Ironing out the details: Fe homeostasis in the shoot and root is regulated by distinct actions of BTS/BTSL1/2 and bHLH IVc subgroup transcription factors.

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.

Iron (Fe) homeostasis is critical for plant growth, development, and stress responses. Fe levels are tightly controlled by intricate regulatory networks in which transcription factors (TFs) play a central role. A series of basic helix-loop-helix (bHLH) TFs have been shown to contribute to Fe homeostasis, but the regulatory layers beyond bHLH TFs remain largely unclear. Here, we demonstrate that the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) TF SlSPL-CNR negatively regulates Fe-deficiency responses in tomato (Solanum lycopersicum) roots. Fe deficiency rapidly repressed the expression of SlSPL-CNR, and Fe deficiency responses were intensified in two clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-generated SlSPL-CNR knock-out lines compared to the wild-type. Comparative transcriptome analysis identified 47 Fe deficiency-responsive genes the expression of which is negatively regulated by SlSPL-CNR, one of which, SlbHLH101, helps regulate Fe uptake genes. SlSPL-CNR localizes the nucleus and interacts with the GTAC and BOX 4 (ATTAAT) motifs in the SlbHLH101 promoter to repress its expression. Inhibition of SlSPL-CNR expression in response to Fe deficiency was well correlated with the expression of the microRNA SlymiR157. SlymiR157-overexpressing tomato lines displayed enhanced Fe deficiency responses, as did SlSPL-CNR loss-of-function mutants. We propose that the SlymiR157-SlSPL-CNR module represents a novel pathway that acts upstream of SlbHLH101 to regulate Fe homeostasis in tomato roots.

Iron (Fe) is an essential element for plant life. Its deficiency impedes growth and development and excessive iron can cause the toxic effect via the Fenton reaction. Thus, plants have developed various mechanisms to acquire, distribute and utilize Fe for the maintenance of their iron homeostasis at cellular and systemic levels. A basic helix-loop-helix (bHLH) transcription factor family plays essential roles in many regulatory and development processes in plants. In this study, we aimed to understand the roles of bHLH38, bHLH39, bHLH100 and bHLH101 genes for Fe homeostasis in Arabidopsis, tomato, rice, soybean and maize species by using bioinformatics approaches. The gene/protein sequence analyses of these genes demonstrated that all bHLH proteins comprised helix-loop-helix DNA binding domain (PF00010) with varied exon numbers between 2 and 13. The phylogenetic analysis did not reveal a clear distinction between monocot and dicot plants. A total of 61 cis-elements were found in promotor sequences, including biotic and abiotic stress responsiveness, hormone responsiveness, and tissue specific expressions. The some structural divergences were identified in predicted 3D structures of bHLH proteins with different channels numbers. The co-expression network analysis demonstrated that bHLH39 and bHLH101 played more important roles in Fe regulation in Arabidopsis. The digital expression analysis showed various expression profiles of bHLH genes which were identified in developmental stages, anatomical parts, and perturbations. Particularly, bHLH39 and bHLH101 genes were found to be more active genes in Fe homeostasis. As a result, our findings can contribute to understanding of bHLH38, bHLH39, bHLH100 and bHLH101 genes in Fe homeostasis in plants.

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) homeostasis in plants is governed by a complex network of regulatory elements and transcription factors (TFs), as both Fe toxicity and deficiency negatively impact plant growth and physiology. The Fe homeostasis network is well characterized in Arabidopsis thaliana and remains poorly understood in monocotyledon species such as rice (Oryza sativa L.). Recent investigation of the rice Fe homeostasis network revealed OsIRO3, a basic Helix–Loop–Helix (bHLH) TF as a putative negative regulator of genes involved in Fe uptake, transport, and storage. We employed CRISPR-Cas9 gene editing to target the OsIRO3 coding sequence and generate two independent T-DNA-free, loss-of-function iro3 mutants in rice cv. Nipponbare. The iro3 mutant plants had similar phenotype under nutrient-sufficient conditions and had stunted growth under Fe-deficient conditions, relative to a T-DNA free, wild-type control (WT). Under Fe deficiency, iro3 mutant shoots had reduced expression of Fe chelator biosynthetic genes (OsNAS1, OsNAS2, and OsNAAT1) and upregulated expression of an Fe transporter gene (OsYSL15), relative to WT shoots. We place our results in the context of the existing literature and generate a model describing the role of OsIRO3 in rice Fe homeostasis and reinforce the essential function of OsIRO3 in the rice Fe deficiency response.

