Abstract
Gametogenesis, which is essential to the sexual reproductive system, has drastically changed during plant evolution. Bryophytes, lycophytes and ferns develop reproductive organs called gametangia—antheridia and archegonia for sperm and egg production, respectively. However, the molecular mechanism of early gametangium development remains unclear. Here we identified a ‘non-canonical’ type of BZR/BES transcription factor, MpBZR3, as a regulator of gametangium development in a model bryophyte, Marchantia polymorpha. Interestingly, overexpression of MpBZR3 induced ectopic gametangia. Genetic analysis revealed that MpBZR3 promotes the early phase of antheridium development in male plants. By contrast, MpBZR3 is required for the late phase of archegonium development in female plants. We demonstrate that MpBZR3 is necessary for the successful development of both antheridia and archegonia but functions in a different manner between the two sexes. Together, the functional specialization of this ‘non-canonical’ type of BZR/BES member may have contributed to the evolution of reproductive systems.
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Data availability
All data are available in the manuscript or the Supplementary Information. All sequence reads were deposited in the Sequence Read Archive (SRA) under project accession number PRJNA993618 and the DNA Data Bank of Japan (DDBJ) under accession numbers DRA016656 and DRA016671. Sequence and motif data in the manuscript or Supplementary Materials can be found in the databases MarpolBase MpTak v.6.1 (https://marchantia.info) and Pfam in InterPro (https://www.ebi.ac.uk/interpro/). Source data are provided with this paper.
Change history
22 April 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41477-024-01702-2
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Acknowledgements
We thank Y. Ozawa, Y. Fukaya, A. Suizu, A. Hata, R. Wakasugi, M. Tabara, A. Takeda, H. Kato, Y. Sakai, A. Mamiya and C. Goto for assistance and kind support. This work was funded by the Ministry of Education, Culture, Sports, Science and Technology, Japan (Scientific Research on Priority Areas and Scientific Research on Innovative Areas (grants 19H04860 and 20H05780 to S.Y., 20H04884 and 22H04733 to R.N., 19H05675 to T.K., 25113005 to T.A., and 17H06476, 20H05407 and 22H04720 to Y.K.), Japan Society for the Promotion of Science (grants 19K21189, 20K15813 and 23K05811 to T.F., 20K15824 and 22K15149 to N.M., 23H04751 to K.M., 22H00417 to T.K., 21K06236 to M.K., 19H03244 to T.A. and 17H05008, 20K15815 and 22H02647 to Y.K.), Sasakawa Scientific Research Grant from the Japan Science Society (grant 4363 to T.F.), Ohsumi Frontier Science Foundation (to S.Y.), Takeda Science Foundation (research grant to K.M.), JST PRESTO (grant JPMJPR20D9 to K.M.) and by JST FOREST Program (grant JPMJFR224Q to Y.K.)).
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Contributions
T.F. and Y.K. conceived and designed the research, coordinated the project and wrote the manuscript. T.F. performed most experiments and data analyses. Y.T. constructed the knock-in lines. N.S., C.Y. and K.M. performed microscopic observations. N.M. performed immunostaining. M.N. and K. Inoue carried out the RNA-seq analysis of archegonia. S.S. contributed to the experiments and data analyses. T.F., S.Y., R.N., K. Ishizaki, T.U., H. Fukaki, T.K., H. Fukuda, M.K., T.A. and Y.K. supervised the project. All authors reviewed and edited the manuscript.
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Extended data
Extended Data Fig. 1 Expression profiles and overexpression phenotypes of MpBZRs.
a, Expression profiles of MpBZR3 (Mp2g23000) in the RNA-seq data sets were visualised. b, Chromatic expression images of MpBZR3 in the expression database for M. polymorpha, MBEX: Marpolbase Expression. c, Tissue-specific gene expression pattern of MpBZR1 and MpBZR2 in the expression database for M. polymorpha, MBEX: Marpolbase Expression. Dots indicate each replicate. d, Relative transcript levels of MpBZRs in the overexpression lines of MpBZRs. Relative expression levels were calculated relative to the expression of MpEF1α. Detail information of all WT and transgenic plants were listed in Supplementary Table 2. Total RNAs were extracted from thalli grown from gemmae of WT male (Tak-1) and WT male (proMpCYCD;1:MpCYCD;1Nter-tdTomato-NLS, CYCD reporter) for 11 days and explants of the overexpression lines of MpBZRs grown for 20 days. Data represent the mean ± standard deviation (SD). Dots indicate each replicate (n = 3; biological replicates). Significant differences were examined using two-sided Welch’s t test (*P < 0.05, **P < 0.01). e,f, Morphology of thalli grown from gemmae of WT male (Tak-1) and WT male (CYCD reporter) for 10 days. g, h, Morphology of the proMpEF1α:MpBZR3-Citrine-2. i, Morphology of the proMpEF1α:MpBZR2-Citrine-4. j, Morphology of the proMpEF1α:MpBZR1-Citrine-4. Scale bars: 1 mm in e,f,h–j, and 3 mm in h.
