Ⅰ. HIGHLIGHTS OF THE YEAR
RIKEN
Accel. Prog. Rep. 56 (2023)

RIKEN Accel. Prog. Rep. 56 (2023)

Identiﬁcation of a sex determining gene in a dioecious plant Silenelatifolia †Y. Kazama,∗1,∗2 M. Kitoh,∗1 T. Kobayashi,∗1 K. Ishii,∗2,∗3 M. Krasovec,∗4,∗5 Y. Yasui,∗6 T. Abe,∗2 S. Kawano,∗7
and D. A. Filatov∗4
Silene latifolia is a dioecious ﬂowering plant with
XY-type sex chromosomes belonging to the Caryophyllaceae family. Since its discovery in 1923 as one of theﬁrst species with sex chromosomes discovered in higher
plants, it has been used for studies of sex chromosome
evolution and sex-determining mechanisms in plants.1,2)
Two sex-determining genes have been postulated to bepresent on the Y chromosome: the male-promoting gene(SPF) and the female-suppressor gene (GSF). However,the Y chromosome is 1arge (approx. 660 Mb) and mostof it comprises a non-recombining region and occupied
by repetitive sequences, which has prevented genome sequence assembly and rendered the identiﬁcation of sexdetermining genes diﬃcult.
We irradiated S. latifolia with heavy-ion beams to obtain several hermaphroditic (GSF-deﬁcient) and asexual (SPF-deﬁcient) mutants, and mapped the shareddeleted regions of mutants as two sex-determining regions, respectively.3) Genome sequencing and RNA-seq
were performed on two of the hermaphroditic mutants
with small deletion sizes. Sequences were then compared
between male, female, and mutants. Consequently, wefound three genes that were present and expressed onlyin males. One gene was commonly deleted in both ofthe 11 hermaphroditic mutants and females, which wasidentiﬁed as a candidate gene for GSF. Further, a Blast
search revealed that this gene was highly homologous tothe Arabidopsis CLV3 gene, which regulates the size ofthe shoot apical meristems (SAMs) and ﬂower bud primordia.4)
clv3 mutants exhibited increased carpel size, whiletransgenic plants with overexpression of CLV3 showed
suppressed carpel development, suggesting that this geneis a promising candidate for the GSF. Furthermore, inaddition to the Y copy (GSFY ), we found its X-lined
gametolog (GSFX ). RT-PCR results showed that both
GSFY and GSFX were expressed in the shoot apicalmeristems and young ﬂower buds. The translation product of CLV3 is known to act as a peptide with 12 aminoacid residues in the ﬁnal product. Therefore, we compared the amino acid residue sequences of GSFY andGSFX peptides and found that the sixth glycine residuein GSFX was mutated to an alanine residue. As this†
∗1
∗2
∗3
∗4
∗5
∗6
∗7

Condensed from the article in Mol. Biol. Evol. 39, msac195(2022)
Department of Bioscience and Biotechnology, Fukui Prefectural University
RIKEN Nishina Center
National Institutes for Quantum Science and Technology(NIRS)
Department of Biology, Oxford University
CNRS, Sorbonne University
Graduate School of Agriculture, Kyoto University
Graduate School of Frontier Sciences, University of Tokyo

mutation is the same as that of the Arabidopsis clv31/clv3-5 mutant (CLV3m), GSFX was considered to bea loss-of-function type. When these peptides were artiﬁcially synthesized and treated to the SAMs of S. latifolia.
GSFY was found to be active in suppressing SAM size,
similar to CLV3, where GSFX and CLV3m were inactive. Next, when GSFY was introduced into Arabidopsis
thaliana, a hermaphroditic plant, the carpel development
was inhibited. However, introduction of GSFX did notinhibit development of the carpel. Furthermore, treatment of ﬂower buds of S. latifolia with the GSFY peptide
also inhibited the development of the pistils. These results indicate that GSFY, a homolog of the CLV3 gene,is a likely candidate of the sex-determining gene thatsuppresses gynoecium development in S. latifolia, andthat GSFX on the X chromosome is dysfunctional.
We have previously shown that, in contrast to GSFY,SlWUS1, which is considered to function in enlarging thecarpels, is present on the X chromosome but not on theY chromosome in S. latifolia.5) This suggests that thefunction of the X chromosome functions is to promote
female development. In fact, there is a report in theold literature that the creation of XXXXY pentaploid
chromosomes cause the carpels to enlarge and produce
hermaphroditic ﬂowers.6) The manner in which the Y
chromosome acquired the dominant GSFY during theevolution of sex chromosomes has been the greatest mystery; however, this system, wherein the X chromosome
enlarges the carpels and the Y chromosome reduces its
size, can successfully explain the emergence of the dominant GSFY (Fig. 1).

Fig. 1. Model for the evolution of S. latifolia sex chromosomes with GSFX dysfunction and loss of SlWUS1 inthe Y chromosome.

References
1) E. Kejnovsky et al., Cytogenet. Genome Res. 129, 250(2010).
2) D. Charlesworth, Genetics 210, 1143 (2018).
3) Y. Kazama et al., Sci. Rep. 6, 18917 (2016).
4) J. C. Fletcher et al., Science 283, 1911 (1999).
5) Y. Kazama et al., G3 (Bethesda) 2, 1269 (2012).
6) H. E. Warmke, Am. J. Bot. 33, 648 (1946).

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