Iron (Fe) homeostasis in plant cells is crucial for crop productivity and quality. An intricate transcriptional network involving numerous basic Helix-Loop-Helix (bHLH) transcription factors has been proposed to control Fe homeostasis. In the present study, we characterized rice (Oryza sativa) OsbHLH062, a member of the IVb subgroup of the bHLH family, demonstrating that it negatively regulates Fe-deficiency responses. OsbHLH062 represses transcription by recruiting TOPLESS/TOPLESS-RELATED co-repressors (TPL/TPRs) through its ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif. Under Fe deficiency, the expression of OsbHLH062 is upregulated in roots and downregulated in shoots. Overexpression of OsbHLH062 leads to decreased Fe accumulation in the shoot. Furthermore, OsbHLH062 interacts with POSITIVE REGULATOR OF IRON HOMEOSTASIS 1 (OsPRI1) and inhibits its transactivation activity, thereby negatively regulating the expression of many Fe homeostasis-related genes. These results indicate an important role for OsbHLH062 in regulating Fe homeostasis by negatively regulating Fe deficiency responses in rice. This knowledge will aid in the design of Fe-biofortified rice plants that can help to address the global issue of Fe deficiency.

Inhibitor of DNA binding (Id) proteins bind to and inhibit the function of basic helix-loop-helix (bHLH) transcription factors including those that regulate pancreatic development. Moreover, bone morphogenetic proteins (BMPs) regulate the expression of Ids. We hypothesized that BMP4 and Id proteins play a role in the expansion and differentiation of epithelial progenitor cells. We demonstrate that BMP4 induces the expression of Id2 along with the expansion of AR42J pancreatic epithelial cells. Furthermore, neutralization of BMP4 significantly reduced duct epithelial cell expansion in a mouse model of islet regeneration. BMP4 stimulation promotes Id2 binding to the bHLH transcription factor NeuroD, which is required for the differentiation of pancreatic islet cells. Therefore, our results indicate that BMP4 stimulation blocks the differentiation of endocrine progenitor cells and instead promotes their expansion thereby revealing a novel paradigm of signaling explaining the balance between expansion and differentiation of pancreatic duct epithelial progenitors. Understanding the mechanisms of BMP and Id function elucidates a key step during pancreas embryogenesis, which is important knowledge for expanding pancreatic progenitors in vitro.

Iron (Fe) deficiency, a worldwide agricultural problem on calcareous soil with low Fe availability, is also a major human nutritional deficit. Plants induce Fe acquisition systems under conditions of low Fe availability. Previously, we reported that an Fe-deficiency-inducible basic helix-loop-helix (bHLH) transcription factor, OsIRO2, is responsible for regulation of the genes involved in Fe homeostasis in rice. Using promoter-GUS transformants, we showed that OsIRO2 is expressed throughout a plant's lifetime in a spatially and temporally similar manner to the genes OsNAS1, OsNAS2 and TOM1, which is involved in Fe absorption and translocation. During germination, OsIRO2 expression was detected in embryos. OsIRO2 expression in vegetative tissues was restricted almost exclusively to vascular bundles of roots and leaves, and to the root exodermis under Fe-sufficient conditions, and expanded to all tissues of roots and leaves in response to Fe deficiency. OsIRO2 expression was also detected in flowers and developing seeds. Plants overexpressing OsIRO2 grew better, and OsIRO2-repressed plants showed poor growth compared to non-transformant rice after germination. OsIRO2 overexpression also resulted in improved tolerance to low Fe availability in calcareous soil. In addition to increased Fe content in shoots, the overexpression plants accumulated higher amounts of Fe in seeds than non-transformants when grown on calcareous soil. These results suggest that OsIRO2 is synchronously expressed with genes involved in Fe homeostasis, and performs a crucial function in regulation not only of Fe uptake from soil but also Fe transport during germination and Fe translocation to grain during seed maturation.

