Regulatory Mechanisms of bHLH Transcription Factors in Plant Adaptive Responses to Various Abiotic Stresses

1Basic helix-loop-helix proteins (bHLHs), are one of the largest transcription factor (TF) families. They are widely distributed in plants, fungi, and animals. In, the first plant bHLH TF was observed in maize. Since then, a great many bHLHs have been proven to regulate plant responses to various abiotic stresses. bHLHs are involved in regulating the synthesis of flavonoids, which play important roles in the ROS homeostasis under these stresses. As an illustration of the size of the bHLHs family in various plant species, 164 bHLH TFs have been found in Arabidopsis (Arabidopsis thaliana L.), while there are 180 in rice (Oryza sativa L.), 190 in tobacco (Nicotiana tabacum L.), 191 in grapes (Vitis vinifera L.), 102 in walnut (Juglans regia L.), 85 in Ginkgo biloba, 268 in Brassica oleracea, 440 in Brassica napus, and 251 Brassica rapa.

2TFs, also known as trans-acting factors, are a category of proteins that specifically bind to cis-acting elements in the promoter region of eukaryotic genes. They regulate specific physiological or biochemical processes in cells at the transcriptional level. The protein structure of TFs generally contains four functional domains: a DNA-binding domain, a transcriptional regulation domain, an oligomerization site, and a nuclear localization domain. The transcriptional regulatory domain may include both an activation domain and an inhibitory domain.

3The bHLH transcription factor conservatively contains two connected sub-regions, the N-terminal basic region directly followed by the HLH (helix-loop-helix) domain. More than 50% of bHLHs that have been found in plants possess a highly conserved HER motif (His5-Glu9-Arg13) to achieve DNA binding and regulate the transcription of their target genes. The HLH region is composed of 40∼50 amino acid residues, which is required for the formation of dimers.

4The animal bHLHs were first categorized into six subfamilies (A∼F) by based on characteristics of the HLH and other conversed domains. The phylogenetic tree of plant bHLH was first constructed in Arabidopsis thaliana. The bHLH family of Arabidopsis was divided into 12 subfamilies. Later, these subfamilies were expanded into 32 subgroups by phylogenetic analysis based on the 638 bHLH genes extracted from Arabidopsis, Populus trichocarpa, Oryza sativa, Physcomitrella patens, and five algae species. Evolutionary and functional relationships within subfamilies are supported by intron patterns, predicted DNA-binding motifs, and the architecture of conserved protein motifs. Some subfamilies may modulate biological responses critically for the development of terrestrial plants.

5As one of the largest TF families, bHLHs regulate the expression of downstream target genes usually in a binary or ternary complex form (Figure 1). The ternary MBW complex has been analyzed extensively in various biological processes. The bHLH proteins of the IIIf subgroup (TT8, GL3, EGL3, and AtMYC1) can interact with R2R3-MYBs from various subgroups (TT2, PAP1, or PAP2), and form ternary complexes with a WD-repeat protein (TTG1). The MYB5-TT8-TTG1 complex is activated in the endothelium to regulate DFR (dihydroflavonol reductase), LDOX (leucoanthocyanidin dioxygenase), and TT12 expression, whereas the TT2-EGL3/GL3-TTG1 complexes regulate the expression of LDOX, BAN (BANYULS; anthocyanidin reductase), AHA10 (autoinhibited H+-ATPase isoform 10), and DFR in the chalaza. Regulatory complexes composed of MYB10, bHLH3, and WD40 may control the biosynthesis of anthocyanins in peaches. BoPAP2 (MYB), BoTT8, BoEGL3.1, BoMYC1.2 (bHLH), and BoTTG1 (WD40) have been identified as candidate genes for regulating anthocyanin biosynthetic activity in cabbage.

6There are also many studies that address the activation mechanism of the MYB-bHLH binary complex. The C-terminal domain of many bHLHs have been found to contain a MYB-binding site. Through binding to this site, MYB could heterodimerize with bHLHs. For example, VvMYC1 in grapes cannot activate the promoters of CHI, UFGT, and ANR genes in the flavonoid biosynthetic pathway by itself, but the co-transfection of VvMYC1 with a MYB transcription factor can significantly activate the expression of the genes for these three enzymes. Rosea1 (ROS1, MYB-type) and Delila (DEL, bHLH-type) from Snapdragon (Antirrhinum majus) can specifically induce anthocyanin accumulation when co-expressed in tomato fruits. found that CmbHLH2 significantly upregulated CmDFR transcriptional expression and triggered anthocyanin accumulation when co-expressed with CmMYB6. Using transient assays in both Nicotiana tabacum leaves or Actinidia arguta fruits and stable transformation in Arabidopsis, demonstrated that co-expression of AcMYB123 and AcbHLH42 is a prerequisite for anthocyanin production by activating transcription of AcF3GT1 and AcANS or their homologous genes. found that AabHLH1 interacted with AaMYB3 to regulate the accumulation of procyanidine. In addition, DhMYB2 interacted with DhbHLH1 to regulate anthocyanin production in Dendrobium hybrid petals. As well, the bHLH transcription factor PPLS1 interacts with SiMYB85 to control the color of leaf sheath and pulvinus by regulating anthocyanin biosynthesis in Setaria italica.

