GENETICS OF GRACILARIA TIKVAHIAE (RHODOPHYCEAE). X. STUDIES ON A BISEXUAL CLONE12

A male done of the red alga Gracilaria tikvahiae McLachlan spontaneously produced a bisexual frond which remained bisexual in subsequent subcultures. Both male and female components of bisexual fronds were functional; however, some unusual results were obtained in crosses. When bisexual fronds were crossed with a normal haploid male, the resulting carpospores all developed into diploid male gametophytes. When bisexual plants were self fertilized, all the carpospores yielded diploid bisexual gametophytes. Only when bisexual plants were crossed to normal haploid females, did carpospores develop into diploid tetrasporophytes as they normally do. The F₁ gametophyte generation obtained from these tetrasporophytes, however, included not only females and males but also bisexual plants, in a 2:1:1 ratio. These results are consistent with the interpretation that bisexual plants have a recessive mutation of a gene other than the primary sex determining locus, and that this mutation is expressed only in male plants. It is suggested that the altered gene may ordinarily have a regulatory function in the maintenance of the dioecious condition.     

Sporophyte formation in experimentally-induced unisexual female and bisexual gametophytes ofLygodium japonicum

Unisexual female and male and bisexual gametophytes were experimentally induced inLygodium japonicum. A single bisexual gametophyte was isolated in a dish and a female gametophyte was paired with a male one to allow intragametophytic selfing and intergametophytic mating, respectively. About 30% of the females formed sporophytes but no bisexual gametophytes formed them.     

Localization of a Female-Specific Marker on the Chromosomes of the Brown Seaweed Saccharina japonica Using Fluorescence In Situ Hybridization

Background

There is a heteromorphic alternative life in the brown seaweed, Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders ( = Laminaria japonica Aresch.), with macroscopic monoecious sporophytes and microscopic diecious gametophytes. Female gametophytes are genetically different from males. It is very difficult to identify the parent of a sporophyte using only routine cytological techniques due to homomorphic chromosomes. A sex-specific marker is one of the best ways to make this determination.

Methodology/Principal Findings

To obtain clear images, chromosome preparation was improved using maceration enzymes and fluorochrome 4′, 6-diamidino-2-phenylindole (DAPI). The chromosome number of both male and female haploid gametophytes was 31, and there were 62 chromosomes in diploid sporophytes. Although the female chromosomes ranged from 0.77 µm to 2.61 µm in size and were larger than the corresponding ones in the males (from 0.57 µm to 2.16 µm), there was not a very large X chromosome in the females. Based on the known female-related FRML-494 marker, co-electrophoresis and Southern blot profiles demonstrated that it was inheritable and specific to female gametophytes. Using modified fluorescence in situ hybridization (FISH), this marker could be localized on one unique chromosome of the female gametophytes as well as the sporophytes, whereas no hybridization signal was detected in the male gametophytes.

Conclusions/Significance

Our data suggest that this marker was a female chromosome-specific DNA sequence. This is the first report of molecular marker localization on algal chromosomes. This research provides evidence for the benefit of using FISH for identifying molecular markers for sex identification, isolation of specific genes linked to this marker in the females, and sex determination of S. japonica gametophytes in the future.     

Molecular differentiation of sex chromosomes probed by comparative genomic hybridization

Comparative genomic hybridization (CGH) was used to identify and probe sex chromosomes in several XY and WZ systems. Chromosomes were hybridized simultaneously with FluorX-labelled DNA of females and Cy3-labelled DNA of males in the presence of an excess of Cot-1 DNA or unlabelled DNA of the homogametic sex. CGH visualized the molecular differentiation of the X and Y in the house mouse, Mus musculus, and in Drosophila melanogaster: while autosomes were stained equally by both probes, the X and Y chromosomes were stained preferentially by the female-derived or the male-derived probe, respectively. There was no differential staining of the X and Y chromosomes in the fly Megaselia scalaris, indicating an early stage of sex chromosome differentiation in this species. In the human and the house mouse, labelled DNA of males in the presence of unlabelled DNA of females was sufficient to highlight Y chromosomes in mitosis and interphase. In WZ sex chromosome systems, the silkworm Bombyx mori, the flour moth Ephestia kuehniella, and the wax moth Galleria mellonella, the W chromosomes were identified by CGH in mitosis and meiosis. They were conspicuously stained by both female- and male-derived probes, unlike the Z chromosomes, which were preferentially stained by the male-derived probe in E. kuehniella only but were otherwise inconspicuous. The ratio of female:male staining and the pattern of staining along the W chromosomes was species specific. CGH shows that W chromosomes in these species are molecularly well differentiated from the Z chromosomes. The conspicuous binding of the male-derived probe to the W chromosomes is presumably due to an accumulation of common interspersed repetitive sequences. Received: 6 January 1999; in revised form: 28 January 1999 / Accepted: 11 February 1999     

