bHLH11 inhibits bHLH IVc proteins by recruiting the TOPLESS/TOPLESS-RELATED corepressors.

Iron (Fe) homeostasis is essential for plant growth and development. Many transcription factors (TFs) play pivotal roles in the maintenance of Fe homeostasis. bHLH11 is a negative TF that regulates Fe homeostasis. However, the underlying molecular mechanism remains elusive. Here, we generated two loss-of-function bhlh11 mutants in Arabidopsis (Arabidopsis thaliana), which display enhanced sensitivity to excess Fe, increased Fe accumulation, and elevated expression of Fe deficiency responsive genes. Levels of bHLH11 protein, localized in both the cytoplasm and nucleus, decreased in response to Fe deficiency. Co-expression assays indicated that bHLH IVc TFs (bHLH34, bHLH104, bHLH105, and bHLH115) facilitate the nuclear accumulation of bHLH11. Further analysis indicated that bHLH11 represses the transactivity of bHLH IVc TFs toward bHLH Ib genes (bHLH38, bHLH39, bHLH100, and bHLH101). The two ethylene response factor-associated amphiphilic repression motifs of bHLH11 provided the repression function by recruiting the TOPLESS/TOPLESS-RELATED (TPL/TPRs) corepressors. Correspondingly, the expression of Fe uptake genes increased in the tpr1 tpr4 tpl mutant. Moreover, genetic analysis revealed that bHLH11 has functions independent of FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR. This study provides insights into the complicated Fe homeostasis signaling network.

bHLH121 Functions as a Direct Link that Facilitates the Activation of FIT by bHLH IVc Transcription Factors for Maintaining Fe Homeostasis in Arabidopsis.

Iron (Fe) deficiency is prevalent in plants grown in neutral or alkaline soil. Plants have evolved sophisticated mechanisms that regulate Fe homeostasis, ensuring survival. In Arabidopsis, FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) is a crucial regulator of Fe-deficiency response. FIT is activated indirectly by basic helix-loop-helix (bHLH) IVc transcription factors (TFs) under Fe deficiency; however, it remains unclear which protein(s) act as the linker to mediate the activation of FIT by bHLH IVc TFs. In this study, we characterize the functions of bHLH121 and demonstrate that it directly associates with the FIT promoter. We found that loss-of-function mutations of bHLH121 cause severe Fe-deficiency symptoms, reduced Fe accumulation, and disrupted expression of genes associated with Fe homeostasis. Genetic analysis showed that FIT is epistatic to bHLH121 and FIT overexpression partially rescues the bhlh121 mutant. Further investigations revealed that bHLH IVc TFs interact with and promote nuclear accumulation of bHLH121. We demonstrated that bHLH121 has DNA-binding activity and can bind the promoters of the FIT and bHLH Ib genes, but we did not find that it has either direct transcriptional activation or repression activity toward these genes. Meanwhile, we found that bHLH121 functions downstream of and is a direct target of bHLH IVc TFs, and its expression is induced by Fe deficiency in a bHLH IVc-dependent manner. Taken together, these results establish that bHLH121 functions together with bHLH IVc TFs to positively regulate the expression of FIT and thus plays a pivotal role in maintaining Fe homeostasis in Arabidopsis.

WRKY2/34-VQ20 Modules in Arabidopsis thaliana Negatively Regulate Expression of a Trio of Related MYB Transcription Factors During Pollen Development.

Male gametogenesis in plants is tightly controlled and involves the complex and precise regulation of transcriptional reprogramming. Interactions between WRKY proteins and VQ motif-containing proteins are required to control these complicated transcriptional networks. However, our understanding of the mechanisms by which these complexes affect downstream gene expression is quite limited. In this study, we found that WRKY2 and WKRY34 repress MYB97, MYB101, and MYB120 expression during male gametogenesis. MYB expression was up-regulated in the wrky2-1 wrky34-1 vq20-1 triple mutant during male gametogenesis. The expression levels of six potential targets of the three MYBs increased the most in the wrky2-1 wrky34-1 vq20-1 triple mutant, followed by the wrky2-1 wrky34-1 double mutant, compared with in wild-type. Yeast one-hybrid and dual luciferase reporter assays indicated that WRKY2 and WRKY34 recognized the MYB97 promoter by binding to its W-boxes. MYB97 overexpression caused defects in pollen germination and pollen tube length, which impacted male fertility. Thus, WRKY2/34-VQ20 complexes appear to negatively regulate the expression of certain MYBs during plant male gametogenesis.

Arabidopsis WRKY2 and WRKY34 transcription factors interact with VQ20 protein to modulate pollen development and function.

Plant male gametogenesis is tightly regulated, and involves complex and precise regulations of transcriptional reprogramming. WRKY transcription factors have been demonstrated to play critical roles in plant development and stress responses. Several members of this family physically interact with VQ motif-containing proteins (VQ proteins) to mediate a plethora of programs in Arabidopsis; however, the involvement of WRKY-VQ complexes in plant male gametogenesis remains largely unknown. In this study, we found that WRKY2 and WKRY34 interact with VQ20 both in vitro and in vivo. Further experiments displayed that the conserved VQ motif of VQ20 is responsible for their physical interactions. The VQ20 protein localizes in the nucleus and specifically expresses in pollens. Phenotypic analysis showed that WRKY2, WRKY34 and VQ20 are crucial for pollen development and function. Mutations of WRKY2, WRKY34 and VQ20 simultaneously resulted in male sterility, with defects in pollen development, germination and tube growth. Further investigation revealed that VQ20 affects the transcriptional functions of its interacting WRKY partners. Complementation evidence supported that the VQ motif of VQ20 is essential for pollen development, as a mutant form of VQ20 in which LVQK residues in the VQ motif were replaced by EDLE did not rescue the phenotype of the w2-1 w34-1 vq20-1 triple-mutant plants. Further expression analysis indicated that WRKY2, WRKY34 and VQ20 co-modulate multiple genes involved in pollen development, germination and tube growth. Taken together, our study provides evidence that VQ20 acts as a key partner of WRKY2 and WKRY34 in plant male gametogenesis.