7The transcriptional activities of bHLHs can also be regulated through dimerization, and a series of post-translational modifications including phosphorylation and ubiquitination (Figure 1; ). The Arabidopsis bHLH transcription factor FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) displays a pivotal role in the Fe-deficiency response, and its activity is regulated by heterodimerizing with bHLH039. Interestingly, this heterodimerization capacity is affected by differential phosphorylation. Specifically, the heterodimerization is activated through Ser and deactivated through Tyr site phosphorylation. Another Arabidopsis bHLH transcription factor, SPEECHLESS (SPCH), initiates the stomatal lineage. Recent research revealed that the protein stability of SPCH could be positively regulated by phosphorylation mediated via the MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascade or GSK3-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) protein phosphatase 2A phosphatase. Chenopodium glaucum CgbHLH001 can positively regulate plant stress tolerance via clearing excessive ROS and accumulating transcripts of stress-related genes. A recent study indicates that CgbHLH001 activity might be affected by phosphorylation through interaction with CgCDPK. Ubiquitination can likewise directly regulate bHLH activity via the 26S proteasome protein degradation pathway (Figure 1). The BTB-BACK-TAZ domain protein MdBT2 in apple can target MdCIbHLH1 for ubiquitination, so as to regulate malate accumulation and vacuolar acidification. Pyrus pyrifolia CONSTITUTIVE PHOTOMORPHOGENIC1 (PpCOP1) could ubiquitinate PpbHLH64 and trigger its proteolysis, further reducing the accumulation of anthocyanins. bHLH activity can also be indirectly regulated by ubiquitination through the degradation of its partners in the MBW complex. The Arabidopsis thaliana MYB Interaction Factor 1 (AtMIF1) is a member of the ubiquitin-protein ligase E3 complex involved in the 26S proteasome protein degradation pathway. AtMIF1 ubiquitinates and degrades AtMYB5, so that transcriptional activation of the MYB/bHLH/WD-repeat (MBW) complex further increases oil content by attenuating GL2 inhibition.

8The latest reports reveal an emerging regulatory mechanism of MBW transcriptional activation by antagonistic interactions with MBW components (Figure 1). MYB paralogs or ROS-related proteins could disrupt the activated MBW complex by competitively interacting with bHLHs. In Medicago truncatula, two R2R3 MYB paralogs, RED HEART1 (RH1) and RH2, play vital roles in patterned pigmentation. These two antagonistic paralogous proteins competitively bind the MtTT8-MtWD40-1 complex to fine tune the phenotypes of leaf anthocyanin spot marking. In Zea mays, the ROS-related protein ZmSRO1e (TO RCD-ONEs) interacts with ZmPL1 (MYB)/AtPAP1 (bHLH) to inhibit the formation of an activated MBW complex, thereby repressing the over-accumulation of anthocyanins under abiotic stress. These two reports provide a multidimensional antagonistic regulatory paradigm for MBW activation. It’s reasonable to speculate that bHLH paralogs or other stress/growth-related proteins might also regulate the MBW activity by competitively interacting with MYB in the complex.

9The bHLH TFs can form homologous or heterologous dimers and bind to target genes on specific cis-acting elements. Among them, the most frequently studied cis-acting elements are the E-box (5′-CANNTG-3′) and the N-box [5′-CACG(A/C)G-3′]. bHLHs DNA binding domains containing at least five basic amino acids in the N-terminal basic region are expected to bind these DNA motifs.

