Showing papers in "Sexual Plant Reproduction in 2003"

Flower development schedule in tomato Lycopersicon esculentum cv. sweet cherry

Abstract: The ontogeny of tomato (Lycopersicon esculentum cv. sweet cherry) flowers was subdivided into 20 stages using a series of landmark events. Stamen primordia emergence and carpel initiation occur at stage 4; archesporial and parietal tissue differentiate at stage 6 and meiosis in anthers begins at stage 9. Subepidermal meristematic ovule primordia are formed on the placenta at stage 9; megasporogenesis begins at stage 11–12 and embryo sac differentiation and ovule curvature take place at stage 14, once the pollen is maturing. We established a correlation between the characteristic cellular events in carpels and stamens and morphological markers of the perianth. The model of tomato flower development schedule was then used to analyse the spatial, temporal and tissue-specific expression of gene(s) involved in the regulation of floral organ development. As an example, the expression pattern of ORFX, a gene controlling cell size in tomato fruits, shows that expression starts very early during the ontogeny of reproductive organs.

Coevolution of apomixis and genome size within the genus Hypericum

Abstract: Trends concerning coevolution of mode of reproduction and genome size were elucidated by screening both components in 71 species/subspecies of the genus Hypericum. Two independent agamic complexes were identified (sections Ascyreia with ten, and Hypericum with five apomictic species). In the phylogenetically younger section Hypericum, the relative DNA content of apomicts is increased solely by polyploidy. The apomicts of the evolutionarily older section Ascyreia have significantly larger genomes than all other species due to polyploidization and higher DNA content per chromosome. An accumulation of retroelements might be one reason for the larger genomes. The male fertility of the apomicts was reduced compared to sexuals, although all apomicts were facultative pseudogamous, forming reduced male gametes. Another form of apomixis (obligate pseudogamous with unreduced male gametes), probably indicating an escape from interspecific sterility, was found in H. scabrum, the only case of asexual seed formation outside of sections Ascyreia and Hypericum. The described scenario for evolution of apomixis in relation to genome size deserves consideration in harnessing of apomixis.

A meiotic time-course for Arabidopsis thaliana

Abstract: A meiotic time-course for Arabidopsis pollen mother cells has been established based on BrdU pulse-labelling of nuclear DNA in the meiotic S-phase. Labelled flower buds were sampled at intervals and the progress of labelled cells through meiosis assessed by anti-BrdU antibody detection. The overall duration of meiosis from the end of meiotic S-phase to the tetrad stage, at 18.5°C, was 33 h, which is about three times longer than the mitotic cell cycle in seedlings. The onset of leptotene was defined by reference to the loading of the axis-associated protein Asy1, and this permitted the detection of a definite G2 stage, having a maximum duration of 9 h. It is likely, from two independent sources of evidence, that the meiotic S-phase has a duration similar to that of G2. The durations of leptotene and zygotene/pachytene are 6 h and 15.3 h, respectively, but the remaining meiotic division stages are completed very rapidly, within 3 h. The establishment of a meiotic time-course provides a framework for determining the relative timing and durations of key molecular events of meiosis in Arabidopsis in relation to cytologically defined landmarks. In addition, it will be important in a broader developmental context for determining the timing of epigenetic mechanisms that are known or suspected to occur during meiosis.

The arrest of development of abortive reproductive organs in the unisexual flower of Vitis vinifera ssp. silvestris

Abstract: During the first stages of development, flowers of most dioecious species are hermaphroditic, with their transition to unisexual flowers being the result of the developmental arrest of one set of reproductive organs. In this work, we describe the development of male and female flowers of the dioecious wild grape species Vitis vinifera ssp. silvestris through scanning electron microscopy analysis and cytological observations, focusing our attention on the transition from bisexual to unisexual development. We divide floral development of the wild grape into eight stages. Differences between male and female flowers appear first at stage 6, when the style and stigma start to differentiate in female but not in male flowers. Cytological analysis of the slowly growing abortive pistil of male flowers shows that megagametophyte formation is, surprisingly, not inhibited. Instead of pistil abortion in the male flower, sexual determination is accomplished through programmed death of external nucellus cells and some layers of integumentary cells. Sterility of male structures in female flowers follows a different pattern, with microspore abnormalities evident from the time of their release from the tetrad. Sterile microspores and pollen grains in female flowers display an abnormal round shape, lacking colpi and possessing uniformly thickened cell walls that impede germination.

