d-old seedlings that were germinated directly on +Fe or –Fe media. (D) Chlorophyll content of leaves on +Fe or –Fe media. Significant differences from the wild-type were determined by Student’s t-test, *P<0.05). (E) Iron reductase activity. Seedlings were germinated and grown on +Fe medium for 10 d and then transferred to –Fe media for 3 d. Significant differences from the wild-type were determined by Student’s t-test, *P<0.05). (F) Time-course quantification of root length of plants from 0 to 4 d after transfer to –Fe medium.

Fig. 2.

Phenotypes of bHLH115-OX transgenic plants. (A) Images of 50-d-old plants that were grown in soil at pH 6.0–6.5. The seventh or eighth leaves are shown in the inset, from left to right: wild-type, bHLH115-OX-14, and bHLH115-OX-21. (B) Relative expression of bHLH115 in the transgenic plants. (C) Images of 10-d-old seedlings that were germinated directly on –Fe media. (D) Chlorophyll content of leaves on –Fe or +Fe media. Significant differences from the wild-type were determined by Student’s t-test, *P<0.05). (E) Relative expression of FER genes in the leaves. Shoots of 10-d-old seedlings germinated directly on +Fe media were used for RNA extraction. Significant differences from the wild-type were determined by Student’s t-test, *P<0.05). (F) Fe concentration in the leaves. Shoots of 10-d-old seedlings germinated directly on +Fe media were used for Fe measurement. Significant differences from the wild-type were determined by Student’s t-test, *P<0.05).

Fig. 3.

Expression of Fe-deficiency-responsive genes in wild-type, mutants, and overexpression plants. Wild-type, mutants, and transgenic plants were grown on +Fe medium for 4 d, then transferred to +Fe or –Fe media for 3 d. RNA was prepared from root tissues. The data represent means (±SD) of three biological replicates. Significant differences from the wild-type were determined by Student’s t-test, *P<0.05).

Fig. 4.

Direct regulation of bHLH38/39/100/101 and PYE by bHLH115. (A) E-box in promoters. The bars indicate the E-box (CANNTG). The 1000-bp upstream sequence from the translation start site of the indicated gene is shown. (B) ChIP-qPCR analyses of the DNA binding ratio of bHLH115 to the promoters of bHLH38/39/100/101 and PYE genes. qPCR was used to quantify enrichment of the indicated promoters and a fragment of the TUB2 promoter containing an E-box motif was used as a negative control. The binding to the TUB2 promoter fragment in the wild-type was set to 1 and used to normalize the DNA binding ratio. Significant differences were determined by Student’s t-test, *P<0.05. (C) bHLH115 activates the promoters of bHLH38/39/100/101 and PYE. In the transient assays, Pro35S:Myc-GUS was expressed as an internal control. GFP transcript abundance was normalized to the GUS transcript. The value obtained with the empty vector as an effector was set to 1. The results are means (±SD) of three biological replicates. Significant differences were determined by Student’s t-test, *P<0.05.

Fig. 5.

Complementation of bhlh115-1 by bHLH34/104/105/115. (A) Images of 10-d-old seedlings that were germinated directly on +Fe or –Fe media. (B) Plants were grown on +Fe media for 4 d, then transferred to +Fe or –Fe media for 3 d. RNA was prepared from root tissues. The data represent means (±SD) of three biological replicates. Significant differences from the wild-type were determined by Student’s t-test, *P<0.05).

Fig. 6.

Interaction between bHLH115 and bHLH34/104/105/115. (A) Yeast two-hybrid assays. Interaction was indicated by the ability of cells to grow on synthetic dropout (SD) medium lacking Leu/Trp/His/Ade. N-terminal truncated bHLH115 was cloned into pGBKT7, and full-length cDNAs of bHLH34, bHLH104, bHLH105, and bHLH115 were cloned into pGADT7. (B) BiFC assays Of N. benthamiana leaves infiltrated with different combinations of the indicated constructs.

Fig. 7.

Expression of a ProbHLH115:GUS fusion. Images of 10-d-old seedlings that were germinated directly on +Fe medium and used for GUS staining. (A) Seedling, (B) hypocotyl and root, (C) connected region between petioles and hypocotyl, (D) root hair zone, (E) emerging lateral root, and (F) root tip.

Fig. 8.

Phenotypes of various mutants. Images of 10-d-old seedlings germinated directly on +Fe or –Fe media.

Fig. 9.

Negative regulation of bHLH115 by BTS. (A) Images of 10-day-old plants that were grown under either Fe-sufficient or Fe-free plus Frz (50 uM) conditions. (B) Expression of Fe-deficiency-responsive genes. Plants were grown on +Fe media for 4 d, then transferred to Fe-sufficient or Fe-free plus Frz media for 3 d. RNA was prepared from root tissues. Significant differences between two samples were determined by Student’s t-test, *P<0.05.