# The arabidopsis bHLH transcription factor family

Fei Gao, Christian Dubos

► To cite this version:

Fei Gao, Christian Dubos. The arabidopsis bHLH transcription factor family. Trends in Plant Science, 2024, 29 (6), pp.668-680. 10.1016/j.tplants.2023.11.022. hal-04837469

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1 **The Arabidopsis bHLH transcription factor family**

2

3 Fei Gao¹,², Christian Dubos³

4

5 ¹ College of Agronomy, Hunan Agricultural University, Changsha 410128, China

6 ² Yuelushan laboratory, Changsha 410128, China

7 ³ IPSiM, Univ. Montpellier, CNRS, INRAE, Institut Agro, Montpellier, France

8 Correspondence: Gao F (gaofei@hunau.edu.cn); Dubos C (christian.dubos@inrae.fr).

9

10 **Keywords:** basic Helix-Loop-Helix, heat, iron, root hair, shade, stomata

11

12 **Abstract**

13 bHLHs are present in all eukaryotes and form one of the largest family of transcription
14 factors (TFs) found in plants. bHLHs function as transcriptional activators and/or
15 repressors of genes involved in key processes for plant growth and development in
16 interaction with the environment (e.g. stomata and root hair development, iron
17 homeostasis, response to heat and shade). Recent studies have improved our
18 understanding of bHLH TFs functioning in complex regulatory network where a series
19 of post-translational mechanisms play critical roles in regulating their subcellular
20 localization, DNA-binding capacity, transcriptional activity and/or stability (e.g.
21 protein-protein interaction, phosphorylation, ubiquitination, sumoylation). Further
22 elucidating the function and regulation of bHLHs will provide knowledge for the
23 biology of plants in general and new tools for crop improvement.
24 The plant bHLH transcription factor family

25

26 In 1989, the first plant bHLH (basic Helix-Loop-Helix) transcription factor (TF),
27 Lc, was identified in maize (Zea mays) as the causal gene of the R loci whose function
28 in anthocyanin biosynthesis as remained elusive for several decades until it was cloned
29 following transposon tagging experiments and DNA library screenings [1]. Within the
30 past three decades, bHLH TFs from several plant species were identified and
31 characterized, highlighting the preponderant role played by this family of TFs in
32 regulating various biological processes. It also emerged that bHLHs form one of the
33 largest family of TFs found in plants. For instance, 183, 231, 571 and 602 bHLHs were
34 identified in rice (Oryza sativa), maize, wheat (Triticum aestivum) and rapeseed
35 (Brassica napus) genomes, respectively [2, 3].

36

37 Typical bHLHs are composed of a highly conserved bHLH (basic Helix-Loop-
38 Helix) domain of about 60 amino acids that consists of two functionally distinct regions:
39 a basic region (b) at the N-terminal side and a Helix-Loop-Helix (HLH) region at the
40 C-terminal side [4]. The basic region is composed of 13-17 amino acids (with a high
41 proportion of basic amino acids such as arginine and lysine) and is involved in the
42 binding to DNA [5]. The HLH region is usually composed of two amphipathic α-helices
43 separated by a variable loop. The HLH region mediates the formation of homo- and/or
44 heterodimers between bHLH proteins through hydrophobic contacts between the
45 helices [4, 6]. HLH domain dimerization allows specific binding of the basic region to
46 DNA cis-regulatory sequences (Box 1).

47

48 Beside the bHLH domain, the overall consensus amino acid sequence between
49 bHLH proteins is poorly conserved. This is partly because of the presence of additional
50 domains necessary to modulate their activity and/or DNA-binding specificity such as
51 the leucine-zipper motif, the MYB-interacting region (MIR) or the aspartate kinase,
52 chorismate mutase, and TyrA-like (ATC-like) domain (Box 1) [7, 8].

53

54 In 2003, 133 bHLH family members were reported in *Arabidopsis thaliana* and
55 divided into 12 major groups (I to XII) and subdivided into 25 subfamilies [8]. Seven
56 years after this first classification, 15 major bHLH groups (I to XV) subdivided into 26
57 subfamilies were described, thanks to the accurate identification of bHLH TFs within
58 different plant genomes [6]. For this purpose, 155 bHLH sequences from Arabidopsis
59 together with 389 sequences from rice and more ancestral plants (including algae) were
60 analysed [6]. If small differences were observed between these two classifications, it is
61 noteworthy that this second study allowed the identification of three new subgroups (i.e.
62 XIII to XV; Table S1). This is now 171 bHLHs that were identified in Arabidopsis and
63 categorized into 17 major groups (I to XVII) and 31 subfamilies following the
64 nomenclature model proposed by [6] and [8] (Figure 1; Table S1).

