Download Expression of the Floral B-function Gene SLM2 in Female Flowers of

Plant Cell Physiol. 46(5): 806–811 (2005)
doi:10.1093/pcp/pci080, available online at www.pcp.oupjournals.org
JSPP © 2005
Short Communication
Expression of the Floral B-function Gene SLM2 in Female Flowers of Silene
latifolia Infected with the Smut Fungus Microbotryum violaceum
Yusuke Kazama 1, Ayako Koizumi, Wakana Uchida 2, Amr Ageez and Shigeyuki Kawano
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, FSB-601, 5-1-5 Kashiwanoha, Kashiwa, Chiba,
277-8562 Japan
;
In higher plants, the floral primordia are arranged in four
different whorls, containing sepals, petals, stamens and carpels. The regulation of floral organ identity has been explained
by the ABC model (Coen and Meyerowitz 1991, Weigel and
Meyerowitz 1994), in which the A-function gene specifies
sepal formation in whorl 1, the combination of A- and B-function genes specifies the formation of petals in whorl 2, B- and
C-function genes specify stamen formation in whorl 3, and the
C-function gene determines the formation of carpels in whorl
4. Most of these genes encode MADS-box genes (Theissen et
al. 2000). In S. latifolia, nine MADS-box genes have been
identified: SLM1-5 (Hardenack et al. 1994), SlAP3A and
SlAP3Y (Matsunaga et al. 2003), and SlSEP1 and SlSEP3
(Matsunaga et al. 2004b). The C-function gene SLM1 and the
B-function genes SLM2 and SLM3, which are orthologs of
AGAMOUS (AG), PISTILLATA (PI) and APETALA3 (AP3),
respectively, show different patterns of expression in male and
female flower buds (Hardenack et al. 1994). Because there are
three AP3 homologs, i.e. SLM3 in the eu-AP3 lineage and
SlAP3A and SlAP3Y in the TM6 lineage, they are thought to be
functionally differentiated (Matsunaga et al. 2003). On the
other hand, the PI homolog is duplicated not in higher eudicots
but in lower eudicots and monocots (Kramer et al. 1998). The
S. latifolia PI homolog SLM2 is also a single copy gene
(Hardenack et al. 1994). The PI homolog would not be functionally differentiated in the higher eudicots such as S. latifolia.
The different expression patterns of these B-function genes in
male and female flower buds appear to be controlled by a sex
determination factor linked to the Y chromosome. In this study,
we analyzed the expression of SLM2 using in situ hybridization and determined that the control of this B-function gene
was independent of the presence of the Y chromosome.
In male (XY) flowers of S. latifolia, 10 stamens develop,
and the growth of carpels is suppressed (Fig. 1A). In female
(XX) flowers, a gynoecium composed of five fused carpels
develops, and the growth of stamens is suppressed (Fig. 1B).
However, when female plants are infected with the smut fungus, development of the 10 stamens is induced (Fig. 1C). Grant
et al. (1994) defined 12 stages in the floral development of
male and female plants, and scanning electron microscopy has
Silene latifolia is a dioecious plant in which sex is
determined by X and Y chromosomes. Expression of the Bfunction gene SLM2, an ortholog of PISTILLATA (PI) in
Arabidopsis, was examined by in situ hybridization. SLM2
was not expressed in suppressed stamens of female flowers,
but was expressed in developing stamens of smut-infected
female flowers. These results indicate that the control of
SLM2 is independent of the presence of the Y chromosome.
Smut-infected females provide a useful system for clarifying the relationship between the B-function gene and the
sex determination factor.
Keywords: Dioecious plant — Flower development — Microbotryum violaceum — PISTILLATA — Silene latifolia — Smut
fungus.
Abbreviations: DIG, digoxigenin; PBS, phosphate-buffered
saline; PCR, polymerase chain reaction.