Extended Data Fig. 2 Classification of each subgroup of BZR/BES transcription factors.
a,BZR/BES transcription factors in various species were classified based on the comparison of their amino acid sequences. Details of all proteins are listed in Supplementary Data 2. b, Comparison analysis of sequence motif in BZR/BES transcription factors in Arabidopsis thaliana (At), Solanum lycopersicum (Sl), Oryza sativa (Os) Ceratopteris richardii (Cr), Selaginella moellendorffii (Sm), Physcomitrium patens (Pp), Marchanita polymorpha (Mp), Anthoceros agrestis (Aa), and Penium margaritaceum (Pm) was performed in SALADA DB.
Extended Data Fig. 3 Alignments for a domain and motif of BZR/BES transcription factors.
a, Multiple sequence alignment of the DNA-binding domain of BZR/BES transcription factors in Arabidopsis thaliana (At), Solanum lycopersicum (Sl), Oryza sativa (Os) Ceratopteris richardii (Cr), Selaginella moellendorffii (Sm), Physcomitrium patens (Pp), Marchanita polymorpha (Mp), Anthoceros agrestis (Aa), and Penium margaritaceum (Pm) was constructed using the MAFFT software. Motif 1 and motif 2 detected in analysis using SALADA DB are involved in the DNA-binding domain. Feature of secondary structures in AtBZR1 is also indicated. b, Multiple sequence alignment of the type-B specific N-terminal motif (motif 16) detected in analysis using SALADA DB is indicated. The motif 16 is conserved in the type-B subgroup of BZR/BES transcription factors in land plants.
Extended Data Fig. 4 Phenotypes of the β-oestradiol-inducible MpBZR3 overexpression lines.
a–c Transgenic plants, proMpE2F:XVE»MpBZR3-2 male, -3 female, -10 male, -12 male, -19/Mpbnbko male, -22/Mpbnbko male, -13 female, -14 female, -23/Mpbnbko female, and -25/Mpbnbko female were grown from gemmae for 4 days. And then these plants were treated with or without 10 µM β-oestradiol for 1 day. Relative expression levels were normalized by the expression of MpEF1α and calculated relative to comparison to proMpE2F:XVE»MpBZR3-2 male without β-oestradiol treatment for a or proMpE2F:XVE»MpBZR3-10 male without β-oestradiol treatment for b,c, respectively. Data represent the mean ± standard deviation (SD). Dots indicate each replicate (n = 3; biological replicates). Significant differences were examined using two-sided Welch’s t test (*P < 0.05, **P < 0.01). d, Morphology of the β-oestradiol-inducible MpBZR3 overexpression lines, proMpE2F:XVE»MpBZR3-2 female and -3 male. Transgenic plants grown from gemmae for 4 days were treated with or without 10 µM β-oestradiol for 0, 8, 12, and 16 days. e, Visualisation of induced gametangium-like structures in the proMpE2F:XVE»MpBZR3-2 female and -3 male treated with 10 µM β-oestradiol for 16 days. Gametangia and induced gametangium-like structures were treated with ClearSee solution and stained by SCRI Renaissance 2200 (Blue) and SYBR Green I (Green). f, Visualisation of developing sperm-like structures in the proMpE2F:XVE»MpBZR3-2 treated with 10 µM β-oestradiol for 14 days. Sperm-like structures were immunostained by α-AC-tubulin (Green) and stained by Hoechst 33342 (Blue). Left and right panels show a blight field image and a fluorescent image, respectively. Scale bars: 3 mm in d, 100 µm in e, 10 μm in f.