10The E-box is one of the most common targets of the bHLH subfamilies 1 and 27. According to three-dimensional structural analysis of bHLHs, Glu-13 and Arg-16 are essential for E-box-binding recognition. This cis-acting element is usually located in the promoter of genes involved in multiple physiological pathways. found that the yew bHLH TcJAMYC can bind to the E-box in the promotor of paclitaxel-related genes to activate their transcriptional expression. demonstrated that the Poncirus trifoliata PtrbHLH can bind to an E-box in the promoter region of the POD gene. showed that the rice OsbHLH062 can bind to an E-box in the promoter of ion transport genes such as OsHAK21. found that PnbHLH1 can interact with the E-box core sequence in Panax notoginseng, a traditional Chinese medicine, to induce the triterpenoid saponins. experimentally showed that CsbHLH18 bound to the promoter of the sweet orange (Citrus sinensis) CsPOD gene through the E-box. Recent studies have found that GhbHLH18 strongly binds to the E-box in the promoter region of the GhPER8 gene coding for a peroxidase from cotton. Finally, demonstrated through DNA-protein interactions both in vitro and in vivo that the bHLH transcription factor MYC2 can bind to the cis-acting E-box element in the HY5 promoter to negatively regulate HY5 expression.

11A specific member of the E-box family, the G-box (5′-CACGTG-3′) could be recognized by about 81% of bHLHs mainly from subfamilies 4, 5, 10, 11, 13, 14, 24, 25, and 26. confirmed the specific interaction between the pea bHLH transcription factor PsGBF and the G-box of PsCHS1 by transient expression experiments in tobacco. In Arabidopsis, the bHLH transcription factor PIL5 has a high affinity with the G-box as well. Arabidopsis bHLH106 was characterized through its association with salt tolerance. Transcriptional analysis of an AtbHLH106 overexpression line showed 198 genes positively were regulated and 36 genes were negatively regulated; these genes possessed one or more G-box sequences in their promoter regions, and many of them are associated with the abiotic stress response. isolated CrMYC2 from a Catharanthus roseus cDNA library and found that its corresponding protein could specifically bind to G-box of the ORCA3 gene. In addition, tobacco NtMYC2 and NtbHLH123 bind directly to the G-box region of the PMT2 and CBF, respectively, and activated their transcription.Besides, Tamarix hispida ThbHLH1 specifically binds to the G-box of P5CS and the ALDH gene. The apple MdMYC2 homodimer also binds to the G-box motif of the AtJAZ3 gene. Moreover, the Hevea brasiliensis bHLHs HbMYC2, HbMYC3, and HbMYC4 of interacted with the G-box of the HbPIP2 promotor.

12The N-box is associated with bHLHs possessing an enhancer activity. bHLHs that bind to the N-box usually have a proline in the N-terminal basic domain and a “WRPW” sequence at its C-terminus. OsPIF14 binds to the OsDREB1B promoter on two N-boxes [CACG(A/C)G]. Diterpenoid phytoalexin factor (DPF) is a rice bHLH transcription factor which positively regulates CPSCPS2 transcription through N-box binding. DPF can also regulate CYP99A2 through the N-box, thereby affecting the biosynthesis of diterpenoid phytoalexins (DP). In addition, some bHLHs can bind to other cis-acting elements, for example, AtbHLH112 can not only bind the E-box, but also bind the GCG-box [5′-GG(G/T)CC(G/T)(GA)(TA)C-3′].

13Heterodimers can be formed between different bHLHs. discovered that two bHLH TFs, PIF3/AtbHLH008 and HFR1, could heterodimerize. This interaction effectively prevented monomer PIF3/AtbHLH008 from binding to the E-box of phytochrome A (phyA), thereby modulating phyA signaling. In addition, bHLHs can heterodimerize with other TFs, such as R2R3-MYB/BZR1-BES1 as well as other signal transduction proteins. The formation of heterodimers makes it possible for different TFs to interact with each other to co-regulate the expression of target genes.

14In summary, bHLHs bind directly to cis-elements in the promoter of target genes or form heterodimers to co-regulate their expression. To date, E-box cis-acting elements, in particular, the G-box of this family, are the widest binding targets of bHLHs (Table 1). We found that most bHLHs in subfamilies 1, 2, 12, 13, 15, 19, 24, 25, 26, 27 can bind to the G-box; and members in subfamilies 1, 2, 13, 15, 22, 26, 27 can recognize other cis-elements in the E-box family, except for the G-box; members of subgroup 15 could also bind to the GCG-box.

15Previous studies found that bHLHs were involved in a variety of pathways regulating the adaptation to stress in plants, including resistance to mechanical damage, drought, high salt, oxidative stress, low temperature stresses, heavy metal stress, iron deficiency, and osmotic stress. One of the most common responses of plants to stress is to enhance the biosynthesis of different types of compatible organic solutes. In general, this mechanism protects plants from stresses in multiple different ways through regulating cell osmotic homeostasis, eliminating excessive ROS, maintaining the integrity of the plasma membrane, and stabilizing enzymes and structural proteins.