Silene latifolia is a dioecious plant that possesses X and Y
sex chromosomes. XY plants develop male flowers with
stamens and an undifferentiated filamentous structure (a
suppressed carpel). Conversely, XX plants develop female
flowers with a normal gynoecium and arrest stamen development at an early stage. Sex determination in S. latifolia is regulated by the Y chromosome, which contains a putative sex
determination factor (for reviews, see Matsunaga and Kawano
2001, Negrutiu et al. 2001, Vyskot and Hobza 2004) and has
been shown to be insensitive to exogenous plant hormones
(Heslop-Harrison 1963, Ruddat et al. 1991).
The smut fungus Microbotryum violaceum infects S. latifolia and produces teliospores in the anthers of the host plant
(Antonovics and Alexander 1992). When M. violaceum infects
female flowers, suppression of stamen development does not
occur, and the development of anthers is induced (Fischer and
Holton 1957, Uchida et al. 2003), suggesting that most genes
directly responsible for male organ maturation are X and/or
autosome linked.
1
2
Corresponding author: E-mail, [email protected]; Fax, +81-4-7136-3674.
Present address: Molecular Membrane Biology Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.
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SLM2 gene expression in smut-infected female plants
807
Fig. 1 Morphology of healthy flowers of Silene latifolia and female flowers infected with the smut Microbotryum violaceum. Flowers were dissected with fine forceps. Some sepals and petals were removed to reveal the internal sex organs. (A) A healthy male flower lacking styles. (B) A
healthy female flower lacking stamens. (C) An infected female flower with styles, stamens, and smut-induced anthers. Bar = 1 cm.
shown that stamen development is initiated in the infected
female flower at stage 8 (Uchida et al. 2003).
To investigate the control of SLM2 expression, we performed in situ hybridization using a 439 bp fragment beginning downstream of the MADS domain of SLM2 as a probe.
Flower buds were fixed in 4% paraformaldehyde and embedded in paraffin. The digoxigenin (DIG)-labeled SLM2 probe
was hybridized to paraffin sections. The transcripts of SLM2
were first detectable at stage 3 in healthy males, healthy
females and infected females (Fig. 2A–C, respectively). SLM2
transcripts were not detected on the central dome of the meristem, but were limited to both whorl 2 and whorl 3 regions
where petal and stamen primordia, respectively, would later
develop (Fig. 2A–C, between the white arrowheads and black
arrowheads). These results from the healthy plants are in agreement with those of Hardenack et al. (1994).
We also examined the expression of SLM2 during later
stages in healthy and infected plants. SLM2 continued to be
expressed in whorl 2 and 3 regions during the later stages of
development. Fig. 2C shows the expression of SLM2 in an
infected female at stage 4. Expression of SLM2 was observed
in the petal and stamen primordia of healthy males, healthy
females and infected females until stage 7 (Fig. 2D–F). SLM2
transcripts were detected in developing petals and developing
stamens in healthy males at stage 8 (Fig. 2G). In the stamens
where SLM2 was expressed, anthers became distinguishable
from stamen filaments in male flowers. In healthy females at
stage 8, the developing pistil showed SLM2 signals, while the
stamen primordia did not (Fig. 2H); these stamen primordia did
not develop and the stamen filament did not elongate. In contrast to healthy females, infected females did not suppress the
expression of SLM2 in the stamens and showed SLM2 signals
in both the developing stamens and petals (Fig. 2I). The inability of infected females to suppress the expression of SLM2 may
have caused the elongation of the stamens.
In other plants, the expression of the PI homologs persists
during maturation of stamens and pistils (Tröbner et al. 1992,
Goto and Meyerowitz 1994, Kanno et al. 2003, Park et al.