Exportin1 genes are essential for development and function of the gametophytes in Arabidopsis thaliana

Gametes are produced in plants through mitotic divisions in the haploid gametophytes. We investigated the role of EXPORTIN1 (XPO1) genes during the development of both female and male gametophytes of Arabidopsis. Exportins exclude target proteins from the nucleus and are also part of a complex recruited at the kinetochores during mitosis. Here we show that double mutants in Arabidopsis XPO1A and XPO1B are gametophytic defective. In homozygous–heterozygous plants, 50% of the ovules were arrested at different stages according to the parental genotype. Double-mutant female gametophytes of xpo1a-3/+; xpo1b-1/xpo1b-1 plants failed to undergo all the mitotic divisions or failed to complete embryo sac maturation. Double-mutant female gametophytes of xpo1a-3/xpo1a-3; xpo1b-1/+ plants had normal mitotic divisions and fertilization occurred; in most of these embryo sacs the endosperm started to divide but an embryo failed to develop. Distortions in male transmission correlated with the occurrence of smaller pollen grains, poor pollen germination, and shorter pollen tubes. Our results show that mitotic divisions are possible without XPO1 during the haploid phase, but that XPO1 is crucial for the maternal-to-embryonic transition.     

Characterization of backcross generations obtained under field conditions from oilseed rape-wild radish F1 interspecific hybrids: an assessment of transgene dispersal

Gene flow from glufosinate-resistant transgenic oilseed rape to wild radish was studied over two backcross generations. Under field conditions,?seed production from oilseed rape-wild radish F₁ hybrids due to pollination by wild radish was always low: on average 0.12 and 0.78 seeds per 100 flowers and per plant, respectively. The cytogenetics of the resulting «BC₁» plants can be explained in the main by three different genomic constitutions: either ACRrRr, 2n=37, ACRr, 2n=28 (the same chromosome number as the mother plant), or by the amphidiploid AACCRrRr, 2n=56. The probability of gene exchange through chromosome pairing was high only in plants with 2n=28 or 37 chromosomes. Due to the viability of unreduced or partially reduced female gametes, most of the «BC₁» plants (81.9%) were Basta resistant whereas the analysis of oilseed rape specific loci indicated that their transmission varied with the locus. In spite of low male fertility (8.7%), an improvement of the female fertility over the F₁ hybrids was observed with an average production of 1.4 and 11 seeds per 100 flowers and per plant, respectively. At the following «BC₂» generation, the bar gene transmission (57.2% of Basta-resistant plants) decreased as did the chromosome number, with a majority of plants having between 24 and 27 chromosomes, with 10.5% similar to wild radish (2n=18). The lower the chromosome number, the better the fertility of the «BC₂» plants. On average, 7.9 and 229.3 seeds per 100 flowers and per plant were produced. Gene-flow assessment is discussed based on these data.     