16To uncover the potential roles of bHLHs from different subfamilies in various stress responses, most of the functionally annotated bHLH TFs were categorized into the abovementioned 32 subfamilies (Figure 2 and Table 2). The response function of different bHLH subfamilies to abiotic stress is different. We found that nearly half of the bHLHs subfamilies participated in abiotic stress responses. Among them, there are nine subfamilies of bHLHs involved in drought response, including subfamilies 1, 2, 4, 5, 13, 15, 24, 26, and 30, in which most members are from subfamilies 1, 2, and 15. As well, there are nine subfamilies of bHLHs participating in salinity resistance, including subfamilies 1, 2, 4, 5, 7, 13, 15, 26, and 27. The majority of salinity responsive bHLHs are from subfamilies 1 and 4. In addition, five subfamilies were found to regulate the cold tolerance of plants, including 1, 2, 13, 15, and 26. Interestingly, members in subfamily 1 were found to be able to take over the regulation of chilling responses. Furthermore, bHLHs of subfamilies 1, 3, 4, 12, 28, and 31 are involved in the response to iron deficiency. In short, bHLHs of subfamily 1 seem to participate in most kinds of abiotic stresses, such as drought, salinity, chilling and iron deficiency. Moreover, members of subfamily 2 mainly participate in drought, salt, and cold stress, while that of subfamily 4 mainly regulates responses to drought, salt, and iron deficiency stress.

17The effects of drought stress on plants are mainly manifested in decreased photosynthesis efficiency, disordered hormone metabolism, and reduced enzyme activities, causing irreversible damage to plant growth and yield. The bHLH TFs regulate the plant drought-tolerant responses mainly through modulation of the sensitivity to Abscisic acid (ABA) or by regulating the development of stomata, leaf trichome, and root hair. found that the expression of the bHLH-type transcription factor AtAIB was temporarily induced by ABA, and that plants overexpressing AtAIB showed a stronger drought tolerance. found that AtbHLH68 may play a role in responding to drought stress, likely through an ABA-dependent pathway by either directly or indirectly regulating components of ABA signaling and/or metabolism. demonstrated that heterologous expression of tartary buckwheat FtbHLH3 in Arabidopsis could positively regulate drought/oxidative stress tolerance in an ABA dependent manner. reported that a large number of bHLHs are involved in ABA signaling and positively regulated stress resistance in Lycium ruthenicum.

18In Arabidopsis, AtbHLH006/rd22BP1/AtMYC2 can regulate the expression of RD22. Interestingly, AtbHLH006 can bind to different cis-acting elements under different conditions. AtbHLH006 binds to the E-box (but not G-box) on the PLETHORA gene promoter in JA mediated regulation of the Arabidopsis root stem cell niche; it could also bind to the G-box motif of the SAG29 promoter to activate its expression during JA mediated leaf senescence. Another bHLH transcription factor, ZmPTF1 in maize, regulates drought tolerance by promoting root development and the synthesis of ABA. found that plants overexpressing Populus euphratica PebHLH35 had significantly lower stomatal density and decreased stomatal opening than wild-type plants, so their transpiration was significantly reduced. illustrated that Apple MdbHLH130 acts as a positive regulator of water stress response by regulating stomatal closure and ROS-scavenging in tobacco.