Structural and transcriptional analysis of S-locus F-box genes in Antirrhinum

Abstract: A class of ribonucleases termed S-RNases, which control the pistil expression of self-incompatibility, represents the only known functional products encoded by the S locus in species from the Solanaceae, Scrophulariaceae and Rosaceae. Previously, we identified a pollen-specific F-box gene, AhSLF (S locus F-box)-S 2, very similar to S 2 -RNase in Antirrhinum, a member of the Scrophulariaceae. In addition, AhSLF-S 2 also detected the presence of its homologous DNA fragments. To identify these fragments, we constructed two genomic DNA libraries from Antirrhinum self-incompatible lines carrying alleles S 1 S 5 and S 2 S 4, respectively, using a transformation-competent artificial chromosome (TAC) vector. With AhSLF-S 2-specific primers, TAC clones containing both AhSLF-S 2 and its homologs were subsequently identified (S 2 TAC, S 5 TACa, S 4 TAC, and S 1 TACa). DNA blot hybridization, sequencing and segregation analyses revealed that they are organized as single allelic copies (AhSLF-S 2, -S 1, -S 4 and -S 5) tightly linked to the S-RNases. Furthermore, clusters of F-box genes similar to AhSLF-S 2 were identified. In total, three F-box genes (AhSLF-S 2, -S 2 A and -S 2 C) in S 2 TAC (51 kb), three (AhSLF-S 4, -S 4 A and -S 4 D) in S 4 TAC (75 kb), two (AhSLF-S 5 and -S 5 A) in S 5 TACa (55 kb), and two (AhSLF-S 1 and -S 1 E) in S 1 TACa (71 kb), respectively, were identified. Paralogous copies of these genes show 38–54% identity, with allelic copies sharing 90% amino acid identity. Among these genes, three (AhSLF-S 2 C, -S 4 D and -S 1 E) were specifically expressed in pollen, similar to AhSLF-S 2, implying that they likely play important roles in pollen, whereas three AhSLF-SA alleles showed no detectable expression. In addition, several types of retroelements and transposons were identified in the sequenced regions, revealing some detailed information on the structural diversity of the S locus region. Taken together, these results indicate that both single allelic and tandemly duplicated genes are associated with the S locus in Antirrhinum. The implications of these findings in evolution and possible roles of allelic AhSLF-S genes in the self-incompatible reaction are discussed in species like Antirrhinum.

Characterization of ethylene effects on sex determination in cucumber plants

Abstract: Sex differentiation in cucumber plants (Cucumis sativus L.) appears to be determined by the selective arrest of the stamen or pistil primordia. We investigated the influence of an ethylene-releasing agent (ethephon) or an inhibitor of ethylene biosynthesis (aminoethoxyvinyl glycine) on sex differentiation in different developmental stages of flower buds. These treatments influence sex determination only at the stamen primordia differentiation stage in both monoecious and gynoecious cucumbers. To clarify the relationships between the ethylene-producing tissues and the ethylene-perceiving tissues in inducing female flowers in the cucumber, we examined the localization of mRNA accumulation of both the ACC synthase gene (CS-ACS2) and the ethylene-receptor-related genes (CS-ETR1, CS-ETR2, and CS-ERS) in flower buds by in situ hybridization analysis. CS-ACS2 mRNA was detected in the pistil primordia of gynoecious cucumbers, whereas it was located in the tissues just below the pistil primordia and at the adaxial side of the petals in monoecious cucumbers. In flower buds of andromonoecious cucumbers, only CS-ETR1 mRNA was detected, and was located in the pistil primordia. The localization of the mRNAs of the three ethylene-receptor-related genes in the flower buds of monoecious and gynoecious cucumbers overlap but are not identical. We discuss the relationship between the mRNA accumulation patterns and sex expression in cucumber plants.