65

66 Biological role of plant bHLH transcription factors

67

68 To date, for more than 72% (124 of 171) of the Arabidopsis bHLH TFs, loss-of-
69 function mutants were characterized (Table S1). Such studies highlighted that these
70 bHLH TFs are regulating many key biological processes such as plant development,
71 cell fate determination, plant nutrition, specialized metabolism, and the response to
72 abiotic and biotic stresses (Table S1). Herein, the aim is not to compile the tremendous
73 amount of data that was accumulated on the biological roles played by these bHLH TFs.
74 It is instead to highlight, through few examples, how recent studies have improved our understanding of bHLH TFs functioning in complex regulatory network and in regulating key processes for plant growth and development in interaction with the environment.

78
79 Cell differentiation allows acquiring specialized functions necessary for optimal growth and development. Cell differentiation into guard cells in the aerial tissues or into root hairs belowground is of central importance to plants for up taking water and nutrients.

80
81
82 The differentiation of stomatal cells in Arabidopsis involves three closely related bHLH members of the Ia subfamily that sequentially drive the initiation, the proliferation and the differentiation of stomata by, notably, modulating the expression of genes involved in the cell cycle machinery [9, 10] (Figure 2A). SPEECHLESS (SPCH/bHLH98) promotes the meristemoid mother cell formation and initiates stomatal lineage by driving the first asymmetric cell division of these cells to generate meristemoids [11, 12]. MUTE (bHLH45) is required for the transition of the meristemoid into guard mother cell and for driving terminal symmetric cell division of the guard mother cell to generate guard cells [13-15]. Last, FAMA (bHLH97) and the R2R3-MYB FOUR LIPS (FLP), whose expressions are directly induced by MUTE, complete the final differentiation of guard cells [15-18]. The transition time between SPCH and MUTE is a key parameter that explains, at least in part, the diversified patterns of stomatal development observed in various plant species since it affects the meristemoid cell behaviour and thus stomatal development and the overall leaf epidermal morphology [19]. The activity of SPCH, MUTE and FAMA necessitates their heterodimerization with two closely related bHLHs from the IIIe subfamily, INDUCER OF CBF
99 EXPRESSION 1 and 2 (ICE1/SCREAM/bHLH116 and ICE2/SCREAM2/bHLH33)
100 [20-22] (Figure 2A). In addition, the interaction of ICEs with FLP also promotes the
101 meristemoid to guard mother cell transition [23]. Interestingly, the high degree of
102 conservation for this bHLH-dependent regulatory module, from bryophytes to dicots,
103 provides strong evidences for a common origin of stomata development in land plants
104 [24].

105 The first bHLH TFs involved in root hair specification were characterized in
106 Arabidopsis about two decades ago and named GLABROUS 3 (GL3/bHLH1) and
107 ENHANCER OF GLABROUS 3 (EGL3/bHLH2), two bHLH from the IIIf subfamily
108 [25]. Another member of this subfamily, MYC1 (bHLH12), is also involved in the early
109 stage of root hair specification. Since then, this is altogether 12 bHLH TFs, from three
110 distinct subfamilies, that were found to regulate, in a spatio-temporal manner, root hair
111 cell development, from root epidermal cell patterning to root hair elongation. For
112 instance, root hair initiation and development involves the combined activity of six
113 VIIIc bHLH subfamily members [26, 27] (Figure 2B). ROOT HAIR DEFECTIVE 6
114 (RHD6/bHLH83) together with ARABIDOPSIS THALIANA RHD 6-LIKE 1
115 (RSL1/bHLH86) are required for the early stages of root-hair cell differentiation [28-
116 30]. RSL2 (bHLH85) and RSL4 (bHLH54), act downstream of RHD6 and RSL1, and
117 control root hair cell growth and size [31-34]. RSL3 (bHLH84) and RSL5 (bHLH139)
118 are also positive regulators of root hair growth and development [26]. Root hair
119 elongation involves the redundant functions of three members of the bHLH XI
120 subfamily, namely Lj-RHL1-LIKE1 (LRL1/bHLH66), LRL2 (bHLH69) and LRL3
121 (bHLH82) [35-37]. This relatively high number of bHLH TFs involved in the control
122 of root hair development (about 7% of the total number of bHLH TFs present in
123 Arabidopsis) might reflects the necessity for the plant to ensure proper root hair
124 development to maintain optimal hydro-mineral nutrition.

125

126 Iron is an important micronutrient for plant growth and development since it is a
127 cofactor for several metalloproteins involved in numerous physiological processes (e.g.
128 respiration, photosynthesis). Iron homeostasis is also under the tight control of bHLH
129 TFs. This is for instance about 10% of the total number of bHLH TFs present in
130 Arabidopsis (18 genes from 7 subfamilies) that are involved in the regulation of iron
131 homeostasis [38, 39] (Figure 3). These bHLH TFs function in an intricate regulatory
132 cascade, as homo- and heterodimers, to modulate the expression of genes involves in
133 iron uptake, transport, assimilation and storage. This bHLH network is composed of
134 two interconnected regulatory modules. In Arabidopsis, the first module is centred on
135 FER-LIKE IRON DEFICIENCY INDUCED TRANSCRIPTION FACTOR
136 (FIT/bHLH29; subfamily IIIa) that is the functional homolog of the tomato (Solanum
137 lycopersicum) FER, the first TF identified as directly involved in the regulation of iron
138 homeostasis in plants [40]. FIT is a direct positive regulator of iron uptake genes. FIT
139 interacts with four bHLH members of the Ib subfamily (i.e. bHLH38, bHLH39,
140 bHLH100 and bHLH101) forming heterodimers with partially overlapping functions.