19High salt stress caused by ionic and osmotic stressors eventually results in the suppression of plant growth and a reduction in crop productivity. Some bHLH TFs have a regulatory effect under saline conditions. Under salt stress, plant cells successively face the challenges of osmotic stress, ion toxicity, and oxidative stress. As discussed previously, bHLH TFs usually enhance a plant’s resistances to these secondary stresses. AtNIG1 in Arabidopsis was the first bHLH transcription factor shown to be involved salt stress signaling pathways from plants. The survival rate and dry weight of the atnig1 mutant decreased significantly under salt stress. showed that the expression level of the wheat (Triticum aestivum L.) TabHLH13 gene was significantly up-regulated with the increase of salt ion concentration in the environment. showed that OsbHLH062 could regulate ion transport genes, such as OsHAK21, and modulate the Jasmonic acid (JA) signaling pathway, so as to endow plants with some resistance to salt stress. One of the most important mechanisms by which bHLH TFs participate in salt tolerance is to regulate ROS balance by directly regulating the expression of a group of peroxidase genes. found that AtbHLH92 elevated Arabidopsis tolerance to salt and osmotic stresses through a partial dependence on ABA and SOS2. The beet homolog of AtbHLH92, BvbHLH92, was found to be expressed in both the beet root and leaf in response to salt stress. In Tamarix hispida, ThbHLH1 was highly expressed under high salt induction, significantly increasing peroxidase (POD) and superoxide dismutase (SOD) activities. AtbHLH112 increased the expression of the POD and SOD genes, while concomitantly reduced the expression of the P5CDH and ProDH genes, so as to enhance the resistance of Arabidopsis to high salt. In addition, bHLHs could also regulate the accumulations of resistance-related secondary metabolites in order to improve plant salinity tolerance. found that salt stress activated AtMYC2 through a mitogen-activated protein kinase (MAPK) cascade. Then, the AtMYC2 could bind to the promotor of rate-limiting enzyme P5CS1 in the biosynthesis of proline, thereby regulating the biosynthesis of proline, and thus regulating salt tolerance. AtbHLH122 inhibited the expression of CYP707A3 gene under NaCl stress. Furthermore, identified AtMYC2 and AtbHLH122 as upstream regulators of the ABA-mediated AtNHX1 and AtNHX6, which are both Na+/H+ exchangers, by chromatin immunoprecipitation. Moreover, overexpression of EcbHLH57 can enhance the resistance of tobacco to salt stress by elevating the expression of stress responsive genes such as LEA14, rd29A, rd29B, SOD, APX, ADH1, HSP70, and also PP2C. OsbHLH068 has a similar effect as AtbHLH112 in regulating the salt stress response. In Arabidopsis, heterogenic overexpression of OsbHLH068 can reduce salt-induced accumulation of H2O2.

20Cold stress is a severe threat for plant productivity and crop production, particularly so when it occurs during the growth phase. The DREB1/CBF (dehydration-responsive element binding/C-repeat binding factors) gene is considered as the main regulator of plant’s cold stress response. Under low temperature stress, OsDREBL and OsTPP1 genes were significantly up-regulated at the transcriptional level after overexpression of RsICE1 (inducer of CBF expression) in rice, indicating that RsICE1 is involved in the DREB1/CBF cold regulation signaling network. In Arabidopsis the AtICE1/AtbHLH116 protein binds to the CBF promoter region at low temperatures to affect transcription initiation, and AtICE1/AtbHLH116 overexpressing plants showed higher tolerance to cold. The bHLH transcription factor DlICE1 from Dimocarpus longan Lour has a positive regulatory effect on cold tolerance. Overexpression of DlICE1 in Arabidopsis conferred enhanced cold tolerance via increased proline content, decreased ion leakage, and reduced malondialdehyde (MDA) and reactive oxygen species (ROS) accumulation. The expressions of the ICE1-CBF cold signaling pathway genes, including AtCBF1/2/3 and cold-responsive genes (AtRD29A, AtCOR15A, AtCOR47, and AtKIN1), were also significantly higher in DlICE1-overexpressing lines than in wild-type (WT) plants under cold stress. ICE1 proteins in other plants have similar functions. PuICE1 of can elevate the transcriptional expression of PuDREBa by interacting with PuHHP1, thus improving the cold resistance of Pyrus ussuriensis. The overexpression of BcICE1 in tobacco can positively regulate the expression of stress-related genes such as CBFs (C-repeat binding factor) and enhance the antioxidant activity and osmotic ability of plants. showed that the transgenic Arabidopsis with overexpressed Zoysia japonica ZjICE1 showed an enhanced tolerance to cold stress with an increase in SOD, POD, as well as higher free proline content and decreased MDA content. They also upregulated the transcript abundance of cold-responsive genes (CBF1, CBF2, CBF3, COR47A, KIN1, and RD29A). ZjICE2 from Zoysia japonica enhanced the tolerance of transgenic plants to cold stress by activating DREB/CBF regulators and enhancing reactive oxygen species scavenging. In addition to ICE proteins, some bHLH proteins are also responsible for plant resistance to low temperatures. As a homolog of ICE1, the rice OrbHLH001 could enhance the tolerance of transgenic Arabidopsis to freezing stress. However, the function of OrbHLH001 was different from that of ICE1 and is independent of a CBF/DREB1 cold-response pathway. The grape VvbHLH1 and VabHLH1 are positive regulators of the cold stress response, and the overexpression of these two genes could enhance the expression level of the COR gene. The apple (Malus domestica Borkh.) MdCIbHLH1 gene was recently identified as a hub in the transcriptional regulation of bark freezing tolerance. Moreover, demonstrated that NtbHLH123 is a transcriptional activator that plays a positive regulatory role in cold tolerance by activating the reactive oxygen species scavenging-related gene NtCBF. found that CsbHLH18 can regulate ROS homeostasis at least partially by directly regulating the antioxidant gene CsPOD, thus playing an active role in cold tolerance.