Extended Data Fig. 5 Genetic relationship between MpBZR3 and MpBNB.
a, Relative expression levels of MpBNB and MpBZR3 in WT and Mpbnbko. Plants were grown from gemmae under the white light condition for 10 days, then these were cultured under the condition for reproductive induction, white light supplemented with far-red light, for 14 and 21 days. Each sample contains all apical notches without gametangiophores or with gametangiophore up to stage 1 taken from a single plant. Notches with gametangiophore at stage 2 or later were removed. Relative expression levels were calculated relative to the expression of MpEF1α. n = 3 plants from independent gemma from a single genotype. b, The β-oestradiol-inducible MpBZR3 overexpression lines, proMpE2F:XVE»MpBZR3-12 male, -22/Mpbnbko male, -13 female and -25/Mpbnbko female, grown from gemmae for 4 days were treated with or without 10 µM β-oestradiol for 12 days. c,d, Visualisation of induced gametangium-like structures in proMpE2F:XVE»MpBZR3-10 male, -19/Mpbnbko male, -13 female, and -23/Mpbnbko female treated with 10 µM β-oestradiol for 16 days. Gametangia and induced gametangium-like structures were treated with ClearSee solution and stained by SCRI Renaissance 2200 (white). Dotted lines and blue areas in c indicate the outlines of ectopic antheridium-like structures and spermatogenous-like cells in ectopic antheridium-like structures, respectively. Red area in d show a large cavity in ectopic archegonium-like structures. Scale bars: 3 mm in b, 100 µm in c,d.
Extended Data Fig. 6 The construction and observation of MpBZR3-Citrine knock-in lines.
a, Structures of MpBZR3 loci in WT and MpBZR3-Citrine knock-in lines and a part of the knock-in vector sequences. Black arrowheads indicate primer positions for genotyping using in b. b, Genotyping of WT and MpBZR3-Citrine knock-in lines. Black and white arrowheads indicate the predicted sizes of the PCR products from WT and MpBZR3-Citrine knock-in lines, respectively. Asterisk indicates non-specific amplification. The V-chromosome-linked DNA marker rbm27 and the U-chromosome-linked DNA marker rhf73 were used for sexual genotyping. c, Expression of MpBZR3 or MpBZR3-Citrine transcripts in WT and MpBZR3-Citrine knock-in lines. Total RNAs were extracted from stage 3 of archegoniophores or antheridiophores. Expression levels of MpEF1α were also indicated as a control. d, Relative transcript levels of MpBZR3 and MpBNB in WT and MpBZR3-Citrine knock-in lines. Relative expression levels were calculated relative to the expression of MpEF1α. Total RNAs were extracted from stage 3 of archegoniophores or antheridiophores. Data represent the mean ± standard deviation (SD). Dots indicate each replicate (n = 3; biological replicates). P values by two-sided Welch’s t test are indicated. e, Citrine signals in stage 1 antheridiophore or archegoniophore of MpBZR3-Citrineki male and female, respectively. Citrine and Calcofluor White signals are shown in yellow and blue, respectively. f, g, Citrine signals in developing antheridia and archegonia of MpBZR3-Citrineki male and female, respectively. Citrine and Calcofluor White signals are shown in yellow and blue, respectively. Red and orange arrowheads in f indicate predicted antheridium initial cells and developing antheridia at the early phase, respectively. Red, orange, and white arrowheads in g indicate archegonium initial cells, developing archegonia at the early phase, and archegonia at the late phase, respectively. Parts of pictures are using in Figs. 4 and 5. Scale bars: 1 mm in e, 20 µm in f, g.
Extended Data Fig. 7 Construction and observation of the Mpbzr3 genome-edited lines.
a, Genomic structure of the encoding region for the MpBZR3 gene. The coding regions are indicated as black boxes. Orange box indicates the location of the coding region of DNA-binding domain. Black arrowhead indicates the location of target sites of gRNAs for genome editing. b, A part of genomic sequences of MpBZR3 in the genome-edited lines. The wild type (Tak-1) sequence is shown together with the PAM sequence (orange) and the target sequence (underline). The genome-edited line Mpbzr3-1ge have a 1 bp insertion, while Mpbzr3-2ge has a 4 bp deletion respectively (blue). c, Partial amino acid sequences of MpBZR3 in the WT (Tak-1) and two genome-edited lines. The genome-edited lines have frame shift mutation (blue). d, Morphology of thalli grown for 10 days from gemmae of the wild-type lines, WT female (Tak-2) and WT male (Tak-1) and Mpbzr3 genome-edited lines, Mpbzr3-1ge female and Mpbzr3-2ge male. e, Each stage of archegoniophore in WT female (Tak-2) and Mpbzr3 genome-edited lines, Mpbzr3-1ge female and that of antheridiophore in WT male (Tak-1) and Mpbzr3 genome-edited lines, Mpbzr3-2ge male. Stages are defined according to Higo et al. 201611. Scale bars: 1 mm in d, 3 mm in e.