141 These interactions are antagonized by the interaction of FIT with MYC1 that acts as a
142 negative regulator of the iron deficiency responses [41]. The interaction of FIT with Ib
143 bHLHs is also antagonized by its interaction with the four bHLH members of the IVa
144 subfamily (i.e. bHLH18, bHLH19, bHLH20 and bHLH25), which promotes the
145 degradation of FIT via the 26S proteasome [42]. MYC2/bHLH6 (IIIe subfamily) is also
146 involved in this first module as it indirectly regulates the expression of *bHLH18* and
147 *bHLH25* (activation) as well as *bHLH19*, *bHLH20* and *FIT* (repression) [42].

148 When iron availability is low, the second module activates the expression of both *bHLH*
Ib (direct) and *FIT*. Whether the regulation of *FIT* expression by this module is direct or not is still a matter of debate [43-46]. It involves the four bHLH members of the IVc subfamily, namely IAA-LEUCINE RESISTANT 3 (ILR3/bHLH105), IRON DEFICIENCY TOLERANT 1 (IDT1/bHLH34), bHLH104 and bHLH115, together with UPSTREAM REGULATOR OF IRT1 (URI/bHLH121; IVb subfamily) [43-46].

To achieve their function, these five bHLH TFs interact in the form of homo- and heterodimers. In contrast, the interaction of bHLH11 with the four IVc bHLH leads to the inhibition of both FIT function (indirect) and *bHLH Ib* expression (direct) [47].

POPEYE (PYE/bHLH47) is a transcriptional repressor of genes involves in iron storage whose function relies on its interaction with ILR3 [48]. *PYE* expression is also repressed by the PYE-ILR3 heterodimer in a negative feedback regulatory loop [48]. It is noteworthy that in this network, ILR3 acts as both a transcriptional activator of iron uptake and a repressor of iron storage.

As a whole, the function of such complex regulatory network might prevent plants from iron deficiency when its availability is low and iron overload, and thus toxicity, when its availability is high.

Heat stress is one of the major environmental factors associated with the predicted global climate change that threatens agriculture since it negatively impacts plant growth and crop yield.

Recently, PHYTOCHROME-INTERACTING FACTOR 4 (PIF4/bHLH9, VII subfamily), a bHLH TF initially known for its role in the light-responsive development of Arabidopsis seedlings has emerged as a key regulator of different thermoresponsive signalling pathways [49]. For instance, morphological acclimation responses to temperature elevation, also known as thermomorphogenesis, rely on the activity of
174 PIF4 whose expression is induced when plants are exposed to heat stress. It includes
175 the promotion of hypocotyl elongation [50, 51], leaf hyponasty [52] and flowering time
176 [53], and the restriction of leaf size [54]. Stomatal development, and therefore leaf
177 transpiration, is also a PIF4-dependent thermoresponsive process in which PIF4
178 directly regulate the expression of SPCH [55, 56].

179 Plant response to heat stress involves as well other PIF than PIF4. For instance, it was
180 recently reported that PIF7 (bHLH72, VII subfamily) also plays a major role in
181 thermomorphogenesis and that the promotion of leaf senescence under heat stress relies
182 on the activity of both PIF4 and its homolog PIF5 (bHLH65, VII subfamily) [57-59].

183 How these PIFs act synergistically to regulate thermomorphogenesis is still an open
184 question. Answering it might offer an opportunity to generate climate-smart plants to
185 sustain crop productivity in the context of global climate change [60, 61].

186

187 **Post-translational regulation of plant bHLH transcription factors**
188

188

189 Post-translational modifications allow modulating in an effective manner the
190 activity of TFs and therefore the expression of their target genes. Among the post-
191 translational modifications known to modulate the activity of plant bHLH TFs, the
192 interaction between TFs as well as the phosphorylation, the ubiquitination and the
193 sumoylation are the most studied. Such post-translational modifications modulate
194 bHLH protein activity by modifying their stability, subcellular localization or DNA
195 binding specificity (Figure 4).