21Excessive heavy metals in agricultural lands cause declines in crop productivity. found that excessive expression of GmbHLH30 in tobacco can enhance its resistance to aluminum toxicity through the maintenance of osmotic pressure. Manganese (Mn) toxicity is also an important factor for limiting crop production in acidic soils. found that ZmbHLH105 may improve maize tolerance to Mn stress by regulating antioxidant mechanism-mediated ROS clearance and the expression of Mn/Fe-related transporters in plants.

22The key bHLH transcription factor in iron (Fe) uptake, FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), is critical for adjusting Fe acquisition to plant growth and environmental constraints. found that the bHLH133 transcription factor in rice can regulate the transport of Fe from roots to young leaves, revealing the important role of bHLH proteins in maintaining iron homeostasis in plant cells. found that the bHLH transcription factor FER from tomato played a role in the regulation of plant root iron nutrition. found that the FIT/bHLH29 (FER-like iron deficiency-induced transcription factor) gene played an important role in maintaining intracellular Fe balance through regulating the expression of downstream iron absorption genes. The plant hormone ethylene is one of the signals that trigger iron deficiency responses at both the transcriptional level and the post-transcriptional level. Through ethylene signal transduction, the FIT/bHLH29 protein is protected from degradation by proteasomes. Increased FIT levels subsequently leads to the high level of expression of genes required for Fe acquisition. found that the bHLH18, bHLH19, bHLH20, and bHLH25 in the Iva subgroup are FIT/bHLH29 interactors, which can promote JA induced FIT/bHLH29 protein degradation. Previously,, demonstrated that four Ib bHLH genes (AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101) played important roles in the iron-deficiency responses, though they are not induced by FIT/bHLH29 under iron deficiency conditions. Actually, these four Iva bHLHs mainly antagonized Ib bHLHs, so as to regulate the stability of the FIT/bHLH29 protein under iron deficiency conditions. Furthermore, found that bHLH121 interplayed with another bHLH transcription factor in the Ivc subgroup to positively regulate FIT/bHLH29 expression, thus playing a key role in maintaining Fe homeostasis in Arabidopsis. identified the Arabidopsis AtbHLH104 as a member in subfamily Ivc and demonstrated that bHLH104 acted as a key component positively regulating Fe deficiency responses via targeting Ib subgroup bHLH genes and PYE/bHLH47 expression. supplemented this study and proposed that bHLH34, bHLH104, and bHLH105 (IAA-LEUCINE RESISTANT3) can be used as homologous dimers or heterodimers to regulate the stable state of Fe without redundancy. ILR3/bHLH105, alone was shown to stimulate Fe uptake by inhibiting ferritin expression. showed through chromatin immunoprecipitation-on-chip analysis and transcriptional profiling that PYE/bHLH47 helped maintain iron homeostasis by regulating the expression of known iron homeostasis genes. In addition, AtbHLH115 is also a positive regulator of iron deficiency responses, being negatively regulated by the E3 ligase BTS. In poplar, transgenic line (TL2) overexpressing PtFIT showed a higher chlorophyll content and Chl a/b ratio than the control plant under the conditions of iron deficiency, indicating that PtFIT was involved in the iron deficiency reaction. found that overexpression of OsIRO2/OsbHLH056 could promote the absorption of iron in rice. demonstrated that Chrysanthemum CmbHLH1 promoted iron absorption through H+-ATPase mediated rhizosphere acidification. Recent research demonstrated that the overexpression of GmbHLH57 and GmbHLH300 up-regulated the iron absorption genes and increased the iron content of transgenic soybean plants. Moreover, overexpression of NtbHLH1 results in longer roots, altered rhizosphere pH, and increased ferric-chelate reductase activity under iron deficient conditions. characterized the role of a novel rice bHLH type transcription factor OsbHLH156 in Fe homeostasis and found that OsbHLH156 is mainly expressed in roots and transcription is greatly increased by iron deficiency. Loss of function of OsbHLH156 resulted in Fe-deficiency-induced chlorosis and reduced Fe concentration in the shoots under upland or Fe(III) supplied conditions.