麦秆蹄盖蕨（Athyrium fallaciosum）配子体的发育规律及播种密度对配子体性别分化的影响

在组织培养条件下,对麦秆蹄盖蕨（Athyrium fallaciosum）配子体发育的连续过程进行了详细观察。结果表明:麦秆蹄盖蕨孢子为四面体型; 孢子萌发为书带蕨型（Vittaria-type）;原叶体发育为铁线蕨型（Adiantum-type）,成熟原叶体为对称的心形;精子器近圆球形,成熟颈卵器细长,常向原叶体基部倾斜或弯曲。常规播种条件下,发现麦秆蹄盖蕨配子体有雌配子体、雄配子体、雌雄同体配子体和无性配子体类型。配子体的性别随密度不同而呈现一定的变化趋势,雄配子体随密度增大呈上升趋势;雌配子体随密度增大先上升后下降;雌雄同体配子体和无性配子体随密度变化不大。雌配子体和雌雄同体配子体具颈卵器数目一般为10～15个;精子器数目随密度的增大逐渐减少,雄配子体中具有约50个精子器,雌雄同体配子体具有约20个精子器。     

AN ELECTROPHORETIC INVESTIGATION OF INTRAGAMETOPHYTIC SELFING IN EQUISETUM ARVENSE

Because homosporous pteridophytes (Psilotophyta, Arthrophyta, most Microphyllophyta and Pteridophyta) produce bisexual gametophytes, it was maintained that high levels of inbreeding would characterize these plants. Electrophoretic evidence was used to estimate the frequency of intragametophytic selfing in Equisetum arvense (Arthrophyta). A total of 669 samples from 17 populations was examined from western North America. Although some populations exhibited as many as seven or eight genotypes, 10 populations were each characterized by only a single genotype; eight of these populations were heterozygous for one or more loci. For most populations, estimates of intragametophytic self-fertilization are 0.000, indicating that virtually all matings involve different gametophytes. Genetic data corroborate predictions based on earlier field and laboratory investigations of Equisetum gametophytes. These detailed studies demonstrated that in many species, including E. arvense, gametophytes are initially either male or female; only later and in the absence of fertilization do some gametophytes become bisexual. Our findings join a growing electrophoretic data base which demonstrates that homosporous pteridophytes are not highly inbreeding as previously suggested.     

Gametophytic phase of Alsophila odonelliana (Cyatheaceae)

The gametophytic morphology and development of Alsophila odonelliana (Alston) Lehnert, have been studied through in vitro cultures. This species grows in southern Bolivia and northwestern Argentina. The spores are uniform in structure, but not in size; a certain percentage being smaller than the average. 16 spores per sporangium were found. The germination is of the Cyathea type. It was found that spores stored at 4°C can maintain their viability for over two years. The maximum value of germination depends on spore age. The filamentous gametophytes are 4–16 cells long. Young gametophytes have 1–2 branches that give rise to new gametophytes. Male, female, bisexual and neuter gametophytes were found. Propagules were frequently found in neuter gametophytes, and female and bisexual gametophytes were found to have chlorophyll containing scales. The antheridia are made up of five cells and produce non‐viable spermatozoids. The archegonia have necks formed by four columns with four cells each. Most of the gametophytic phase is documented with photomicrographs.     

Temporal variations of vegetative features, sex ratios and reproductive phenology in a Dictyota dichotoma (Dictyotales, Phaeophyceae) population of Argentina

This paper addresses the phenology of a Dictyota dichotoma population from the North Patagonian coasts of Argentina. The morphology of the individuals was characterized, and analyses of the temporal variations of vegetative features, diploid and haploid life cycle generations and sex ratios are provided. Individuals, represented by growing sporophytes and gametophytes, occurred simultaneously throughout the year. Morphological variables showed temporal variation, except the width and height of medullary cells, which did not vary between seasons. All vegetative variables were significantly correlated with daylength. Besides, frond length, frond dry mass and apical and basal branching angles were significantly correlated with seawater temperatures. Vegetative thalli were less abundant than haploid and diploid thalli. Sporophytes were less abundant than male and female gametophytes. Male gametophytes dominated in May, August, October and January, and female gametophytes were more abundant in September, November, December, February and March. The formation of female gametangia showed a significant correlation with daylength, and the highest number of gametangia was registered in spring. In general, the male/female sex ratio varied between 1:2 and 1:1. Apical regions were more fertile than basal regions. Our data about frequency in the formation of reproductive structures and male/female ratios are the first recorded in the Dictyota genus and thus could not be compared with populations from other regions of the world. Significant morphological variation was observed in thalli of both life cycle generations, regarding length and dry mass, number of primary branches and branching basal angle. In general, all variables analyzed varied seasonally except cortical cell width.     