Class A basic Helix-Loop-Helix transcription factors in early stages of chick neural tube development: evidence for functional redundancy

The basic-helix-loop-helix (bHLH) transcription factors Ascl1/Mash1, Hes1, and Olig2 regulate the fate choice of neurons, astrocytes, and oligodendrocytes, respectively; however, these factors are coexpressed in self-renewing multipotent neural stem cells (NSCs) even before cell fate determination. This fact raises the possibility that these fate determination factors are differentially expressed between self-renewing and differentiating NSCs with unique expression dynamics. Real-time imaging analysis utilizing fluorescent proteins is a powerful strategy for monitoring expression dynamics. Fusion with fluorescent reporters makes it possible to analyze the dynamic behavior of specific proteins in living cells. However, it is technically challenging to conduct long-term imaging of proteins, particularly those with low expression levels, because a high-sensitivity and low-noise imaging system is required, and very often bleaching of fluorescent proteins and cell toxicity by prolonged laser exposure are problematic. Furthermore, to analyze the functional roles of the dynamic expression of cellular proteins, it is essential to image reporter fusion proteins that are expressed at comparable levels to their endogenous expression. In this review, we introduce our recent reports about the dynamic control of bHLH transcription factors in multipotency and fate choice of NSCs, focusing on real-time imaging of fluorescent reporters fused with bHLH transcription factors. Our imaging results indicate that bHLH transcription factors are expressed in an oscillatory manner by NSCs, and that one of them becomes dominant during fate choice. We propose that the multipotent state of NSCs correlates with the oscillatory expression of several bHLH transcription factors, whereas the differentiated state correlates with the sustained expression of a single bHLH transcription factor.

Transcription factors of the basic helix–loop–helix (bHLH) class are involved in the determination of precursor cells towards neural (proneural) and other cell fates. The proneural members of this class constitute a particular subset of bHLH proteins that are arranged in a cascade that affects different stages of neuronal commitment and differentiation, and act in a combinatorial code along with other transcription factors to specify neuronal identity. Proneural proteins themselves are a target of regulation by ID and HES proteins among others, and are involved in setting up a negative feedback pathway of lateral inhibition involving NOTCH and DELTA signalling. In addition, the activities of bHLHs are also subject to control by posttranslational modification and degradation. Finally, recent analyses have begun to identify the direct downstream targets of bHLH genes in the nervous system, and this is shedding light on the mechanisms ensuring specificity of the individual bHLH proteins. Key concepts: bHLH proneural transcription factors have multiple direct downstream transcriptional targets including other transcription factors, signal transducers and cytoskeletal modifiers. bHLH proneural transcription factors act in a combinatorial code to confer neuronal subtype identity. bHLH transcription factors are regulated posttranslationally by mechanisms including phosphorylation and ubiquitin‐mediated proteolysis. bHLH proneural transcription factors have multiple downstream targets that orchestrate neural differentiation. Negative regulators of bHLH proneural proteins such as HES and ID proteins act by dimerizing with and sequestering E protein partners. Lateral inhibition mediated by the NOTCH – DELTA pathway limits the number of neurons that differentiate from the pool of neuronal precursors.

BackgroundBasic helix-loop-helix (bHLH) transcription factors (TFs), which are widely distributed in eukaryotic organisms, play crucial roles in plant development. However, no comprehensive analysis of the bHLH family in wheat (Triticum aestivum L.) has been undertaken previously.ResultsIn this study, 225 bHLH TFs predicted from wheat using genomic and RNA sequencing data were subjected to identification, classification, phylogenetic reconstruction, conserved motif characterization, chromosomal distribution determination and expression pattern analysis. One basic region, two helix regions and one loop region were found to be conserved in wheat bHLH TFs. The bHLH proteins could be separated into four categories based on sequences in their basic regions. Neighbor-joining-based phylogenetic analysis of conserved bHLH domains from wheat, Arabidopsis and rice identified 26 subfamilies of bHLH TFs, of which 23 were found in wheat. A total of 82 wheat bHLH genes had orthologs in Arabidopsis (27 TFs), rice (28 TFs) and both of them (27 TFs). Seven tissue-specific bHLH TF clusters were identified according to their expression patterns in endosperm, aleurone, seedlings, heading-stage spikes, flag leaves, shoots and roots. Expression levels of six endosperm-specifically expressed TFs measured by qPCR and RNA-seq showed a good correlation.ConclusionThe 225 bHLH transcription factors identified from wheat could be classed into 23 subfamilies, and those members from the same subfamily with similar sequence motifs generally have similar expression patterns.

