The evolution of special types of cells requires the acquisition of new gene regulatory networks controlled by transcription factors (TFs). In stomatous plants, a TF module formed by subfamilies Ia and IIIb basic helix–loop–helix TFs (Ia-IIIb bHLH) regulates stomatal formation; however, how this module evolved during land plant diversification remains unclear. Here we show that, in the astomatous liverwort Marchantia polymorpha, a Ia-IIIb bHLH module regulates the development of a unique sporophyte tissue, the seta, which is found in mosses and liverworts. The sole Ia bHLH gene, MpSETA, and a IIIb bHLH gene, MpICE2, regulate the cell division and/or differentiation of seta lineage cells. MpSETA can partially replace the stomatal function of Ia bHLH TFs in Arabidopsis thaliana, suggesting that a common regulatory mechanism underlies setal and stomatal formation. Our findings reveal the co-option of a Ia-IIIb bHLH TF module for regulating cell fate determination and/or cell division of distinct types of cells during land plant evolution.
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Public RNA-seq data can be downloaded from SRA repository (https://www.ncbi.nlm.nih.gov/sra) under accession PRJNA350270, PRJDB6579, PRJDB4420, PRJDB9329 and PRJNA265205. All data supporting the conclusions in the paper are available within the paper or the supplementary materials. Sequence data and gene ID can be found in the database, as follows: MarpolBase (https://marchantia.info), Phytozome v.13 (https://phytozome-next.jgi.doe.gov/), OneKP (https://db.cngb.org/onekp/), TAIR (http://www.arabidopsis.org/) and NCBI (https://www.ncbi.nlm.nih.gov/genome/?term=PRJNA701193). Source data are provided with this paper.
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We thank T. Nakagawa (Shimane University, Japan), S. Mano (National Institute for Basic Biology, Japan), S. S. Sugano (National Institute of Advanced Industrial Science and Technology, Japan) and K. U. Torii (The University of Texas at Austin, USA) for sharing the materials. We also thank K. Nakajima (Nara Institute of Science and Technology, Japan) for sharing the figures of plants in Fig. 5b. We are grateful to J. Raymond for his critical readings of this manuscript. This work was supported by Ministry of Education, Culture, Sports, Science and Technology/Japan Society for the Promotion of Science (MEXT/JSPS) KAKENHI grants to M.S. (JP19K06722 and JP20H05416), K.T. (JP26711017 and JP18K06283), Y.O. (JP18K19964), T.M. (JP20H05905 and JP20H05906), I.H.-N. (JP15H05776), R.N. (JP20H04884) and T.S. (JP18K06284); Grants-in-Aid JSPS Fellows to K.C.M. (JP21J14990) and M.S. (JP12J05453); and the Takeda Science Foundation, the Kato Memorial Bioscience Foundation and the Ohsumi Frontier Science Foundation to M.S. J.L.-M. and Y.-T.L. were supported by PhD studentships from the Darwin Trust of Edinburgh.
The authors declare no competing interests.
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Extended Data Fig. 1 Comparison of the domain architecture of Ia bHLHs in land plants.
a, A diagram of the domain architecture of MpSETA (M. polymorpha), PpSMF1, PpSMF2 (P. patens), AtSPCH, AtMUTE, and AtFAMA (A. thaliana). While no PEST domain was identified, MpSETA has a bHLH domain and SMF domain conserved at the C-terminus like other Ia bHLH proteins. SMF domain is structurally considered to be the ACT-like domain, which is a putative domain for protein-protein dimerization. b, Sequence alignment of the bHLH domain of Ia bHLH proteins. Ia bHLHs are surrounded by a black box, and others are Ib(1) bHLHs. Asterisks indicate amino acids that are assumed to be important for binding to the E-box (CANNTG), and the triangles indicate amino acids that are assumed to be important for the dimerization of the bHLH domain. The yellow box indicates the LxCxE motif, which is a binding motif with Retinoblastoma-related (RBR). c, Sequence alignment of the C-terminal SMF domain of Ia bHLH proteins.
Extended Data Fig. 2 Function of MpSETA in A. thaliana Ia bHLH mutants.
a, Confocal images of A. thaliana abaxial cotyledons of wild type (Col-0), spch-3, and proAtSPCH:MpSETA spch-3 at 9 days after stratification (DAS). b, Confocal images of A. thaliana abaxial cotyledons of wild type (Col-0), fama-1, and proAtFAMA:MpSETA fama-1 at 9 DAS. Brackets and arrows indicate fama tumors and stomatal-lineage cells, respectively. c, Quantitative data of the distribution of the number of cell divisions that occurred in the stomatal lineage in each genotype. (n > 320 cells per genotype, 9 DAS cotyledons). d, Y2H assays in which the MpSETA fused with the GAL4 DNA-binding domain (DBD) was used as bait, and the AtICE1 and AtSCRM2 fused with the GAL4 activation domain (AD) were used as prey. DBD alone and AD alone were used as negative controls. e, BiFC assays showing the interaction between MpSETA and AtICE1 or AtSCRM2 in N. benthamiana leaf epidermal cells. MpSETA was fused to the N-terminal fragment of EYFP (nYFP), whereas AtICE1 or AtSCRM2 was fused to the C-terminal fragment of EYFP (cYFP). nYFP alone and cYFP alone were used as the negative controls. Nuclei were stained with DAPI. The experiments in this figure were repeated at least three times with similar results. Bars, 10 µm (e), and 100 µm (a,b).
Extended Data Fig. 3 Expression analysis of MpSETA in the gametophytic tissues.