The resurgence of haploids in higher plants

The life cycle of plants proceeds via alternating generations of sporophytes and gametophytes. The dominant and most obvious life form of higher plants is the free-living sporophyte. The sporophyte is the product of fertilization of male and female gametes and contains a set of chromosomes from each parent; its genomic constitution is 2n. Chromosome reduction at meiosis means cells of the gametophytes carry half the sporophytic complement of chromosomes (n). Plant haploid research began with the discovery that sporophytes can be produced in higher plants carrying the gametic chromosome number (n instead of 2n) and that their chromosome number can subsequently be doubled up by colchicine treatment. Recent technological innovations, greater understanding of underlying control mechanisms and an expansion of end-user applications has brought about a resurgence of interest in haploids in higher plants.     

HAPLOID PARTHENOGENETIC SPOROPHYTES OF LAMINARIA JAPONICA (PHAEOPHYCEAE)1

Parthenogenetic sporophytes were obtained from three strains of Laminaria japonica Areschoug. These sporophytes grew to maturity in the sea, producine spores that all grew into female gametophytes. These female gametophytes gave rise to another generation of parthenogenetic sporophytes during the next year, so that by the year 1990 parthenogenetic sporophytes had been cultivated for 12, 9, and 7 generations, respectively, for the three strains. When female gametophytes from parthenogenetic sporophytes were combined with normal male gametophytes, normal sporophytes that reproduced and gave rise to both female and male gametophytes were obtained. The parthenogenetic sporophytes were shorter and narrower than the normal sporophytes of the same strain. Chromosome counts on mature sporophytes showed that normal sporophytes (from fertilized eggs) were diploid (2n = approximately 40) and that the spores they produced were haploid (n = approximately 20), while nuclei from both somatic and sporangial cells in parthenogenetic sporophytes were haploid. All gametophytes were haploid. Young sporophytes derived from cultures with both female and male gametophytes were diploid, while young, sporophytes obtained from female gametophytes from parthenogenetic sporophytes had haploid, diploid, or polyploidy chromosome numbers. Polyploidy was associated with abnormal cell shapes. The presence of haploid parthenogenetic sporophytes should be use in breeding kelp strains with useful characteristics, since the sporophyte phenotype is expressed from a haploid genotype which can be more readily selected.     

A positive feedback loop mediated by CsERF31 initiates female cucumber flower development

Sex determination is a crucially important developmental event that is pervasive throughout nature and enhances the adaptation of species. Among plants, cucumber (Cucumis sativus L.) can generate both unisexual and bisexual flowers, and the sex type is mainly controlled by several 1-aminocyclopropane-1-carboxylic acid synthases (CsACSs). However, the regulatory mechanism of these synthases remains elusive. Here, we used gene expression analysis, protein–DNA interaction assays, and transgenic plants to study the function of a gynoecium-specific gene, ETHYLENE RESPONSE FACTOR31 (CsERF31), in female flower differentiation. We found that in a predetermined female flower, ethylene signaling activates CsERF31 by CsEIN3, and then CsERF31 stimulates CsACS2, which triggers a positive feedback loop to ensure female rather than bisexual flower development. A similar interplay is functionally conserved in melon (Cucumis melo L.). Knockdown of CsERF31 by RNAi causes defective bisexual flowers to replace female flowers. Ectopic expression of CsERF31 suppresses stamen development and promotes pistil development in male flowers, demonstrating that CsERF31 functions as a sex switch. Taken together, our data confirm that CsERF31 represents the molecular link between female–male determination and female–bisexual determination, and provide mechanistic insight into how ethylene promotes female flowers, rather than bisexual flowers, in cucumber sex determination.

A key regulator promotes female flower development by triggering a positive feedback loop during cucumber sex determination.     