Floral development of the model legume Medicago truncatula: ontogeny studies as a tool to better characterize homeotic mutations

Abstract: This work provides new evidence of the complex genetic regulation necessary to accomplish flower development in legumes. Using scanning electron microscopy (SEM) analysis, we have characterized the early developmental events of the wild type Medicago truncatula flower and selected morphological characters as markers to break it down into eight different developmental stages. The order of floral organ initiation in M. truncatula and pea (Pisum sativum L.), in contrast to Arabidopsis and Antirrhinum, is unidirectional in all whorls starting from the abaxial position of the flower with a high degree of overlap. Another main difference is the existence of four common primordia from which petals and stamens differentiate. The formation of common primordia, as opposed to discrete petal and stamen primordia, has been described in many legume and non-legume plants. The main differences between pea and M. truncatula floral ontogeny are in carpel and fruit development. We also used these morphological markers as tools to characterize early alterations in the flower development of a male-sterile M. truncatula floral homeotic mutant named mtapetala. This mutant displays a phenotype resembling those of weak class B mutants with homeotic conversions of floral organ whorls 2 and 3 into sepaloid and carpelloid structures, respectively. Ontogeny studies of the mtapetala mutant flowers showed similarities with the effects of previously described loss-of-B-function mutations. Differences between ontogeny of wild type and mtapetala flowers could not be detected during the first stages (1–5) of flower development. In late stage 5, abnormal-shaped petals with acute lobes and trichomes as well as abnormal-shaped stamens were visible in whorls 2 and 3. At stage 6, the morphology of petals began to change, developing enlarged sepaloid structures bearing trichomes and first the antesepalous stamens and then the antepetalous stamens began to differentiate carpelloid anthers from filaments. Third whorl organs presented different degrees of carpelloidy. The present study should provide tools for the characterization and comparative analyses of new Medicago floral homeotic mutants and could be useful in elucidating how floral organ identity functions work in legumes.

WAG, a wheat AGAMOUS homolog, is associated with development of pistil-like stamens in alloplasmic wheats

Abstract: Pistillody, homeotic transformation of stamens into pistil-like structures, has been reported in cytoplasmic substitution (alloplasmic) lines of common wheat (Triticum aestivum) with Aegilops crassa cytoplasm. The induction of pistillody is suppressed by the Rfd1 gene detected on the long arm of chromosome 7B in wheat cultivar 'Chinese Spring' (CS). Because of the absence of Rfd1, the alloplasmic line CS ditelosomic 7BS [(cr)-CSdt7BS] lacking the long arm of chromosome 7B exhibits pistillody in all florets, whereas the euplasmic CS ditelosomic 7BS (CSdt7BS) with normal cytoplasm forms normal stamens. To study the molecular mechanism of pistillody caused by nuclear-cytoplasm interaction in alloplasmic wheat, we cloned and characterized a wheat AGAMOUS homolog, WAG. The WAG gene copy number in the wheat genome was estimated at three, located on the homoeologous group 1 chromosomes. Northern blot analysis with a normal wheat line revealed that the transcription level of WAG was lower in young spikes and increased during spike development. The highest expression was observed in spikes at the booting to heading stage. The 1.1 kb WAG transcript accumulated in both the reproductive (pistil and stamen) and non-reproductive (palea and lemma) portions of spikes at the heading stage, whereas an extra transcript of different molecular size (1.3 kb) was observed in pistil-like stamens of line (cr)-CSdt7BS as well as in pistils of CSdt7BS and (cr)-CSdt7BS. These results suggest that the product of the 1.3 kb WAG transcript is involved in pistil development and is associated with pistillody caused by a nuclear-cytoplasm interaction in alloplasmic wheat.

Breakdown of self-incompatibility in a natural population of Petunia axillaris caused by a modifier locus that suppresses the expression of an S-RNase gene

Abstract: We had previously studied the cause of breakdown of self-incompatibility in a natural population (designated U1) of Petunia axillaris subsp. axillaris (Solanaceae) identified in Uruguay. This population contained mostly self-incompatible and some self-compatible plants. We had found that an S-haplotype, S 13 , carried by three self-compatible individuals (U1-2, U1-16, and U1-22) was functional in the pollen, but not in the style, and that S13-RNase was not detected in the styles of these individuals. This defective haplotype was designated S 13 sps , with "sps" standing for "stylar-part suppression". In this work, we further investigated the molecular and genetic basis of this suppression. We isolated and sequenced cDNAs for S13-, S1- and S15-RNases, and used them as probes to show that the transcript of the S13-RNase gene was not detectable in U1-2 (S 1 S 13 sps ), U1-16 (S 13 sps S 15 ), or U1-22 (S 13 sps S 13 sps ), but the transcript levels of the S1- and S15-RNase genes in U1-2 and U1-16, respectively, were normal. Genomic blotting analysis revealed that the S13-RNase gene was present in these three self-compatible plants. The S-genotypes obtained in bud-selfed and normally selfed progenies of these three plants were determined. Analysis of these S-genotypes led us to propose a model invoking a modifier locus, named MDF, to explain the suppression of the expression of the S13-RNase gene in the genetic background of the self-compatible plants carrying the S 13 sps -haplotype.