2004). This continual expression is necessary to establish and
maintain petal and stamen identity (Zachgo et al. 1995). Therefore, we examined the expression pattern of SLM2 in both
healthy and infected plants during later maturation stages. The
elongation of stamens is initiated and anther locules are
rounded in healthy male plants at stage 9. To investigate the
expression patterns of SLM2 in the anthers after development
of the anther locules, we carried out in situ hybridization of
flower buds at stage 10. In healthy males, the transcripts of
SLM2 were observed in the developing petals and stamens
(Fig. 3A). Healthy females showed SLM2 expression only in
the developing petals (Fig. 3B). The stamens in which SLM2
expression was suppressed remained to be aborted. In the
flower bud of the infected females at stage i10, corresponding
to stage 10 in the healthy male (Uchida et al. 2003), transcripts
of SLM2 were detected in the developing petals and stamens
(Fig. 3C). In the anther locules of healthy males at stage 10,
meiotic tetrads were surrounded by developing tapetums, and
the epidermis differentiated. SLM2 was expressed in all of
these tissues (Fig. 3D), especially in the tapetums. In the anther
locules of infected females at stage i10, the tapetums and pollen mother cells degenerated, and many teliospores developed.
Therefore, the internal tissues of the anther locules resembled
meshwork in the paraffin sections, and the difference between
the epidermis and the mesh-like structure was less clear. SLM2
signals were observed in the whole tissues of the anther locules (Fig. 3E). The expression of SLM2 is probably necessary
for anther development, but is not sufficient for the differentiation of the internal tissue of the anther locules in infected
females.
Scanning electron micrography has revealed that the abortion of stamen development in female flowers of S. latifolia
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SLM2 gene expression in smut-infected female plants
Fig. 2 Distribution of SLM2 transcripts in healthy and infected flowers. Longitudinal sections of each stage of healthy males (A, D and G),
healthy females (B, E and H) and infected females (C, F and I) were subjected to in situ hybridization with digoxigenin-labeled SLM2 antisense
RNA probes. The developmental stages of healthy and infected flowers followed Grant et al. (1994) and Uchida et al. (2003), respectively. (A) A
healthy male flower at stage 3. (B) A healthy female flower at stage 3. (C) An infected female flower at stage 4. In the early stages, the width of
the central dome between the SLM2 signals in infected females is similar to that in healthy females. Filled arrowheads and closed arrowheads
indicate the boundary between the third and fourth whorls, and the boundary between the second and third whorls, respectively. (D) A healthy
male flower at stage 7. (E) A healthy female flower at stage 7. (F) An infected female flower at stage 7. Expression signals were detected both at
the petal primordia and at the developing stamens in all plants. (G) A healthy male flower at stage 8. The SLM2 transcript was detected at the
petal and stamen primordia. (H) A healthy female flower at stage 8. The expression of SLM2 was suppressed at the stamen primordia. (I) An
infected female flower at stage i8. The SLM2 was expressed both at the pistil primordia and at the developing stamen. (J) A healthy male flower at
stage 9. SLM2 expression continued both at the stamen and at the pistil primordia. (K) A healthy female flower at stage 9. SLM2 signals were
observed only at the pistil primordia. (L) An infected female flower at stage i9. SLM2 was expressed both at the stamen and at the pistil primordia., a, developing anther; dg, developing gynoecium; sg, suppressed gynoecium; p, developing pistil; ds, developing stamen; ss, suppressed stamen. Bars = 100 µm.
occurs at stage 8 (Grant et al. 1994, Uchida et al. 2003). At the
same time, SLM2 expression was suppressed in the stamen primordia of female flowers (Fig. 2H). The stage in which the
expression of SLM2 was suppressed corresponded to the stage
in which stamen development was aborted. Matsunaga et al.
(2004a) reported that the expression levels of a cyclin A1
homolog (SlCycA1) and a histone H4 homolog (SlH4) were
reduced in the stamen primordia of female flowers at stage 10.
It is likely that the suppression of SLM2 expression at stage 8
triggered the decline of cell division rates at stage 10. In contrast, the stamens of infected female flowers retained SLM2
expression, resulting in the elongation of the stamens. We suggest that the Y chromosome is not required to induce the SLM2
expression by which the maturation of stamens may be initiated.