Many basic Helix-Loop-Helix (bHLH) transcription factors precisely regulate the expression of Fe uptake and translocation genes to control iron (Fe) homeostasis, as both Fe deficiency and toxicity impair plant growth and development. In rice, three clade IVc bHLH transcription factors have been characterised as positively regulating Fe-deficiency response genes. However, the function of OsbHLH057, another clade IVc bHLH transcription factor, in regulating Fe homeostasis is unknown. Here, we report that OsbHLH057 is involved in regulating Fe homeostasis in rice. OsbHLH057 was highly expressed in the leaf blades and lowly expressed in the roots; it was mainly expressed in the stele and highly expressed in the lateral roots. In addition, OsbHLH057 was slightly induced by Fe deficiency in the shoots on the first day but was not affected by Fe availability in the roots. OsbHLH057 localised in the nucleus exhibited transcriptional activation activity. Under Fe-sufficient conditions, OsbHLH057 knockout or overexpression lines increased or decreased the shoot Fe concentration and the expression of several Fe homeostasis-related genes, respectively. Under Fe-deficient conditions, plants with an OsbHLH057 mutation showed susceptibility to Fe deficiency and accumulated lower Fe concentrations in the shoot compared with the wild type. Unexpectedly, the OsbHLH057-overexpressing lines had reduced tolerance to Fe deficiency. These results indicate that OsbHLH057 plays a positive role in regulating Fe homeostasis, at least under Fe-sufficient conditions.

ABSTRACTThe basic helix–loop–helix (bHLH) transcription factors (TFs) are essential in development and disease. Their function is regulated at multiple levels, including the structuring of homo‐ or heterodimeric forms among members of the family. Because most bHLH TFs have numerous dimerization partners, the commonly used overexpression or deletion experimental approaches in humans often generate results influenced by the altered regulatory balance of the TF network. To study the direct transcriptional role of two human bHLH TFs, we expressed them in an isolated system (yeast) with no additional tissue‐specific bHLH TFs. The transcriptional effect was measured utilizing a GFP reporter controlled by human regulatory sequences containing different amounts of the bHLH TF consensus binding sites, the E‐boxes. The individual transcriptional contributions of heterodimeric SCX‐E47 or homodimeric E47 were compared over two human regulatory regions implicated in fibrosis: COL1A2 and TGFB1. Briefly, the heterodimeric SCX‐E47 was the best activating form. The COL1A2 regulatory region showed the most significant transcriptional changes despite having fewer E‐boxes (five) than the TGFB1 region (13). Finally, the context of the nearby TF binding sites and the core promoter was also relevant for the final individual transcriptional effect of the bHLH TFs tested.

Plants maintain iron (Fe) homeostasis under varying environmental conditions by balancing processes such as Fe uptake, transportand storage. In Arabidopsis, POPEYE (PYE), a basic helix-loop-helix transcription factor (TF), has been shown to play a crucial role in regulating this balance. In recent years, the mechanisms regulating Fe uptake have been well established but the upstream transcriptional regulators of Fe transport and storage are still poorly understood. In this study, we report that ELONGATED HYPOCOTYL5 (HY5), a basic leucine zipper (bZIP) TF which has recently been shown to play a crucial role in Fe homeostasis, interacts with PYE. Molecular, geneticand biochemical approaches revealed that PYE and HY5 have overlapping as well as some distinct roles in the regulation of Fe deficiency response. We found that HY5 and PYE both act as a repressor of Fe transport genes such as YSL3, FRD3, NPF5.9, YSL2, NAS4and OPT3. HY5 was found to directly bind on the promoter of these genes and regulate intercellular Fe transport. Further analysis revealed that HY5 and PYE directly interact at the same region on PYE and NAS4 promoter. Overall, this study revealed that HY5 regulates Fe homeostasis by physically interacting with PYE as well as independently.