Correlation between development of female flower buds and expression of the CS-ACS2 gene in cucumber plants

Ethylene plays a key role in sex determination of cucumber flowers. Gynoecious cucumber shoots produce more ethylene than monoecious shoots. Because monoecious cucumbers produce both male and female flower buds in the shoot apex and because the relative proportions of male and female flowers vary due to growing conditions, the question arises as to whether the regulation of ethylene biosynthesis in each flower bud determines the sex of the flower. Therefore, the expression of a 1-aminocyclopropane-1-carboxylic acid synthase gene, CS-ACS2, was examined in cucumber flower buds at different stages of development. The results revealed that CS-ACS2 mRNA began to accumulate just beneath the pistil primordia of flower buds at the bisexual stage, but was not detected prior to the formation of the pistil primordia. In buds determined to develop as female flowers, CS-ACS2 mRNA continued to accumulate in the central region of the developing ovary where ovules and placenta form. In gynoecious cucumber plants that produce only female flowers, accumulation of CS-ACS2 mRNA was detected in all flower buds at the bisexual stage and at later developmental stages. In monoecious cucumber, flower buds situated on some nodes accumulated CS-ACS2 mRNA, but others did not. The proportion of male and female flowers in monoecious cucumbers varied depending on the growth conditions, but was correlated with changes in accumulation of CS-ACS2 mRNA in flower buds. These results demonstrate that CS-ACS2-mediated biosynthesis of ethylene in individual flower buds is associated with the differentiation and development of female flowers.     

Sex-Biased Temporal Gene Expression in Male and Female Floral Buds of Seabuckthorn (Hippophae rhamnoides)

Seabuckthorn is an economically important dioecious plant in which mechanism of sex determination is unknown. The study was conducted to identify seabuckthorn homologous genes involved in floral development which may have role in sex determination. Forty four putative Genes involved in sex determination (GISD) reported in model plants were shortlisted from literature survey, and twenty nine seabuckthorn homologous sequences were identified from available seabuckthorn genomic resources. Of these, 21 genes were found to differentially express in either male or female flower bud stages. HrCRY2 was significantly expressed in female flower buds only while HrCO had significant expression in male flowers only. Among the three male and female floral development stages (FDS), male stage II had significant expression of most of the GISD. Information on these sex-specific expressed genes will help in elucidating sex determination mechanism in seabuckthorn.     

Isolation of a partial sequence of a putative nucleotide sugar epimerase, which may involve in stamen development in cucumber (Cucumis sativus L.)

Sex determination is the most widely studied subject in cucumber. The sex of cucumber plants can be monoecious, hermaphrodite, gynoecious, androecious, or andromonoecious. Besides environmental factors, three major genes, F/f, M/m, and A/a mainly govern the sex types in cucumber. Regardless of their sex all floral buds are bisexual at the early bud stage. A stage specific arrest of either stamen or carpel leads to unisexual flower development. The possible downstream product of the interaction of the sex determining genes that may directly allow the growth or selectively arrest stamen or pistil is not yet identified. Therefore, in the current study, we performed suppression subtractive hybridization using floral buds from nearly isogenic gynoecious and hermaphrodite cucumber plants and identified for the first time a cDNA homologous to nucleotide sugar epimerase. The expression level of the isolated putative nucleotide sugar epimerase is weak in female floral buds but strong in bisexual and male flowers. The weak level of the putative nucleotide sugar epimerase may be an indication for its improper functioning, which may influence stamen development in cucumber plants.     

Characterization of three anther-specific genes isolated from Chinese cabbage

Two cDNA libraries were constructed from poly(A)+ RNAs isolated from each of immature flowers (less than 2.0 mm long buds) and anthers (2.0-5.0 mm long buds) of Chinese cabbage (Brassica campestris L. ssp. pekinensis). Using dot-differential hybridization, three cDNA clones, designated BIF38, BAN54, and BAN237, have been isolated from the constructed cDNA libraries and sequenced completely in both directions. Northern blot analyses indicate that all three cDNA clones are abundantly expressed in anther, but not in leaf or other floral organs. The deduced amino acid sequences of BIF38, BAN54, and BAN237 showed high identity with those of known anther-specific genes. Especially the deduced amino acid sequence of BIF38 has 98% identity with that of a phospholipid protein gene (E2) from Brassica napus. Also, the deduced amino acid sequences of BAN54 and BAN237 are similar to the sequences of microspore-specific genes (Bp4A and Bp4C) and pollen oleosins (I3, pol3 and C98), respectively. Southern blot analyses revealed that all three genes belong to multiple gene families in the Chinese cabbage genome.     