196

197 bHLH TFs homo- or heterodimerization through their bHLH domains might be
198 considered as the primary post-translational modification to modulate bHLH proteins
199 activity. For instance, as we have seen earlier, within the transcriptional network that
200 regulate iron homeostasis, bHLH from the IVc bHLH family can form homo- and
201 heterodimers to regulate the expression of their target genes, including FIT (Figure 4A).
202 The heterodimerization of FIT with IVa bHLH leads to its degradation whereas its
203 interaction with Ib bHLHs is necessary to activate the expression of its target genes [38,
204 39, 42]. The later interactions are also necessary to translocate Ib bHLHs from the
205 cytosol to the nucleus [62-64]. The regulation of cell proliferation in meristems is a
206 well-documented developmental process that necessitates also the combined action of
207 bHLH TFs in the form of heterodimers [65] (Figure 4B). It is noteworthy that some
208 atypical bHLHs, referred as (b)HLHs, have been discovered and characterized in
209 Arabidopsis. They showed significant divergence in the basic region and usually lacked
210 the key sequence features for proper DNA binding [4]. These (b)HLHs, together with
211 typical bHLHs, form the antagonistic (b)HLH/bHLH system that plays critical roles in
212 regulating diverse facets of plant development [66] (Figure 4C, Box 2).
213 bHLH TFs also interact with other type of regulatory proteins to modulate the
214 expression of their target genes. The interaction of FIT with gibberellin signalling
215 DELLA repressors is a well-described mechanism that modulates bHLH activity as a
216 function of iron availability (Figure 4D) [67, 68]. Another well-described example in
217 plants is the formation of MBW ternary protein complexes composed of bHLH and
218 R2R3-MYB TFs together with WD40 repeat proteins. Such complexes were first
219 identified in maize and shown to regulate flavonoid biosynthesis (i.e. antocyanins and
220 proanthocyanidins) (Figure 4E) [69-71]. In Arabidopsis, the function of these
221 complexes extent to the regulation of the differentiation of trichomes and root hair cells
222 as well as the biosynthesis of the seed coat mucilage [72]. The bHLH TFs involved in
223 the MBW complexes are from the IIIf subfamily (e.g. R and B from maize; GL3, EGL3,
224 MYC1 and TRANSPARENT TESTA 8: TT8/bHLH42 from Arabidopsis) and interact
225 with their R2R3-MYB partners through their MYB-interacting region (MIR) present in
226 their N-terminal part [70, 72].

227 About 30% of flowering plants' bHLHs possess an aspartate kinase, chorismate mutase,
228 and TyrA (ACT)-like domain that participates in homodimer formation [73, 74]. In
229 maize, R dimerization via the ACT-domain allows recruiting R-INTERACTING
230 FACTOR 1 (RIF1) at the bHLH sites, inhibiting the binding affinity of R to E-boxes
231 because of the absence of dimerized HLH domains. This ACT-dependent dimerization
232 enables the binding of the C1-R-RIF1 complex to MYB cis-regulatory sequences, and
233 thus the activation of the expression of a set of genes involved in anthocyanin
234 biosynthesis [75, 76]. In contrast, R dimerization via the bHLH domain permits C1-R
235 complex tethering to E-box elements and activates the expression of another set of
236 anthocyanin biosynthetic genes [75, 76]. If such regulatory mechanism explains how
237 genes with different cis-regulatory sequences can be co-ordinately regulated by the
238 same set of bHLHs, the role of the WD40 repeat protein (i.e. PALE ALEURONE 1,
239 PAC1) in this regulatory mechanism remains to be elucidated. In Arabidopsis, ICE1's
240 also possess an ACT-like domain that is necessary for its heterodimerization with
241 MUTE and therefore for the transition from meristemoids to guard mother cells (Figure
242 2A) [20]. Similarly, in DYSFUNCTIONAL TAPETUM 1 (DYT1/bHLH22), the ACT-
243 domain participates in dimerization with other bHLH TFs, as well as in modulating its
244 nuclear localization and transcriptional activity, and thus the development of anthers
245 [77, 78].

246

247 Phosphorylation is an important post-translational modification modulating bHLH
248 proteins activity through different mechanism. Among them, phosphorylation was
249 shown to modulate bHLH activity by modifying their subcellular localization, and in
250 particular their accumulation into the nucleus. For instance, within the iron homeostasis
251 regulatory network, the phosphorylation of FIT by the CBL-INTERACTING
252 PROTEIN KINASE 11 (CIPK11) promotes its nuclear accumulation, thereby
253 activating the expression of iron uptake genes [79] (Figure 4F). Conversely, the
254 phosphorylation of FLOWERING BHLH 4 (FBH4/bHLH130) by the cellular fuel
255 sensor SNF1-RELATED KINASE 1 (SnRK1) limits FBH4 nuclear accumulation
256 therefore inhibiting the expression of genes involved in the flowering signalling
257 pathway [80].

258 Phosphorylation was also shown to affect bHLH protein stability. This is for instance
259 the case during the guard cell differentiation process where the phosphorylation states,
260 and thus the stability, of SPCH is fine-tuned by the balanced activity of different kinases
261 and phosphatases. For instance, the activity of both the CYCLIN-DEPENDENT
262 KINASE A;1 (CDKA;1) and the PROTEIN PHOSPHATASE 2A (PP2A) promotes
263 SPCH stability and thus stomatal development [81]. Conversely, the stability of SPCH
264 interacting partner ICE1 is negatively regulated upon its phosphorylation by the kinases
265 MITOGEN-ACTIVATED PROTEIN KINASE 3 and 6 (MPK3 and MPK6) [82].