23Plants must maintain the homeostasis of Fe and Cu. When plants are under Fe-deficiency conditions, bHLH IVC not only directly activates bHLH Ib expression but also promotes bHLH Ib and FIT gene expression through interaction with bHLH121. FIT and bHLH Ib members initiate transcription of Fe-uptake genes (IRT1 and FRO2) and Cu-uptake genes (COPT2, FRO4, and FRO5). An increase in Cu concentration alleviates Fe-deficiency stress. Under Cu-deficiency, Cu-uptake genes (COPT2, FRO4, and FRO5) are activated in response to SPL7. CITF1 also regulates the expression of COPT2, FRO4, and FRO5. SPL7 not only regulated the Cu homeostasis signaling pathway, but also suppressed (IPON MANs) IMAS and bHLH Ib of the Fe homeostasis signaling pathway.

24Proper osmoregulation is important for plant response to environmental changes. Drought, high salinity, low temperature, and other conditions will affect the water content of plants, thus leading to osmotic stress. Therefore, osmotic stress is often accompanied by other types of environmental stresses. Under osmotic stress, RD29A can be regulated through both ABA-independent and ABA-dependent pathways, thus improving plant stress resistance. found that the expression level of RD29A was upregulated in transgenic plants overexpressing TabHLH39. The overexpression of the wild rice gene OrbHLH2 (a homolog protein of ICE1) in Arabidopsis up-regulates the expression of stress response genes DREB1A/CBF3, RD29A, COR15A, and KIN1, thus enhancing tolerance to osmotic stress. A MYC-type ICE1-like transcription factor SlICE1a in tomatoes was induced in response to osmotic stress.

25In vegetative tissues, the flavonoid pathway is usually induced in response to physiological and environmental fluctuations as a protective mechanism against oxidative stresses induced by pathogen infections, high-light, UV, extreme temperature, drought, salt, and deficiency of N, P, or C nutrition. The bHLH transcription factor family is important for regulating the biosynthetic pathway of flavonoids. Flavonoids are generally divided into six categories: flavone, flavonol, isoflavone, flavanone, flavanol, and anthocyanidin. In the Lc protein was initially isolated from maize and showed transcriptional activity on genes involved in anthocyanin synthesis. Heterologous overexpression of the Lc gene significantly enhanced the accumulation of flavonoids in mature fruits of cherry tomato. Since then, the vital roles of bHLH in flavonoid synthesis have attracted growing attention. Flavonoids have strong biological activity and significant antioxidant capacity, and they play protective roles when plants are subjected to single or multiple stresses such as ultraviolet radiation, salt, temperature, and drought. The level of protection is determined by the position of free hydroxyl groups in the structure of flavonoids and the carbon-carbon double bond in the C ring.

26To reveal the relationship between bHLH subfamilies and flavonoid biosynthesis, all the functional annotated bHLHs were categorized into the 32 known subgroups in this research (Figure 2 and Table 3). It seems that only bHLHs in subfamilies 1, 2, 5, 13, 14, 15 are involved in the regulation of flavonoid metabolism (Table 3). Among them, more than half of the annotated bHLHs involved in flavonoid metabolism regulation come from subfamilies 2 and 5. As flavonoids are important metabolites involved in plant responses to abiotic stresses, bHLHs of subfamilies 2 and 5 may regulate a series of physiological stress responses. The typical bHLH transcription factor in the IIIf subfamily [equivalent to the subfamily 5] AtTT8/AtbHLH042 can regulate the expression of DFR and BAN, two flavonoid late biosynthetic genes, thus affecting the synthesis of anthocyanins and procyanidins. AtTT8/AtbHLH042 constitutes a major regulatory step in the specific activation of the expression of flavonoid structural genes. Similarly, MtTT8 in Medicago truncatula is considered to be the central component of a ternary complex (MYB-bHLH-WD40) that controls the biosynthesis of anthocyanins and procyanidins. In addition to AtTT8, three other bHLH genes are also categorized into the IIIf clade: AtGL3, AtEGL3, and AtMYC1. They are also known to be involved in biosynthesis of flavonoids. Together with MYB factors, especially PAP2 (AtMYB90), GL3 seems to be a partner of bHLHs in anthocyanin accumulation under nitrogen-deficient conditions. In contrast, nightshade GLABRA3 (SlGL3), the homolog to AtGL3, inhibited anthocyanin accumulation in Arabidopsis.