Epigenetic control of sexual phenotype in a dioecious plant,Melandrium album

Melandrium album (syn.Silene latifolia) is a model dioecious species in which theY chromosome, present only in heterogametic males, plays both a male-determining and a strict female-suppressing role. We showed that treatment with 5-azacytidine (5-azaC) induces a sex change to androhermaphroditism (andromonoecy) in about 21% of male plants, while no apparent phenotypic effect was observed in females. All of these bisexual androhermaphrodites (with the standard male 24,AA +XY karyotype) were mosaics possessing both male and hermaphrodite flowers and, moreover, the hermaphrodite flowers displayed various degrees of gynoecium development and seed setting. Southern hybridization analysis with a repetitive DNA probe showed that the 5-azacytidine-treated plants were significantly hypomethylated in CG doublets, but only to a minor degree in CNG triplets. The bisexual trait was transmitted to two successive generations, but only when androhermaphrodite plants were used as pollen donors. The sex reversal was inherited with incomplete penetrance and varying expressivity. Based on the uniparental inheritance pattern of androhermaphroditism we conclude that it originated either by 5-azaC induced inhibition ofY-linked female-suppressing genes or by a heritable activation of autosomal female-determining/promoting genes which can be reversed, on passage through female meiosis, by a genomic imprinting mechanism. The data presented indicate that female sex suppression inM. album XY males is dependent on methylation of specific DNA sequences and can be heritably modified by hypomethylating drugs.     

大叶铁线莲大小孢子发生及雌雄配子体发育

铁线莲属植物在花部形态和结构方面存在较大差异, 遗传背景相对复杂。因此, 在杂交育种前对其进行胚胎学研究具有重要意义。利用石蜡切片技术对大叶铁线莲(Clematis heracleifolia)大小孢子发生及雌雄配子体发育过程进行研究, 结果显示, 大叶铁线莲具雄株和两性花植株。雄花中, 雄配子体发育偶见败育现象; 而两性花中多数花粉发育异常, 形成功能性雌花。正常发育的两性花中, 雄蕊较雌蕊先发育完全。花药4室, 具腺质绒毡层, 偶见变形绒毡层。胞质分裂为同时型, 以四面体型四分体为主, 偶见左右对称型。成熟花药中, 花药壁由纤维状加厚的表皮及药室内壁构成, 花粉粒为2-细胞型, 近球状, 散沟型。子房1室, 内含少量退化胚珠及1个发育正常的胚珠, 倒生, 单珠被, 薄珠心, 蓼型胚囊, 具线形大孢子四分体及双核反足细胞。大叶铁线莲可能处于相对进化的过渡地位。在杂交育种中, 建议以雄花植株作为父本, 两性花植株仅用作母本; 在两性花花芽大小为0.5-0.8 cm时进行去雄处理。     

大叶铁线莲大小孢子发生及雌雄配子体发育

铁线莲属植物在花部形态和结构方面存在较大差异, 遗传背景相对复杂。因此, 在杂交育种前对其进行胚胎学研究具有重要意义。利用石蜡切片技术对大叶铁线莲(Clematis heracleifolia)大小孢子发生及雌雄配子体发育过程进行研究, 结果显示, 大叶铁线莲具雄株和两性花植株。雄花中, 雄配子体发育偶见败育现象; 而两性花中多数花粉发育异常, 形成功能性雌花。正常发育的两性花中, 雄蕊较雌蕊先发育完全。花药4室, 具腺质绒毡层, 偶见变形绒毡层。胞质分裂为同时型, 以四面体型四分体为主, 偶见左右对称型。成熟花药中, 花药壁由纤维状加厚的表皮及药室内壁构成, 花粉粒为2-细胞型, 近球状, 散沟型。子房1室, 内含少量退化胚珠及1个发育正常的胚珠, 倒生, 单珠被, 薄珠心, 蓼型胚囊, 具线形大孢子四分体及双核反足细胞。大叶铁线莲可能处于相对进化的过渡地位。在杂交育种中, 建议以雄花植株作为父本, 两性花植株仅用作母本; 在两性花花芽大小为0.5-0.8 cm时进行去雄处理。     

Cytokinin receptors in sporophytes are essential for male and female functions in Arabidopsis thaliana