266 Phosphorylation might also affect the ability of bHLH TFs to bind their target DNA
267 sequences, as it is the case for URI. URI is a master regulator of the iron homeostasis
268 regulatory network, whose phosphorylation enhances its DNA binding ability under
269 iron deficiency conditions to promote iron uptake [38, 46].

270

271 Ubiquitination is an important mechanism to regulate the stability, and thus the
272 amount and activity, of bHLH TFs. The transcriptional regulation of iron homeostasis
273 best exemplifies the importance of ubiquitination for the fine-tuning of physiological
and developmental processes. Within this network, a set of hemerythrin E3-ubiquitin ligases target key bHLH TFs, leading to their degradation via the 26S proteasome pathway (Figure 4G). In Arabidopsis, BRUTUS (BTS) targets ILR3, bHLH115 and URI whereas BRUTUS LIKE 1 and 2 (BTSL1 and BTSL2) target FIT [46, 83-85]. The degradation of these bHLH TFs hampers plants from iron overload, and thus toxicity. Conversely, the binding of iron on the hemerythrin domains of the E3-ubiquitin ligases leads to their degradation via the 26S proteasome pathway, preventing plants from iron deficiency. Such regulatory loop allowing adapting iron uptake to plant needs.

Sumoylation is another important mechanism to modulate bHLH TFs activities [86]. For example, the transcriptional activity of PIF3 (bHLH8, VII subfamily) is reduced upon its sumoylation since it reduces PIF3 DNA binding affinity to its target gene promoters [87]. Conversely, sumoylation of MYC2 inhibits its interaction with the ubiquitin E3 ligase PLANT U-BOX 10 (PUB10), thereby increasing its accumulation and therefore enhancing its function in blue light signalling [88] (Figure 4H).

Concluding remarks and future perspectives

Since the characterization of the first bHLH gene in maize in 1989 [1], the use of Arabidopsis as model plant species highlighted that bHLH proteins play key roles in complex transcriptional regulatory network controlling several facets of plant growth and development in interaction with the environment (Table S1).

It has also emerged that the activity of the bHLH TFs is tightly controlled at the
299 post-translational level notably via the deposition of specific modification marks (i.e. 300 phosphorylation, ubiquitination and sumoylation). Nevertheless, our knowledge on the 301 post-translational network that modulates the activity of bHLH TFs through these 302 modifications is still fragmented and efforts will be required to identify all the actors 303 involved (e.g. ubiquitin ligases, kinases, phosphatases, proteases). This will necessitate 304 as a prerequisite identifying the post-translational changes associated with each of the 305 bHLH TFs. 306

306
307 Dimerization of bHLH TFs plays critical roles in determining their functions
308 whether this is with typical bHLH or atypical (b)HLH proteins. Nevertheless, we still
309 have a partial understanding of the plant bHLH interaction code and several potential
310 paths might be followed to explore further this important question.

311 One path would be to study at the cell specific level the formation of bHLH homo- and 312 heterodimers. For instance, recent work suggests that cell-type-specific dimerization 313 could provide bHLH TFs peculiar functions, which could partially explain the 314 specificity and functional redundancy of phylogenetically related bHLH TFs from a 315 given subfamily [65]. Therefore, it is of outmost importance to identify *in planta*, at the 316 cell specific level, the formed bHLH homo- and hetero-dimers (for e.g. using *in planta* 317 FRET-FLIM method). Combining such strategy with single cell genomic approaches 318 (e.g. scRNA-seq, scATAC-seq and scChIP-seq) would be valuable to confirm the 319 redundant and distinct roles played by the bHLH proteins.

320 Another path would be to study further the role of (b)HLH. To date, the (b)HLH/bHLH 321 antagonistic system was shown to play a key role in the regulation of cell elongation in 322 response to shade. However, whether these (b)HLHs are involved in the regulation of 323 other signalling pathways is still an open question. High-throughput prediction and
experimental validation of heterodimerization between (b)HLH and bHLH TFs are still required to substantiate the potential antagonistic mechanisms of (b)HLHs in other regulatory networks.

327

The study of bHLH TFs in Arabidopsis has produced a wealth of functional data valuable for predicting the function of potential functional orthologs in crop plants. Although the study of bHLH TFs in crops becomes more extensive in recent years, more in-depth studies and further elucidation of the functions and regulatory roles of bHLH TFs in cultivated plants are still required. Therefore, further studies need to focus on the identification and functional characterization of bHLH genes in cultivated plants to pave the way for future crop genetic improvement.

335

336

## Acknowledgement

338

We apologize to our colleagues whose works and original articles could not be cited because of space limitations and the request to cite mainly no more than five-year-old references. CD was supported by the ANR DYNAFER (ANR-22-CE20-0006). FG was supported by the National Natural Science Foundation of China (32200227), Hunan Provincial Natural Science Foundation of China (2023JJ40312) and the Scientific Research Fund of Hunan Provincial Education Department (22B0182).