Spatial expression dynamics of Men-9 delineate the third floral whorl in male and female flowers of dioecious Silene latifolia

Sex determination in Silene latifolia is controlled by heteromorphic sex chromosomes. Female flowers have five fused carpels and ten arrested stamen primordia. The male-determining Y chromosome overrides female development to suppress carpel formation and promote stamen development. The isolation and characterization of two S. latifoliaM ale en hanced cDNAs, Men-9a and Men-9b, which probably represent different alleles of a novel gene are reported here. Men-9a and Men-9b share 91.8% coding sequence nucleotide identity, yet only 85.4% amino acid identity. The Men-9 cDNAs are related to the previously reported MROS3 cDNA from S. latifolia. However, MROS3 is not present in the S. latifolia population used in these studies and the expression dynamics of Men-9a and Men-9b contrast dramatically with those reported for MROS3. Men-9 cDNAs are expressed primarily in anthers of young male flowers, with highest expression in 1–2 mm buds. Men-9 expression is also observed at a low level in female flowers. In situ hybridization analysis reveals two phases of Men-9 expression. The first phase is during a common stage of early stamen development in male and female flowers prior to stamen arrest in female flowers. The second phase of Men-9 expression is maximal in the epidermis and endothecium of Y chromosome- and Ustilago violacea-induced stamens; expression in male and female flowers extends to the epidermis of the staminal nectaries with strict boundaries at the second and fourth whorls. Men-9 gene expression therefore delineates the boundaries of the third floral whorl in S. latifolia flowers.     

Two novel transcripts expressed in pea dormant axillary buds

To elucidate the molecular mechanism of apical dominance, the expression patterns of genes that are preferentially expressed in dormant axillary buds of pea (Pisum sativum L. cv. Alaska) seedlings were investigated. We isolated two cDNA clones, cPsAD1 and cPsAD2 whose corresponding genes were named PsAD1 and PsAD2, from a cDNA library of dormant axillary buds using the differential display method. The deduced amino acid sequence of PsAD1 contains 87 residues and is rich in glycine residues in the amino terminal region. A search of the protein databases failed to find any sequences similar to PsAD1 protein except for the glycine-rich region. Northern blot analyses showed that PsAD1 mRNA mainly accumulated in dormant axillary buds and that its amount rapidly decreased after decapitation of the terminal bud. In situ hybridization analyses indicated that PsAD1 mRNA was localized in the apical meristem, procambia, and leaf primordia in dormant axillary buds that were competent to grow out but whose growth was temporarily suspended. That is, the expression of the PsAD1 gene is closely associated with the dormancy of axillary buds. The deduced amino acid sequence of PsAD2 contains 98 amino acid residues and is not similar to those of previously characterized proteins. PsAD2 mRNA accumulated in dormant axillary buds, roots, mature leaflets and elongated stems, suggesting that PsAD2 is involved in not only the dormancy of axillary buds but also the non-growing state in various tissues.     

Male-fertility genes expressed in male flower buds of Silene latifolia include homologs of anther-specific genes

