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Reinvention of hermaphroditism via activation of a RADIALIS-like gene in hexaploid persimmon

Nature Plants volume 8, pages 217–224 (2022)Cite this article

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Abstract

In flowering plants, different lineages have independently transitioned from the ancestral hermaphroditic state into and out of various sexual systems1. Polyploidizations are often associated with this plasticity in sexual systems2,3. Persimmons (the genus Diospyros) have evolved dioecy via lineage-specific palaeoploidizations. More recently, hexaploid D. kaki has established monoecy and also exhibits reversions from male to hermaphrodite flowers in response to natural environmental signals (natural hermaphroditism, NH), or to artificial cytokinin treatment (artificial hermaphroditism, AH). We sought to identify the molecular pathways underlying these polyploid-specific reversions to hermaphroditism. Co-expression network analyses identified regulatory pathways specific to NH or AH transitions. Surprisingly, the two pathways appeared to be antagonistic, with abscisic acid and cytokinin signalling for NH and AH, respectively. Among the genes common to both pathways leading to hermaphroditic flowers, we identified a small-Myb RADIALIS-like gene, named DkRAD, which is specifically activated in hexaploid D. kaki. Consistently, ectopic overexpression of DkRAD in two model plants resulted in hypergrowth of the gynoecium. These results suggest that production of hermaphrodite flowers via polyploidization depends on DkRAD activation, which is not associated with a loss-of-function within the existing sex determination pathway, but rather represents a new path to (or reinvention of) hermaphroditism.

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Fig. 1: Transcriptomic profiles during the development of hermaphrodite flowers from male flowers in hexaploid D. kaki.
Fig. 2: ABA treatment induces hermaphroditism and DkRAD expression specifically in polyploid persimmon.
Fig. 3: Functional validation of DkRAD in two model species.
Fig. 4: Model of the contributions of polyploidization or genome/gene duplication events to the various transitions between sexual systems in the genus Diospyros.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. All sequence data generated in the context of this manuscript have been deposited in the appropriate DNA Database of Japan: Illumina reads for mRNA-seq in the Short Read Archives (SRA) database (SRA Submission ID: DRA013154, Run IDs: DRR332477–332618).

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Acknowledgements

We thank T. Saito, N. Onoue and R. Matsuzaki (Grape and Persimmon Research Station, NIFTS, Japan) for some plant materials; and Ho-Wen Yang (University of Illinois) for experimental support. This work was supported by PRESTO from Japan Science and Technology Agency (JST) (grant nos. JPMJPR20D1 to T.A.); Grant-in-Aid for Scientific Research on Innovative Areas from JSPS (grant nos. 19H04862 to T.A. and 20H05391 to Y.I.); the Joint Usage/Research Center, Institute of Plant Science and Resources, Okayama University (to Y.I. and T.A.); and Grant-in-Aid for JSPS Fellows (grant nos. 19J23361 to K.M.).

Author information

Authors and Affiliations

  1. Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan

    Kanae Masuda, Yasutaka Kubo, Koichiro Ushijima & Takashi Akagi

  2. Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama, Japan

    Yoko Ikeda & Takakazu Matsuura

  3. Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan

    Taiji Kawakatsu

  4. Graduate School of Agriculture, Kyoto University, Kyoto, Japan

    Ryutaro Tao

  5. Department of Plant Biology and Genome Center, University of California Davis, Davis, CA, USA

    Isabelle M. Henry

  6. JST-PRESTO, Saitama, Japan

    Takashi Akagi

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Contributions

T.A. conceived the study and designed the experiments. K.M., Y.I., T.M., T.K. and T.A. conducted the experiments. K.M. and T.A. analysed the data. Y.I., T.K., R.T., Y.K., K.U., I.M.H. and T.A. contributed to plant resources and facilities. K.M., Y.I., T.K., I.M.H. and T.A. drafted the manuscript. All authors approved the manuscript.

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Correspondence to Takashi Akagi.