345

346

# Box 1. bHLH DNA binding and biological specificity

The bHLH domain is composed of a basic region involved in the binding to half
site of the E-box (CANNTG with N=A,C,G,T), or the canonical G-box (CACGTG), cis-
regulatory sequences and the HLH (Helix-Loop-Helix) domain is involved in bHLH
homo- or heterodimerization [6]. Each bHLH partner providing binding specificity
towards half of the E-box, dimerization may allow a given bHLH to tether to a wider
set of cis-regulatory sequences than as a homodimer [89]. Nonetheless, because of
structural conformation changes upon dimerization, the E-box that is recognized by a
given bHLH dimer might not be the sum of the half sites recognized by each partner
[89]. bHLH dimerization might involves additional domains. A conserved leucine
zipper motif located immediately C-terminal to the HLH domain is necessary for the
dimerization of bHLH from subfamilies IVc and IVd [8]. In contrast, dimerization via
the ACT-like domain inhibits the binding affinity to E-boxes because of the absence of
dimerized HLH domains [75]. bHLHs, such as MYC2/bHLH6 (subfamily IIIe) or
PIF4/bHLH9 (subfamily VII), can also interact in the form of homotetramers to bind
tandem E-boxes allowing enhancing gene expression [90, 91]. Nevertheless, flexibility
exists in bHLH DNA recognition since binding to other types of cis-elements (e.g.
GCG-box motifs) has been reported [92].

An important question arising from these studies is what provides the biological
specificity to bHLH function if most of the bHLHs recognize similar DNA sequences.
Part of the answer is the specificity of bHLH gene expression that is restricted to certain
tissues, cell types, developmental stages and/or in response to environmental cues.
Posttranslational modifications such as ubiquitination/phosphorylation/sumoylation
also affect bHLHs DNA binding capacity, subcellular localization and/or stability.
Nevertheless, several active bHLHs might still be present at the same time in a given
cell nucleus. The cooperative binding to DNA of bHLHs with other types of interacting
TFs (directly or through bridging cofactors) is an important mechanism that provides
374 bHLH TFs specificity towards their target genes. This is for instance the case between
375 PIF1/PIL5/bHLH15 (subfamily VII) and ABSCISIC ACID INSENSITIVE 5 (ABI5,
376 bZIP) [93]. Here, ABI5 binding to G-box coupling elements (GCEs) allows PIF1
377 targeting both G-box and other types of cis-regulatory sequences (e.g. TGTC-
378 containing sequences). In addition to the above-described mechanisms, the degree of
379 chromatin compaction and the density of nucleosome in euchromatic regions are
380 important parameters that also influence bHLH binding to its DNA targets.

381

382

383 Box 2. Antagonistic (b)HLH/bHLH system in plants

384

385 (b)HLHs are atypical bHLHs that lack key sequence features in the basic region
386 for proper DNA binding and that act antagonistically to bHLHs [4]. The regulation of
387 cell elongation in response to shade, a mechanism known as shade avoidance, best
388 exemplified the role of (b)HLHs in plants [66]. Shade avoidance involves a large set of
389 typical bHLH TFs that act in concert among which are PIFs (i.e. PIF1/PIL5/bHLH15,
390 PIF3/bHLH8, PIF4/bHLH9, PIF5/PIL6/bHLH65; subfamily VII), BR ENHANCED
391 EXPRESSIONs (i.e. BEE1/bHLH44, BEE2/bHLH58 and BEE3/bHLH50; subfamily
392 XII), ACTIVATOR FOR CELL ELONGATIONs (i.e. ACE1/bHLH49, ACE2/bHLH74
393 and ACE3/bHLH77; subfamily XII) and BES1-INTERACTING MYC-LIKEs
394 (BIM1/bHLH46, BIM2/bHLH102 and BIM3/bHLH141; subfamily Va) TFs [94, 95]
395 (Figure 2D, 4C).

396 HFR1/bHLH26, an atypical bHLH that contains mutations within its basic region,
397 forms heterodimers with the PIFs to limit their function [96, 97]. PAR1/bHLH165 and
398 PAR2/bHLH166 are two (b)HLH that lack important amino acid residues in the basic
399 region that are responsible for DNA-binding activity. PARs form competitive
400 complexes with the PIF, BEE and BIM bHLH members and inhibit their DNA-binding
401 activity, thereby blocking the promotion of cell elongation by these bHLH TFs [98, 99].
402 Likewise, AIF subfamily members also negatively regulate cell elongation by forming
403 competitive complexes with bHLH TFs, but only with BEEs and not with PIFs or BIMs
404 [100].

405 PACLOBUTRAZOL RESISTANCE TFs (i.e. PRE1/bHLH136, PRE2/bHLH134,
406 PRE3/bHLH135, PRE4/bHLH161, PRE5/bHLH164 and PRE6/bHLH163; subfamily
407 XV) are (b)HLH that lack the basic amino acid residues that are responsible for the
408 binding to DNA [101]. Instead of inhibiting the activity of typical bHLHs by forming
409 (b)HLH/bHLH complexes, PREs act as secondary tier repressors [100, 102]. In these
410 tri-antagonistic TFs system, PREs competitively interact with AIFs, HFR1 and the
411 PARs, and therefore counteract their ability to affect the activity of PIFs, BIMs and
412 BEEs [95]. In other words, PREs restores the transcriptional activity of PIFs, BIMs and
413 BEEs by releasing them from the (b)HLH/bHLH complexes [95, 100, 102].