Arabidopsis has three cytokinin receptors genes: CRE1, AHK2 and AHK3. Availability of plants that are homozygous mutant for these three genes indicates that cytokinin receptors in the haploid cells are dispensable for the development of male and female gametophytes. The triple mutants form a few flowers but never set seed, indicating that reproductive growth is impaired. We investigated which reproductive processes are affected in the triple mutants. Anthers of mutant plants contained fewer pollen grains and did not dehisce. Pollen in the anthers completed the formation of the one vegetative nucleus and the two sperm nuclei, as seen in wild type. The majority of the ovules were abnormal: 78% lacked the embryo sac, 10% carried a female gametophyte that terminated its development before completing three rounds of nuclear division, and about 12% completed three rounds of nuclear division but the gametophytes were smaller than those of the wild type. Reciprocal crosses between the wild type and the triple mutants indicated that pollen from mutant plants did not germinate on wild-type stigmas, and wild-type pollen did not germinate on mutant stigmas. These results suggest that cytokinin receptors in the sporophyte are indispensable for anther dehiscence, pollen maturation, induction of pollen germination by the stigma and female gametophyte formation and maturation.Key words: cytokinin, cytokinin receptor, female gametophyte, male gametophyte, stigma     

REGENERATION AND SEXUAL DIFFERENTIATION OF GRIFFITHSIA JAPONICA (CERAMIACEAE,RHODOPHYTA) THROUGH SOMATIC CELL FUSION1

Hybrid cells were obtained from somatic cell fusion among male, female, and tetrasporangial plants in Griffithsia japonica Okamura by a wound-healing process. Isolated fusion cells regenerated new mature plants with mixed reproductive structures. The plants regenerated from hybrid cells between male and female plants developed into 1) spermatangiate, 2) carpogonial, 3) bisexual with spermatangia and carpogonial branches, 4) mixed-phase with spermatangia and tetrasporangia, or 5) bisexual/mixed-phase plants with spermatangia, carpogonial branches, and tetrasporangia. About 70% of the plants regenerated from hybrid cells between male and female plants produced tetrasporangia that were always formed with spermatangia on a single cell. Some of those tetrasporangia released tetraspores, six of which gave rise to mature plants. The plants regenerated from hybrid cells between male and tetrasporangial plants developed into spermatangiate, tetrasporangiate, or mixed-phase plants with spermatangia and tetrasporangia. The plants regenerated from hybrid cells between female and tetrasporangial plants developed into carpogonial, tetrasporangiate, or mixed-phase plants with carpogonial branches and tetrasporangia. All types of reproductive structures we re functional.     

CULTURE AND HYBRIDIZATION STUDIES ON PETROCELIS (RHODOPHYTA) FROM ALASKA AND CALIFORNIA1,2

Cultured tetraspores of Petrocelis middendorffii (Ruprecht) Kjellman from Amchitka Island, Alaska, gave rise to foliose, dioecious gametophytes similar to cultured gametophytes of P. franciscana Setchell & Gardner. A 1:1 ratio male:female gametophytes was obtained. Fertilized female plants produced cystocarps and carpospores that gave rise to crustose plants anatomically similar to field-collected Petro-celis sporophytes. Cultured male gametophytes of P. middendorffii were interfertile with cultured female blades of field-collected Gigartina pacifica Kjellman. Cultured P. middendorffii gametophytes from Amchitka were interfertile with cultured gametophytes of P. franciscana from 2 localities in California. Hybrid carpospores gave rise to crustose sporophytes that have not reproduced. Anatomical comparisons of P. middendorffii from Amchitka with P. franciscana from California showed no important differences in the characters originally used to separate these species. The interfertility of cultured Petrocelis gametophytes from california and Amchitka as well as the similarities of the history and anatomy suggests that a single species is involved. P. franciscana is reduced to a synonym of P. middendorfii.     

Cytological studies on Lemanea fluviatilis L. in the River Usk

Seasonal studies of nuclear phases in clonal and naturally occurring plants of Lemanea fluviatilis L. in the River Usk have shown that N = 18–19 and 2N = 36–38.

Carposporophytes, carpospores, the Chantransia-stage and lower cells of gametophyte thalli arising from the latter were all diploid. Meiosis occurred in the apical cells of the gametophytes and in some short, non-sporing gonimoblast branches, particularly induced during periods of low water temperatures. The advantages of using easily transportable rock units for such studies are discussed.