Basic helix-loop-helix transcription factors and epidermal cell fate determination in Arabidopsis

Abstract

Cell fate determination is an important process in multicellular organisms. Plant epidermis is a readily-accessible, well-used model for the study of cell fate determination. Our knowledge of cell fate determination is growing steadily due to genetic and molecular analyses of root hairs, trichomes, and stomata, which are derived from the epidermal cells of roots and aerial tissues. Studies have shown that a large number of factors are involved in the establishment of these cell types, especially members of the basic helix-loop-helix (bHLH) superfamily, which is an important family of transcription factors. In this mini-review, we focus on the role of bHLH transcription factors in cell fate determination in Arabidopsis.

A fundamental aspect of the development of multicellular organisms is the specification of different cell types at the appropriate time and place.¹ During higher plant development, cells descended from the same zygote adopt different cell fates and experience diverse differentiation programs to form an integrated plant.² Most of our knowledge of cell fate determination is derived from the study of Arabidopsis epidermis, which consists of a few well-characterized cell types. Root epidermis differentiates into hair and non-hair cells, while the aerial epidermis is composed of trichomes and stomata scattered at regular intervals between pavement cells.^1,2 A great many transcription factors participate in epidermal cell type specification, including bHLH transcription factors.^2-6

bHLH Transcription Factors in Arabidopsis

The bHLH transcription factor superfamily is composed of a large number of proteins found in almost all organisms, including fungi, plants, and animals.⁷ One of the largest transcription factor families in Arabidopsis, the bHLH family has 147 members.⁸ As genes from the same subfamily tend to have related functions, a structural analysis of this superfamily was performed; as a result, the bHLH superfamily was divided into 12 subfamilies.⁹

In Arabidopsis, bHLH members are involved in a broad range of growth and developmental signaling pathways, including light signaling,^10-19 brassinosteroid and abscisic acid signaling,^20-27 gynoecium development,^28,29 abiotic stress responses,^30,31 flavonoid biosynthesis,^32,33 axillary meristem formation,³⁴ flowering time control,³⁵ trichome and root hair differentiation,^36-42 and stomatal patterning.^4,43-46 Together, these results indicate that the bHLH superfamily is very important in plant development.

bHLH transcription factors function in root and shoot trichoblast cell fate determination and in morphogenesis

Root hairs and trichomes are outstanding models for studying the molecular basis of cell fate determination in Arabidopsis.⁴⁷ Root hairs are unbranched tubular outgrowths of the root epidermis that function to collect water and nutrients, including minerals, from the soil⁴⁷ Trichomes are branched (1–3 branches in wild-type Col-0), single-celled structures on the aerial parts of plants that protect the organism from biotic stress and UV irradiation.²

In Arabidopsis, the first-reported bHLH transcription factors were GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3), which play partially redundant roles in root hair control.⁴¹ gl3 egl3 double mutants have hairy roots due to a failure in non-hair cell specification.^37,41 mRNA and protein expression of these genes was detected in root hair and non-root hair cells, respectively, indicating that cell fate determination in root epidermis occurs in a non-cell autonomous manner.⁴⁰ The molecular mechanism of cell fate determination in trichomes is similar to that in root epidermis,^2,6,48 including a requirement for GL3 and EGL3.^36,39,49 Indeed, gl3-1 mutants show reduced numbers of leaf trichomes and branches,³⁹ although egl3 single mutants have no obvious trichome phenotype.³⁷ gl3 egl3 double mutants have glabrous leaves and inflorescence stems, indicating redundant roles for these genes in trichome specification.³⁷ TT8 is also involved in leaf margin trichome formation.⁵⁰ Unlike the non-cell autonomous control of root cell development, GL3 and EGL3 appear not to be transportable in leaf tissues.⁵¹

bHLH transcription factors, together with R2R3-MYB proteins, including WEREWOLF (WER, for root hair cell fate control) and GLABRA1 (GL1, for trichome differentiation), and the WD-40 factor TRANSPARENT TESTA GLABRA1 (TTG1), form a linear activation complex by using the bHLH protein as a linker; WER and TTG1 interact with bHLH members directly in vitro and in vivo, but no direct interaction has been identified between them.^{6,37,40,41,47,51} The active transcriptional complex R2R3-MYB-bHLH-TTG1 positively regulates the downstream homeobox-leucine zipper gene GLABRA2 (GL2) to promote non-hair cell and trichome differentiation.^{36,41,48,52-54} Compared with R2R3-MYB proteins, six R3-MYB factors, CAPRICE (CPC), TRIPTYCHON (TRY), TRICHOMELESS1 (TCL1), and ENHANCER OF TRIPTYCHON AND CAPRICE 1, 2, and 3 (ETC1, 2, and 3), possess only the DNA-binding domain without a recognizable activation domain.^3,52,55-61 They are activated by the trimeric activation complex, transported to neighboring cells, and integrated competitively into the trimeric complex, rendering it inactive.^{6,47,52,55,58,62} These steps comprise the generally accepted activator-inhibitor model of root hair and trichome cell fate specification.^6,47,63