414

415

416 References

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666 **Figure legends**

667

668 **Figure 1.** Schematic representation of the phylogenetic relationships between the different bHLH transcription factor subgroups in *Arabidopsis thaliana*. The phylogenetic tree was constructed with MEGA X using the neighbour-joining method with 1000 bootstraps. Full-length amino acid sequence of the 171 *Arabidopsis thaliana* bHLH transcription factor was used. Subgroups were designated as previously reported in [6] and [8]. Three new subfamilies are highlighted in red.

674

675 **Figure 2.** Schematic representation of selected signalling pathways employing bHLH transcription factors in *Arabidopsis thaliana*. (A) Stomatal development requires the sequential activity of five bHLHs belonging to two different subfamilies (i.e. Ia and IIIe). (B) Root hairs development is regulated by 12 bHLHs belonging to three different subfamilies (i.e. IIIf, VIIIc and XI). In non-hair cells (atrichoblasts), MBW complexes activate the expression of *GL2* (*GLABARA2*, homeodomain TF) that in turn repress the expression of bHLHs involved in root hair development. In hair cells (trichoblasts), CPC (CAPRICE, R3-MYB) replaces WER (WERWOLF, R2R3-MYB) within the MBW complexes allowing the expression of bHLHs involved in root hair development. TRENSPARENT TESTA GLABRA 1/TTG1, WD40-repeat protein. (C) In response to high temperatures, PIF4 (subfamily VII) is induced and activate the expressions of *FT* (*FLOWERING TIME*) and growth-related genes, promoting flowering and cell elongation, respectively. However, PIF4 repress the expression *SPCH* (subfamily Ia), thereby inhibiting stomatal production. (D) Cell elongation in response to shade is promoted by the activity of a set of 15 bHLH belonging to four different subfamilies (i.e. Va, VI, VII and XII) whose activity is inhibited by their
691 interaction with (b)HLH (from VII, XVIIa and XVIIb subfamilies). These interactions
692 can be antagonized by another set of (b)HLH (subfamily XV) that act as secondary tier
693 repressors allowing shade-induced cell elongation.

694

695 **Figure 3. Simplified representation of the bHLH regulatory network that controls iron homeostasis in Arabidopsis thaliana.** Eighteen different bHLH transcription factors from seven different subfamilies (i.e. Ib, IIIa, IIIe, IIIf, IVa, IVb, IVc) act within an intricate regulatory network to regulate iron homeostasis. bHLH Ib: bHLH38, bHLH39, bHLH100 and bHLH101; bHLH IVa: bHLH18, bHLH19, bHLH20 and bHLH25; bHLH IVc: IAA-LEUCINE RESISTANT 3 (ILR3/bHLH105), IRON DEFICIENCY TOLERANT 1 (IDT1/bHLH34), bHLH104 and bHLH115; MYC1/bHLH12: IIIf subfamily; MYC2/bHLH6: IIIe subfamily.