SLM2 gene expression in smut-infected female plants
809
Fig. 3 In situ hybridization with digoxigenin-labeled SLM2 antisense RNA probes for the internal structures of anthers of S. latifolia flowers at
stage 10. (A) A longitudinal section of a healthy male flower at stage 10. SLM2 expression was detected at the anther whorl. The tetrads of
meiotic cells showed strong expression of SLM2. (B) A longitudinal section of a healthy female flower at stage 10. (C) A longitudinal section of
an infected female flower at stage i10. The internal structures of the anther did not differentiate. SLM2 expression, however, was observed in the
anther whorl. (D) and (E) are enlarged images of (A) and (C), respectively. ia, induced anther; p, developing pistil; t, tapetum; td, tetrad. Bars =
100 µm.
In Arabidopsis and Antirrhinum, the expression of PI
(Goto and Meyerowitz 1994) and GLOBOSA (Tröbner et al.
1992), which are orthologs of SLM2, persists during maturation of stamen and pistil organs. PI proteins form a transcription factor complex with AP3 and SEPALLATA3 proteins
(Honma and Goto 2001). This activates the expression of a
NAP gene, which is involved in the elongation of stamen cells
(Sablowski and Meyerowitz 1998). The SLM2 gene is therefore likely to be necessary for the maturation of stamens.
Although SLM2 continued to be expressed in the anthers
of infected females, male organogenesis did not occur (Fig.
3E). This result can be explained by at least two different
hypotheses. First, maturation of the smut teliospore could
inhibit male organogenesis. Secondly, a downstream target
gene activated by SLM2 could link to the Y chromosome. The
lack of downstream genes results in the failure of male organogenesis in infected females. Law et al. (2002) induced the
maturation of anthers in female plants with the use of silver
thiosulfate (Ag2S2O3), an ethylene inhibitor. Treatment with
this compound promoted early stamen development in females,
allowing meiosis to be completed, but did not allow for the
development of microspores into mature pollen grains. It is
thought that the genes involved in pollen maturation are linked
to the Y chromosome.
B-function genes have been isolated from a number of
dioecious plants that have been studied in connection with the
sex determination factor. In Rumex acetosa (Ainsworth et al.
1995) and Asparagus officinalis (Park et al. 2003), the expression of the AP3 orthologs RAD1, RAD2 and AODEF is suppressed in the developing stamen primordia of female flowers.
This suppression of B-function genes may be controlled by the
sex determination factor. In S. latifolia, although smut-infected
females do not possess the Y chromosome, SLM2 continues to
be expressed in whorl 3 and in the stamens (Fig. 2I). This suggests that the expression of B-function genes is not directly
controlled by the sex determination factor. On the other hand,
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SLM2 gene expression in smut-infected female plants
the size of whorl 4 in infected females was equal to that in
healthy females, but was larger than that in healthy males (Fig.
2A–C), suggesting that the size of whorl 4 may be unaffected
by the smut infection and may be directly regulated by the Y
chromosome. Although the correlation between B- or C-function genes and the Y chromosome has not been resolved thoroughly, smut-infected females are a useful counterpart for the
elucidation of this correlation.
We are grateful to Dr. R. Sugiyama from Toyama Medical and
Pharmaceutical University for technical support. We thank Dr. Sarah
R. Grant and Ms. Theresa F. Law for the generous gifts of S. latifolia
seeds and advice on infection methods. This work was supported by a
Grant-in-Aid for Scientific Research on Priority Areas to S.K.
(No.15013215) from the Ministry of Education, Science, Culture,
Sports, Science, and Technology of Japan.
Materials and Methods
References
An inbred S. latifolia line, K, was produced by 11 generations of
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(Received December 5, 2004; Accepted February 21, 2005)