Histochemical detection of β-glucuronidase (GUS) activity driven by the MpSETA promoter in the developing antheridia. The experiments in this figure were repeated at three times with similar results. Bars, 1 mm.
Extended Data Fig. 4 Generation and phenotypes of MpSETA knock-out lines.
a, Structure of the MpSETA locus disrupted by homologous recombination. Knock-out lines have a deletion in the bHLH domain coding region. White boxes indicate the exons of the MpSETA coding sequence. DT-A, diphtheria toxin A fragment gene; HgrR, hygromycin- resistance gene. b, Genotyping of the Mpsetako lines used in this study to distinguish sex. rbm27, a male-specific marker; rhf73, a female-specific marker. c, Genotyping of the Mpsetako lines. The position of the primers used for PCR is shown in (a). M, Male; F, Female. d, RT-PCR to confirm the loss of the full-length MpSETA transcript in Mpsetako lines in 21 DPF sporophytes. MpEF1α was used as an internal control. e, Spermatogenesis process in the wild type (WT) and Mpsetako lines. All the images are at the same scale. The experiments in this figure were repeated at least three times with similar results. Bars, 10 μm (e).
Extended Data Fig. 5 Phylogenetic tree of IIIb bHLH TFs.
A maximum-likelihood bHLH phylogenetic tree of subfamilies IIIb, III (a + c) (light blue), and III(d + e) (grey, outgroup) is shown. Numbers at branches indicate bootstrap values calculated from 1,000 replicates. The scale bar indicates the substitution rate per residue. IIIb bHLHs are divided into 2 groups: ICE/SCRM clade (orange) and NFL clade (magenta). Species are abbreviated as follows: Mp, M. polymorpha (liverwort); Lc, L. cruciata (liverwort); Pp, P. patens (moss); Cepur, Ceratodon purpureus (moss); Aagr, Anthoceros agrestis (hornwort); Sm, Selaginella moellendorffii (lycophyte); AmTr, Amborella trichopoda (basal angiosperm); Os, Oryza sativa (monocot); At, A. thaliana (dicot). Arrows indicate MpICE1 (Mp4g04910) and MpICE2 (Mp4g04920). For the phylogenetic construction of subfamilies III(a + c) and III(d + e), we used amino acid sequences from only A. thaliana and M. polymorpha.
Extended Data Fig. 6 Comparison of the domain architecture of IIIb bHLHs in land plants.
a, A diagram of the domain architecture of MpICE1, MpICE2 (M. polymorpha), PpSCRM1 (P. patens), AtICE1, and AtSCRM2 (A. thaliana). MpICE1 and MpICE2 have a bHLH domain and ACT-like domain conserved at the C-terminus like other IIIb bHLH proteins. b, Sequence alignment of the bHLH domain of the IIIb bHLH proteins. IIIb bHLHs are surrounded by a black box, and others are an outgroup. Asterisks indicate amino acids that are assumed to be important for binding to the E-box (CANNTG). c, Sequence alignment of the C-terminal ACT-like domain of the IIIb bHLH proteins.
Extended Data Fig. 7 The expression analysis of MpICE2.
a, Histochemical detection of β-glucuronidase (GUS) activity driven by the MpICE2 promoter in the vegetative thallus. b, Confocal images of the dorsal epidermis of proMpICE2:Citrine-GUS-NLS line. The upper and lower panels indicate the epidermis around the apical notch and the epidermis around the midrib, respectively. Arrows indicate the air pores. c,d, Histochemical detection of GUS activity driven by the MpICE2 promoter in the gametophytic reproductive organs. An antheridiophore (c) and an archegoniophore (d) are shown. e, Expression pattern of MpICE2 in developing sporophytes. f, foot; s, seta; at, archesporial tissue; sp, sporangium; ca, calyptra; p, pseudoperianth (n). Arrowheads indicate the cell wall of the first cell division. The experiments in this figure were repeated at least three times with similar results. Bars, 5 mm (a,c,d), 100 μm (b and e).
Extended Data Fig. 8 Generation of Mpice2 mutants by CRISPR/Cas9.
a, Schematic representation of the MpICE2 gene and the resulting mutations in the obtained CRISPR/Cas9-generated alleles. Gray, white, and blue boxes indicate the coding sequences (CDS), the untranslated regions (UTR), and the bHLH domain coding region, respectively. b, Sequence alignment of putative translational products of wild type (WT) and Mpice2ge mutants. Asterisks indicate the amino acids that are assumed to be important for binding to the E-box.
Extended Data Fig. 9 Functional analysis of MpICE1 and MpICE2 in A. thaliana mutants.
a, Confocal images of A. thaliana abaxial cotyledons of wild type (Ws-4), ice1-2 scrm2-1, and proAtMUTE:MpSETA mute-2 expressing MpICE2 at 9 DAS. Arrowheads and asterisks indicate stomata and hydathode pores, respectively. The experiments were performed once. b, Confocal images of A. thaliana abaxial leaves of wild type (Col-0), ice1-2 scrm2-1, proAtICE1:MpICE1 ice1-2 scrm2-1, and proAtICE1:MpICE2 ice1-2 scrm2-1 at 13 DAS. The experiments were repeated three times with similar results. Bars, 100 µm.
Supplementary Tables 1 and 2.
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Moriya, K.C., Shirakawa, M., Loue-Manifel, J. et al. Stomatal regulators are co-opted for seta development in the astomatous liverwort Marchantia polymorpha. Nat. Plants 9, 302–314 (2023). https://doi.org/10.1038/s41477-022-01325-5