703

704 **Figure 4. Post-translational mechanisms regulating bHLH transcription factor activities in Arabidopsis thaliana.** Dimerization is one of the main post-translational mechanisms that regulate bHLH transcription factors activity. It can be as (A) homodimer (e.g. regulation of iron homeostasis; Figure 3) or as (B) heterodimers (e.g. regulation of cell proliferation in meristems). (C) The antagonistic bHLH/(b)HLH interactions is another example of bHLH hetero-dimerization (e.g. regulation of cell elongation in response to shade). (D) Other proteins than bHLHs can also directly interact with bHLHs to modulate their activity (e.g. regulation of iron homeostasis by the gibberellin signalling DELLA repressors). (E) Interactions with other types of transcription factors than bHLHs also affect their activity (e.g. the formation of MBW protein complexes to regulate proanthocyanidin biosynthesis in the seed coat; Figure
2B). The deposition of post-translational marks also modulates bHLH activity and stability. Among them, (F) the phosphorylation (e.g. the phosphorylation of FIT by CIPK11 protein kinase to promote the uptake of iron), (G) the ubiquitination (e.g. the ubiquitination-dependent degradation of bHLH115 and ILR3 to inhibit the uptake of iron) and (H) the sumoylation (e.g. sumoylation of MYC2 to inhibit its interaction with PUB10 and thus its degradation and promote its role in regulating photomorphogenesis) are the most studied. ACEs: bHLH subfamily XII; BTS (E3-ubiquitin ligase); GL3/bHLH1, EGL3/bHLH2 and TT8/bHLH42: bHLH subfamily IIIf; LHW/bHLH156, LHL1/bHLH173 and LHL2/bHLH155: bHLH subfamily XIII; IBH1/bHLH158: bHLH subfamily XVIIa; PRE1/bHLH136: bHLH subfamily XV; PUB10: E3-ubiquitin ligase; TMO5/bHLH32, T5L1/bHLH30, T5L2/bHLH107: bHLH subfamily Vb; TT2, R2R3-MYB; TTG1, WD-repeat protein.
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Schematic representation of the phylogenetic relationships between the different bHLH transcription factor subgroups in *Arabidopsis thaliana*. The phylogenetic tree was constructed with MEGA X using the neighbour-joining method with 1000 bootstraps. Full-length amino acid sequence of the 171 *Arabidopsis thaliana* bHLH transcription factor was used. Subgroups were designated as previously reported in [5] and [7]. Three new subfamilies are highlighted in red.</figcaption></figure>
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Schematic representation of selected signalling pathways employing bHLH transcription factors in *Arabidopsis thaliana*. (A) Stomatal development requires the sequential activity of five bHLHs belonging to two different subfamilies (i.e. Ia and IIle). (B) Root hairs development is regulated by 12 bHLHs belonging to three different subfamilies (i.e. IIIf, VIIIc and XI). In non-hair cells (atrichoblasts), MBW complexes activate the expression of *GL2* (*GLABARA2*, homeodomain TF) that in turn repress the expression of bHLHs involved in root hair development. In hair cells (trichoblasts), CPC (CAPRICE, R3-MYB) replaces WER (WERWOLF, R2R3-MYB) within the MBW complexes allowing the expression of bHLHs involved in root hair development. TRENSPARENT TESTA GLABRA 1/TTG1, WD40-repeat protein. (C) In response to high temperatures, PIF4 (subfamily VIIa) is induced and activate the expressions of *FT* (*FLOWERING TIME*) and growth-related genes, promoting flowering and cell elongation, respectively. However, PIF4 repress the expression *SPCH* (subfamily Ia), thereby inhibiting stomatal production. (D) Cell elongation in response to shade is promoted by the activity of a set of 15 bHLH belonging to four different subfamilies (i.e. Va, VI, VIIa and XII) whose activity is inhibited by their interaction with (b)HLH (from VIIb, XVIIa and XVIIb subfamilies). These interactions can be antagonized by another set of (b)HLH (subfamily XV) that act as secondary tier repressors allowing shade-induced cell elongation.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 3.** Simplified representation of the bHLH regulatory network that controls iron homeostasis in *Arabidopsis thaliana*. Eighteen different bHLH transcription factors from seven different subfamilies (i.e. Ib, IIIa, IIIe, IIIf, IVa, IVb, IVc) act within an intricate regulatory network to regulate iron homeostasis. bHLH Ib: bHLH38, bHLH39, bHLH100 and bHLH101; bHLH IVa: bHLH18, bHLH19, bHLH20 and bHLH25; bHLH IVc: IAA-LEUCINE RESISTANT 3 (ILR3/bHLH105), IRON DEFICIENCY TOLERANT 1 (IDT1/bHLH34), bHLH104 and bHLH115; MYC1/bHLH12: IIIf subfamily; MYC2/bHLH6: IIIe subfamily.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure 4.** Post-translational mechanisms regulating bHLH transcription factor activities in *Arabidopsis thaliana*. Dimerization is one of the main post-translational mechanisms that regulate bHLH transcription factors activity. It can be as (A) homodimer (e.g. regulation of iron homeostasis; Figure 3) or as (B) heterodimers (e.g. regulation of cell proliferation in meristems). (C) The antagonistic bHLH/(b)HLH interactions is another example of bHLH hetero-dimerization (e.g. regulation of cell elongation in response to shade). (D) Other proteins than bHLHs can also directly interact with bHLHs to modulate their activity (e.g. regulation of iron homeostasis by the gibberellin signalling DELLA repressors). (E) Interactions with other types of transcription factors than bHLHs also affect their activity (e.g. the formation of MBW protein complexes to regulate proanthocyanidin biosynthesis in the seed coat; Figure 2B). The deposition of post-translational marks also modulates bHLH activity and stability. Among them, (F) the phosphorylation (e.g. the phosphorylation of FIT by CIPK11 protein kinase to promote the uptake of iron), (G) the ubiquitination (e.g. the ubiquitination-dependent degradation of bHLH115 and ILR3 to inhibit the uptake of iron) and (H) the sumoylation (e.g. sumoylation of MYC2 to inhibit its interaction with PUB10 and thus its degradation and promote its role in regulating photomorphogenesis) are the most studied. ACEs: bHLH subfamily XII; BTS (E3-ubiquitin ligase); GL3/bHLH1, EGL3/bHLH2 and TT8/bHLH42: bHLH subfamily IIIf; LHW/bHLH156, LHL1/bHLH173 and LHL2/bHLH155: bHLH subfamily XIII; IBH1/bHLH158: bHLH subfamily XVIIa; PRE1/bHLH136: bHLH subfamily XV; PUB10: E3-ubiquitin ligase; TMO5/bHLH32, T5L1/bHLH30, T5L2/bHLH107: bHLH subfamily Vb; TT2, R2R3-MYB; TTG1, WD-repeat protein.</figcaption></figure>