AtMYC1, another bHLH transcription factor belonging to the same subfamily as GL3 and EGL3 (subfamily IIIf), was first cloned in 1996.⁶⁴ Until rescently, its role in root hair and trichome control has been largely demonstrated.^36,49,65Atmyc1 mutants have reduced numbers of non-hair cells and trichomes.^36,49 Genetic analyses have shown partially redundant yet divergent functions between AtMYC1 and GL3/EGL3.³⁶ GL3 and EGL3 can successfully complement the defects observed in Atmyc1; however, AtMYC1 is incapable of recovering the lesions seen in gl3 elg3 double mutants.³⁶

AtMYC1 functions upstream of GL2, through interactions with MYB proteins and TTG1.^36,49 Unlike GL3 and EGL3, which function as homodimers or heterodimers,^37,39 AtMYC1 tends to work as a monomer.³⁶ Besides its reported protein-interacting domain, the arginine at position 173 (R173) in AtMYC1, which is conserved between AtMYC1 and GL3/EGL3, is crucial for its interaction with partner proteins and for its proper function.³⁶ The importance of this residue was demonstrated in an analysis of Atmyc1–1, which carries a point mutation at this position (R173H). Previously, we confirmed that AtMYC1 was responsible for the phenotype of this mutant by transgenic complementation. We also performed crosses between Atmyc1–1 and Atmyc1–2, and between Atmyc1–1 and Atmyc1–3. The F1 progeny behaved like the parental lines (Fig. 1), indicating that Atmyc1–1 is a new allele of Atmyc1. AtMYC1 mRNA is expressed mainly in root hair cells,³⁶ similar to its homologs, while AtMYC1 expression is limited to the same cell files.⁶⁵ In addition, like GL3/EGL3, AtMYC1 is negatively regulated by WER and positively regulated by CPC.^40,65

Figure 1.Atmyc1–1 is a new allele of Atmyc1. A. Leaf trichomes from 12-d-old seedlings of the indicated genotype. F1 plants produced by crossing Atmyc1–1 with Atmyc1–2, and Atmyc1–1 with Atmyc1–3 are shown. Wild-type and Atmyc1 mutant control plants are also shown. B. Trichome number analysis of the first and second pair of leaves in the F1 and control plants. At least 12 plants were analyzed for each genotype. Error bars represent the standard deviation

Recently, a genome-wide transcriptome analysis followed by a detailed functional analysis demonstrated that the bHLH genes bHLH54, bHLH66, and bHLH82, which belong to subfamilies VIIIc and XI, participate in root epidermal cell development in a stage-specific manner.⁶⁵ It is reasonable to assume that these bHLH factors also control trichome development.

Taken together, these results demonstrate the extensive participation of bHLH family members in different stages of root and shoot epidermis development. Consequently, bHLH transcription factors belonging to the IIIf, VIIIc, and XI subfamilies, together with a number of other transcription factors, play crucial roles in epidermis trichoblast cell fate determination in Arabidopsis.

bHLH Transcription Factors are Involved in Stomata Development

Stomata are microscopic pores found in land plants that consist of a pair of specialized, epidermis-derived guard cells.⁴ Stomata play critical roles in gas and water vapor exchange with the atmosphere.⁴ In Arabidopsis, stomata formation provides an exceptional model for studying cell fate determination.^45,66 Stomata are produced through a series of asymmetric cell divisions followed by a single symmetric cell division.⁶⁷ Detailed analyses have shown that stomata formation occurs in four stages: meristemoid mother cell (MMC), meristemoid, guard mother cell (GMC), and terminally-differentiated guard cells.⁴ Three closely related bHLH genes, SPEECHLESS (SPCH), MUTE, and FAMA, function consecutively as positive regulators of stomata formation.^4,45 Indeed, loss-of-function mutants of these genes do not produce stomata.⁴ Spatial and temporal expression pattern analyses and mutant phenotype analysis have demonstrated that SPCH is involved in the transition from MMCs to meristemoids, while MUTE guides the transition from meristemoids to GMCs, and FAMA promotes the shift from GMCs to guard cells.^43,46,68,69

The frequently discussed bHLH genes GL3 and EGL3 are also involved in stomata development in the hypocotyl; gl3 egl3 double mutants show an increased number of stomata.⁴⁰ Together, these results suggest that bHLH genes make a significant contribution to the specification of stomata in Arabidopsis.

Conclusions

As one of the largest superfamilies in Arabidopsis, bHLH transcription factors participate in a broad range of growth and developmental signaling pathways.⁸ However, there are still many bHLH genes whose biological functions are unknown. Thus, more extensive analyses should be performed to identify the biological roles and developmental pathways bHLH transcription factors are involved in. This will enrich our understanding of the bHLH superfamily, and provide new insight into the mechanism of epidermal cell fate determination.