When the female plant of Silene latifolia is infected with the smut fungus Microbotryum violaceum, its rudimentary stamens develop into anthers which contain fungus teliospores instead of pollen. To identify genes required for maturation of anthers in S. latifolia, we performed a cDNA subtraction approach with healthy male buds and female buds infected with M. violaceum. We isolated five cDNA clones, which were preferentially expressed in healthy male buds during stages associated with a burst in tapetal activity. These five cDNAs are predicted to encode a mandelonitrile lyase protein (SlMDL1), a strictosidine synthase protein (SlSs), a glycosyl hydrolase 17 protein (SlGh17), a proline-rich protein APG precursor (SlAPG), and a chalcone-synthase-like protein (SlChs). All five genes showed expression in both healthy and fungus-infected male buds, but not expressed in either healthy or infected female buds. The first three genes were highly expressed in both tapetum and pollen grains while the last two genes were expressed only inside the tapetum of male flower buds. Phylogenetic analysis results showed that SlChs and SlGh17 belong to anther-specific subgroups of chalcone-synthase-like genes and glycosyl hydrolase 17 family genes, respectively. Our results suggest that the isolated five genes are related to the fertility of the anther leading to the development of fertile pollen. The smut fungus was not able to induce the expression of the five genes in the infected female buds. This raises the possibility that these genes are under the control of master gene(s) on the Y chromosome.     

Expression of AODEF,a B-functional MADS-box gene,in stamens and inner tepals of the dioecious species Asparagus officinalis L.

Garden asparagus (Asparagus officinalis L.) is a dioecious species with male and female flowers on separate unisexual individuals. Since B- and C-functional MADS-box genes specify male and female reproductive organs, it is important to characterize these genes to clarify the mechanism of sex determination in monoecious and dioecious species. In this study, we isolated and characterized AODEF gene, a B-functional gene in the development of male and female flowers of A. officinalis. Southern hybridization identified a single copy of AODEF gene in asparagus genome. Northern blot analysis showed that this gene was specifically expressed in flower buds and not in vegetative tissues. In situ hybridization showed that during early hermaphrodite stages, AODEFgene was expressed in the inner tepal and stamen whorls (whorls 2 and 3, respectively), but not in the outer tepals (whorl 1), in both male and female flowers. In late unisexual developmental stages, the expression of AODEF gene was still detected in the inner tepals and stamens of male flowers, but the expression was reduced in whorls 2 and 3 of female flowers. Our results suggest that AODEF gene is probably not involved in tepal development in asparagus and that the expression of AODEF gene is probably controlled directly or indirectly by sex determination gene in the late developmental stages.     

MROS1, a Male Stamen-Specific Gene in the Dioecious Campion Silene latifolia Is Expressed in Mature Pollen

The MROS1 gene, one of the genes that are expressed specificallyin male reproductive organs of a dioecious campion Silene latifolia,was predicted to encode only 36 amino acids but have an intron.In situ hybridization revealed that MROS1 mRNA was localizedin mature pollen grains. (Received January 30, 1997; Accepted February 24, 1997)     

Water-deficit-responsive proteins in maritime pine

We have isolated three receptor-like kinase cDNAs from an Arabidopsis flower cDNA library by PCR using degenerate oligonucleotide primers for conserved domains of protein kinases. Cloning and sequencing of the full-length cDNAs, designated RKF1 to 3 (receptor-like kinase in flowers), showed that the putative extracellular domain of the RKF1 protein contains 13 tandem repeats of leucine-rich sequences and those of RKF2 and RKF3 have no significant homology with other plant sequences. RNA blot analysis revealed that the RKF1 mRNA is highly expressed in stamens while RKF2 and RKF3 mRNAs are present at low levels in all organs examined. In situ localization experiments indicated that the RKF1 mRNA is detectable in early flower primordia and during stamen development. In addition, when fused to a GUS reporter gene, the RKF1 promoter directed high GUS expression in pollen grains. Recombinant RKF1, produced in Escherichia coli, was found to have kinase activity with serine/threonine specificity in vitro.     

Development of distylous flowers and investment of biomass in male and female function in Palicourea padifolia (Rubiaceae)