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Extended data

Extended Data Fig. 1 Morphological characterization of male, NH, and AH flowers at various organ developmental stages.

a-l, Flower cross-sections stained with toluidine blue at stage 2 (a-c) and stage 3 (d-f) (see details in Supplementary Fig. 1). Dissected flower (g-i) and pollen fertility (j-l) at the mature stage. Ovu, ovule; Pe, Petal; Pt, Pollen tube; Sp, Sepal. Scale bars: 0.5 mm for stages 2 and 3, 1.0 mm for the mature stage, and 0.2 mm for pollen germination. o, MeGI expressions in NH and AH flowers were not upregulated from male flowers (P = 0.82-0.97), and significantly lower than female flowers during the organ developmental processes (P < 0.005 at stage 2 and P < 8.0e-4 at stage 3 with a one-sided Student’s t-test; n = total 61 and 72 samples at stage 2 and 3, respectively, see detail in Supplementary Table 1). *** indicate P < 0.005 for statistical significance. P = 0.005, 0.004, 0.003 for M, NH, AH in comparison to F at stage 2, respectively. P = 8.0e-4, 5.0e-4, and 5.0e-4 for M, NH, AH in comparison to F at stage 3, respectively.

Extended Data Fig. 2 Clustering of differentially expressed genes (DEGs) between NH/AH and male flowers in stage 2.

a, Number of differentially expressed genes (DEGs) between NH/AH and male flowers in stage 2 (FDR < 0.1, bias > 1.2-fold). b, Expression patterns of NH and AH upregulated DEGs in stage 2 (n = 507 and 703, respectively). NH upregulated genes were overall downregulated in AH. c, Clustering of the 5,163 DEGs revealed 14 clusters. d, The heatmap shows the distinct expression patterns of the four flower types in stage 2. e, Expression patterns of the four flower types in each of the 14 clusters. NH-specific, AH-specific, and NH/AH-shared upregulated clusters were defined as Clst 10, Clst 4, and Clst 5, respectively. *** indicate P < 2.2e-16 with two-sided Student’s t-test. The boxes spanned the interquartile range (25th to 75th percentiles), the center line indicated the median values, and the whiskers extended to 1.5x interquartile range.

Extended Data Fig. 3 Comparison of the expression patterns of all Clst5 genes and the three transcription factors present in Clst 5 (other than DkRAD), between hexaploid D. kaki and diploid D. lotus.

a, Expression clustering with the Clst 5 genes, in the control male, NH, male treated with 100 mg l−1 ABA, and male treated with 10 ppm CK, both in D. kaki and in D. lotus. Only DkRAD (RADIALIS, highlighted in pink) and glycosyl transferase-like genes exhibited statistically supported upregulation specific to the conversion into hermaphrodite flowers. b-d, All three transcription factors showed upregulation after the treatment with 100 mg l−1 ABA, not only in D. kaki but also in D. lotus. Their expression activation patterns in hexaploid D. kaki were much less correlated to the gynoecium restoration patterns (see Fig. 2d). P-values were obtained by comparing expression values using a one-sided t-test (n = total 69 samples, see details in Supplementary Table 1). The boxes spanned the interquartile range (25th to 75th percentiles), the center line indicated the median values, and the whiskers extended to 1.5x interquartile range.

Extended Data Fig. 4 Evolutionary tree of the RADIALIS family genes in angiosperms.

a, Phylogenetic tree of RADIALIS-like genes from representative eudicot species. We selected outgroup genes in the Arabidopsis thaliana genome (RAD1 and RAD3 clades), based on the previous study on clustering of RADIALIS-like genes (Gao et al. 2017). RADIALIS from Antirrhinum majus, which was originally identified as the RADIALIS gene, and DkRAD and DkRAD2 (from D. kaki) were indicated in green and red, respectively. They were nested into the same clade, the RAD2, with significant statistic support (bootstrap value = 0.93). b, Phylogenetic tree of RADIALIS-like genes within the RAD2 clade, with the genes from various angiosperms species (homology e-value < 1e−30), using CCA1-like genes from A. thaliana as the outgroup. Although this clade was thought to be basically monophyletic, each lineage underwent frequent duplications. DkRAD and DkRAD2 were also derived from a lineage-specific duplication. c, Expression patterns of the two RADIALIS-like genes, DkRAD and DkRAD2, in hexaploid D. kaki and diploid D.lotus. DkRAD expression was not detectable in diploid D. lotus, or in stage 3 in D.kaki, but it was expressed in stage 2 flowers of D. kaki. DkRAD2 exhibited AH-specific upregulation in D. kaki (as evidenced by the fact that belongs to Clst 4, Fig. 1f) and was expressed at some level in all samples examined, including diploid D. lotus.