Extended Data Fig. 8 Phenotypes of the complementation lines.
a, Visualisation of antheridia in the antheridial receptacles in Mpbzr3ge mutants. Antheridial receptacles (stage 4) of the WT male, Mpbzr3-2ge, and the complementation lines, proMpBZR3:MpBZR3resist-3xCitrine/Mpbzr3-2ge (Comp-2 and Comp-3) were treated with ClearSee solution and stained by SYBR Green I. Antheridia were highlighted as strong SYBR Green I signal (Green). b, Relative expression levels of antheridium-expressed genes in antheridial receptacles (stage 4) of the WT male, Mpbzr3-2ge, and the complementation lines, Comp-2 and Comp-3. Relative expression levels were normalised by the expression of MpEF1α and calculated relative to comparison to the expression in WT. Data represent the mean ± standard deviation (SD). Dots indicate each replicate (n = 3; biological replicates). Different lowercase letters indicate significant difference (P < 0.05; one-way ANOVA followed by two-sided Tukey-Kramer test). Scale bars: 1 mm in a.
Extended Data Fig. 9 Phenotypes of Mpbzr3ge mutants.
a, b, Cross-sections of stage 2 of antheridial receptacles of the WT male and Mpbzr3-2ge male. Magnified images in a are indicated in b. c, number of discharged sperms from the antheridiophere of WT male and Mpbzr3-2ge male. An antheridia receptacle was placed in 100 µl H2O. Data represent the mean ± standard deviation (SD). Dots indicate each replicate (n = 4; biological replicates). Significant differences were examined by two-sided Student’s t test (*P < 0.05). d, Cross-sections of stage 4 of archegonial receptacles of the WT female and Mpbzr3-1ge female. e, Morphologies of archegonia from the stage 4 of archegonial receptacles of the WT female and Mpbzr3-1ge female. Scale bars: 1 mm in a, 100 µm in b,d, and 500 µm in e. f, Expression levels of gametangium-related genes in Mpbzr3ge mutants. TPM values of gametangium-related genes in the archegonial receptacles (stage 1 and 4) and antheridial receptacles (stage 1 and 4) in the Mpbzr3ge mutants. Data represent the mean ± standard deviation (SD). Dots indicate each replicate (n = 3; biological replicates). FDR values (q values) calculated using EdgeR are indicated. g, Fertility of sperm from Mpbzr3ge male mutants. Spores from sporophyte by crossing between WT female and Mpbzr3-2ge male were cultured on half-strength B5 agar medium with hygromycin B. Some sporelings had hygromycin resistance. In 14 resistant sporelings, 8 were females and 6 were males. The sequence data of MpBZR3 locus in these female plants is indicated. Four-sevenths of female plants had mutations in a similar sequence as that of Mpbzr3-2ge male plants.
Extended Data Fig. 10 Gene expression pattern of BZR/BES transcription factors in Physcomitrium patents.
a, Tissue-specific gene expression pattern of PpBZRs in the expression database, Physcomitrella eFP Browser. Detail information for PpBZRs is indicated in Supplementary Data 2. b, Chromatic expression images of two type-B BZRs, PpBZR3A and PpBZR3B, in Physcomitrella eFP Brwser.
Supplementary information
Supplementary Information
Supplementary Fig. 1 and Tables 1–4.
Supplementary Data
1. List of the differentially expressed genes in the reproductive organs. 2. List of BZR/BES transcription factors.
Supplementary Video 1
Video of discharged sperm cell masses from antheridiophores of WT.
Supplementary Video 2
Video of discharged sperm cell masses from antheridiophores of Mpbzr3-2ge males.
Source data
Source Data Extended Data Fig. 6
Unmodified gels for Extended Data Fig. 6b,c.
Source Data Extended Data Fig. 8
Statistical source data for Extended Data Fig. 8b.
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Furuya, T., Saegusa, N., Yamaoka, S. et al. A non-canonical BZR/BES transcription factor regulates the development of haploid reproductive organs in Marchantia polymorpha. Nat. Plants (2024). https://doi.org/10.1038/s41477-024-01669-0
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DOI: https://doi.org/10.1038/s41477-024-01669-0