27Homologous or heterologous overexpression of bHLHs can exhibit a significant regulatory effect on the synthesis of various flavonoids. Actually, most bHLH have been found to possess a positive regulatory function on plant flavonoid synthesis. In Arabidopsis, transgenic plants overexpressing AtMYC3 and AtMYC4 showed higher levels of anthocyanin than that of wild-type plants. Heterologous overexpression of the grape VvbHLH1 gene increases the activity of key enzymes in the flavonoid synthesis pathway such as Phenylalanine Ammonia-Lyase (PAL), Chalcone Synthase (CHS), Chalcone Isomerase (CHI), and Flavanone-3-Hydroxylase (F3H) as well as enhancing the salt and drought tolerance of transgenic Arabidopsis seedlings (2016a). VvbHLH003 and VvbHLH007 were also found to be related to anthocyanin or flavonoids synthesis. Likewise, the heterologous expression of eggplant (Solanum melongena) SmbHLH13 can promote anthocyanin biosynthesis by positively regulating the expression of the structural genes SmCHS and SmF3H. The heterologous expression of Arabidopsis PAP2 induces anthocyanin accumulation in tomato, while the heterogeneous overexpression of liverwort (Plagiochasma appendiculatum) PabHLH1 in Arabidopsis can activate the synthesis of both flavonoids and anthocyanins, through the up-regulation of early and late structural genes in the synthesis pathway of flavonoids. The ectopic overexpression of apple MdMYC2 also significantly up-regulated the transcriptional expression of these structural genes in transgenic Arabidopsis lines. Recent studies have shown that both triticale (Triticum × Secale) TsMYC2 and wheat (Triticum aestivum) TaMYC1 can regulate anthocyanin biosynthesis and control the grain properties. On the other hand, several bHLHs could negatively regulate flavonoid synthesis. For example, the wintersweet (Chimonanthus praecox L.) CpbHLH13 was found to reduce the anthocyanin content when ectopically overexpressed in the tobacco inflorescence.

28It is generally believed that the mechanism of action of flavonoids in the stress-resistance process is through their antioxidant properties. Under abiotic stresses, such as low temperature, drought, high salt, or heavy metal exposure, a series of physiological and biochemical changes will occur in plant cells, which are mainly manifested as the reduction of photosynthesis efficiency and the generation of a large number of reactive oxygen radicals, as well as serious damage to cell structure. The oxidative stress generated by these reactive oxygen species can also induce a large amount of flavonoids synthesis, thereby quenching the reactive oxygen species and protecting cells from oxidative damage. found that abiotic stress promotes the synthesis of anthocyanins by inhibiting SPL9 through miR156, thereby facilitating the neutralization of reactive oxygen species (ROS) and simultaneously inducing ZmSRO1e. ZmSRO1e interacts with ZmPL1/AtPAP1 to inhibit the formation of an activated MBW complex, thus repressing the over-accumulation of anthocyanins under abiotic stress. These two pathways balance the relationship between development and abiotic stress tolerance via their control of ROS accumulation. Overexpression of the Antirrhinum majus bHLH gene AmDEL led to the up-regulation of genes related to the biosynthesis of flavonoids, proline biosynthesis, and reactive oxygen scavenging under both salt and drought stress. Moreover, flavonoids can also bind with copper irons to reduce the toxic damage of these ions to cytoplasmic structures and organelles such as chloroplasts. The role of flavonoids in plant stress tolerance is not only limited to the removal of reactive oxygen species, but also can act as signaling molecules.

29As one of the most numerous TFs in eukaryotes, the bHLH family has many members and diverse functions. Many studies have shown that bHLHs can regulate plant resistances to various abiotic stresses. As well, there are many reports that show that flavonoids in plants are synthesized in large quantities to effectively eliminate ROS, to enhance the plant’s tolerances to survive in an adverse environment. This naturally leads to an interesting question: Can bHLHs regulate plant tolerance by regulating the synthesis of flavonoids? A large number of studies have confirmed that bHLHs are involved in the synthesis of flavonoids. Specially, bHLHs in subfamily 1, 2, 13, and 15 could bind to G-box or E-box in the promoter of cold, drought, and salinity responsive genes (Tables 1, 2); members of this subfamily also could modulate the synthesis of some flavonoids (Table 3). Since members of this group share similarly conversed protein motifs (Supplementary Figure 1), it is reasonable to hypothesize that plant bHLHs in subfamily 1, 2, 13, and 15 could bind to G-box or E-box of cold, drought and salt responsive genes to further regulate the synthesis of flavonoids. Similarly, it also makes sense that bHLHs in subfamily 5 could regulate the synthesis of flavonoids to resist salinity and drought stresses (Tables 1-3). However, these hypotheses still need to be verified.

30YQ, TZ, YY, JY, JX, and LG analyzed the phylogenetic relationships of bHLH family members. EP conceived the original idea for the review. All authors wrote the manuscript.

31The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.