Extended Data Fig. 5 Various side effects of the DkRAD over-expression Arabidopsis and Nicotiana tabacum transgenic lines.

a-c, Ectopic over-expression of DkRAD resulted in failure of self-fertilization in A. thaliana. Siliques developed normally after self-pollination in the control, while DkRAD ox. lines were self-sterile. Phenotype of the control line and DkRAD over-expression line #12 were shown. DSd, deficient seeds; Sd, seed; Si, silique; SS, sterile silique; Sty, Style. d, Number of seeds in control and the DkRAD over-expression lines (n = 3–9 biological replicates, see detail in Supplementary Table 8). P-values were detected in comparison to the control #1 using two-sided Student’s t-test. Data were expressed as mean values ± SD. f, Cross-pollination with control lines could produce fertile silique, suggesting that female organs are functional in the DkRAD transgenic lines (n = 3-4 biological replicates). Comparison of the fertile seed numbers between open-pollination and artificial self-pollination or reciprocal crossing with the control in DkRAD ox. #12. All the artificial self-pollination and reciprocal cross-pollination exhibited recovery of the seed fertility from the open-pollination, supporting that physical distance between anthers and stigma would be the cause of sterility in the DkRAD ox. lines. P-values were detected in comparison to DkRAD ox. #12 using two-sided Student’s t-test. Data were expressed as mean values ± SD. g, The control and DkRAD ox. lines showed no substantial differences in ovule development (immediately before the anthesis). Self-sterility of the DkRAD ox. lines could be due to the physical distance between stigma and anthers, caused by the hyper-growth of carpel. h, DkRAD ox. lines showed severe growth retardance, although their flowering timing was not substantially delayed from the control lines. i-j, Precocious flowering in the transgenic lines in comparison to the control. The DkRAD and DkRAD2 over-expression lines often formed flower buds even in selection media immediately after the regeneration in vitro (i). k, The DkRAD2 over-expression lines exhibit enlarged carpel before flowering in comparison to the control. Cp, carpel; FB, flower bud; St, stamen.

Extended Data Fig. 6 Phenotypic variations in carpel development, in the progeny of DkRAD-ox x pSyGI-SyGI lines.

a, Half of the stigma exhibited restoration of normal development. The area on the left (highlighted with a pink asterisk showed the pSyGl-SyGl phenotype with shrunken stigma. b, Weak restoration of stigma development. c-d, Carpel development was severely repressed in pSyGI-SyGl plants in flowering stage (Akagi et al. 2018) (c), while DkRAD overexpression restored the carpel development, often resulting in overgrowth (d). Cp, carpel; Pe, petal; Sg, stigma; St; stamen. e, Characterization of gynoecium development and carpel length in the 4 independent DkRAD ox. x pSyGI-SyGI lines.

Extended Data Fig. 7 Comparison of gene expression patterns in DkRAD overexpressing and control A. thaliana flower, and D. kaki leaves.

a, Detection of differentially expressed genes (DEGs) between the transgenic lines of DkRAD ox. and control developing flowers in A. thaliana (stages 8–10). Distribution of the expression patterns of the DEGs. The X and Y axes correspond to the normalized expression level (RPKM) and DkRAD ox./control flowers ratio, respectively. The 330 upregulated and 187 downregulated DEGs are highlighted in blue circles (FDR < 0.1, Supplementary Table 10). The DEGs with known auxin- and defense/stress-signals functions, or those involved in cell cycle or circadian rhythm regulation functions are indicated with pink circles. b, Visualization of the co-expression network exhibiting upregulation in DKRAD ox. Transcription factors are aligned at the top. c,d, Detection of DEGs between control and transiently DkRAD overexpressing D. kaki leaves. The 1,413 upregulated and 595 downregulated DEGs are shown in blue circles (FDR < 0.1) (c). Ven diagram of the DEGs identified in transiently DkRAD overexpressing flowers (blue circles), NH flowers (deep green circles) and AH flowers (orange circle) in persimmon, compared to control flowers (Extended Data Fig. 2a and Supplementary Table 12) (d). e, Hypothetical regulatory pathway to produce hermaphrodite flowers from male flowers in persimmon. Activation of DkRAD expression, which integrates NH and AH signals, would lead to upregulation of auxin and stress responsive signaling, potentially via MYB73 expression, resulting in promotion of gynoecium growth.

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Supplementary Figs. 1–5 and Tables 1–14.

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Supplementary Table 1

Supplementary Tables 4, 6, 7, 10 and 12.

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Masuda, K., Ikeda, Y., Matsuura, T. et al. Reinvention of hermaphroditism via activation of a RADIALIS-like gene in hexaploid persimmon. Nat. Plants 8, 217–224 (2022). https://doi.org/10.1038/s41477-022-01107-z

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