Knowledge of developmental pathways for achieving differences in style and anther heights, in concert with those of ancillary features accompanied with data in regard to biomass investment to male and female function, provide an excellent opportunity for examining the developmental correlations between primary and ancillary floral traits so as to understand the evolution of heterostyly. The ontogenetic relationships between bud length and anther height and between bud length and style height, and between bud length versus bud width, anther length, and number of pollen grains per anther for long-styled (LS) and short-styled (SS) morphs of P. PADIFOLIA are described. We also described the ontogenetic biomass allocation to male and female function and to corolla with elongation of buds harvested at regular intervals. We observed an early termination of stylar growth in SS buds, whereas LS styles steadily increased in size. Morph differences for relative growth rates were significant for anther height, anther length, and pollen number but not for bud width. Bud width and anther length had a negative allometric relationship with bud elongation. The relationship between bud length and number of pollen grains per anther was positive and morph differences in pollen number were detected at later stages of development. An increase in corolla mass involved a disproportionate allocation to the female function in SS flowers and male allocation was similar for the two morphs over the course of development. Our results are consistent with theoretical and empirical data for distylous species with an approach herkogamous ancestor, and with the more general hypothesis of ontogenetic lability of heterostyly, in which morph differences in style and anther heights are achieved in various ways. Variations observed in sexual investment between floral morphs suggest differences in sex expression during flower development.     

Multicopy genes uniquely amplified in the Y chromosome-specific repeats of the liverwort Marchantia polymorpha

Sex of the liverwort Marchantia polymorpha is determined by the sex chromosomes Y and X, in male and female plant, respectively. Approximately half of the Y chromosome is made up of unique repeat sequences. Here, we report that part of the Y chromosome, represented by a 90-kb insert of a genomic clone pMM2D3, contains five putative genes in addition to the ORF162 gene, which is present also within the Y chromosome-specific repeat region. One of the five putative genes shows similarity to a male gamete-specific protein of lily and is expressed predominantly in male sex organs, suggesting that this gene has a male reproductive function. Furthermore, Southern blot analysis revealed that these five putative genes are amplified on the Y chromosome, but they also probably have homologs on the X chromosome and/or autosomes. These observations suggest that the Y chromosome evolved by co-amplifying protein-coding genes with unique repeat sequences.     

Acropetal disappearance of PsAD1 protein in pea axillary buds after the release of apical dominance

We recently isolated PsAD1 cDNA from pea (Pisum sativum L. cv. Alaska) seedlings, whose mRNA abundantly accumulated in dormant axillary buds and disappeared after decapitation [Madoka and Mori (2000) Plant Cell Physiol. 41: 274]. To further elucidate the function of PsAD1, we investigated the temporal and spatial distribution patterns of PsAD1 protein using Western blot and immunocytochemical analyses. Western blot analyses showed that accumulation patterns of PsAD1 protein in axillary buds after decapitation and in response to IAA and 6-benzyladenine were the same as those of PsAD1 mRNA. Immunocytochemical analyses showed that (1) PsAD1 proteins were localized in the procambia, leaf primordia, apical meristem, and secondary axillary buds in the dormant axillary bud, and this distribution was the same as that of PsAD1 mRNA, (2) PsAD1 proteins acropetally disappeared after decapitation, and (3) the growth of axillary buds occurred in the same manner. These acropetal changes occur in a manner similar to the way in which the procambium differentiates into vascular tissue. These results suggest that PsAD1 plays some role in the inhibition of growth and differentiation, or in the maintenance of the dormant state in axillary buds.     

The Control of Sex Expression in Cucurbits by Ethephon

Application of ethephon to field-grown plants of both bush andtrailing forms of Cucurbita maxima and C. pepo caused leaf epinasty,suppression of male flowers and earlier production and increasein numbers of female flowers. This gave rise to an increasein the ratio of female to male flowers per plant and a decreasein the total number of flowers. Observations of C. pepo showed that even at the two true leafstage there are several nodes present in the unexpanded shoot.Each node has one main and several secondary buds. The sex ofthe main bud at the first five to six nodes is usually determinedat this stage but the secondary buds still have bisexual potential.The change in sex expression was brought about by all male flowerbuds that had formed by the spraying time aborting, and allbuds that developed (both main and secondary) for at least 7days after spraying became female flowers. Thus, nodes fiveand six had male flowers in the controls, whereas in ethephon-sprayedplants the presumptive male flowers aborted at the bud stageat these nodes and secondary primordia developed into functionalfemale flowers. Cucurbita maxima, Cucurbita pepo, sex expression, ethephon, ethylene, flower abortion, flower differentiation