Identification of anther-specific genes in a cruciferous model plant, Arabidopsis thaliana, by using a combination of Arabidopsis macroarray and mRNA derived from Brassica oleracea

Abstract: Arabidopsis thaliana, a member of the Brassicaceae, is a model plant whose genome was the first higher plant genome to be sequenced. Because of the small size of the flowers, it is difficult to dissect and separate reproductive organs (anthers and pistils) at different developmental stages in A. thaliana. In order to perform genome-wide identification of anther-specific genes in A. thaliana, an Arabidopsis cDNA macroarray was hybridized to cDNA derived from anthers and pistils of another crucifer, Brassica oleracea. After scanning the signal intensity for each clone, and cluster analysis, 52 anther-specific genes were identified. These clones contained several anther-specific genes that have already been characterized, as well as novel anther-specific genes. In RT-PCR analysis with mRNA of A. thaliana and B. oleracea, the expression pattern of one-third of the clones was similar to that determined by cDNA macroarray. This system of heterologous hybridization analysis (Arabidopsis cDNA macroarray vs Brassica tissue-specific mRNA) should be applicable to other model species and their close relatives.

The occurrence of phenotypically complementary apomixis-recombinants in crosses between sexual and apomictic dandelions ( Taraxacum officinale )

Abstract: Apomictic seed development in dandelion (Taraxacum officinale) involves (1) restitutional meiosis (diplospory), (2) egg cell parthenogenesis, and (3) autonomous endosperm development. The question is whether these elements of apomixis are controlled by one single gene or by several independent genes. Five triploid non-apomictic hybrids, obtained in diploid sexual × triploid apomict crosses were characterized using cyto-embryological and genetic methods. Nomarski-differential interference contrast microscopy and the transmission of microsatellite markers and ploidy levels indicated that the hybrids combined elements of the apomictic and the sexual developmental pathway. Hybrids form two complementary groups with respect to the presence or absence of parthenogenesis and autonomous endosperm development. The occurrence of complementary apomixis-recombinants suggests that parthenogenesis and autonomous endosperm development in Taraxacum are regulated independently by different genes. This study also indicates that early embryo development is independent of endosperm formation, but that endosperm is essential for later embryo growth.

Hormonal status of the pollen-pistil system at the progamic phase of fertilization after compatible and incompatible pollination in Petunia hybrida L.

Abstract: The hormonal status of the pollen-pistil system in Petunia hybrida L. during the progamic phase of fertilization was investigated. The contents of indolyl-3-acetic acid (IAA), abscisic acid (ABA), and cytokinins, as well as the rate of ethylene production in the pistils and their parts (stigma, style, and ovary) were measured over an 8-h period following compatible and self-incompatible pollination. In both pollinations, the phytohormones were present in various proportions in the stigma, style and ovary: the stigma was the main site of ethylene synthesis and contained 90% of the ABA, while the style contained 80% of the total cytokinin content in the pollinated pistil. Relatively low levels of hormones in the ovary did not influence the hormonal status of the pollen-pistil system. The interaction of the male gametophyte with the stigmatic tissues was accompanied by a 7- to 10-fold increase in ethylene production and a 1.5- to 2.0-fold increase in IAA content in the pollen-pistil system over 0–4 h. Pollen tube growth after self-incompatible pollination, in contrast to compatible pollination, was accompanied by a 3-fold increase in the ABA content in the stigma and style and by a 5-fold higher cytokinin content in the stylar tissues. Thus, the ethylene/ABA status of the stigma may play a role in controlling the processes of adhesion, hydration, and germination of pollen grains during pollination while the auxin/cytokinin status of the style may be involved in controlling pollen tube growth.

Immunocytochemical localization of Cry j 1, the major allergen of Cryptomeria japonica (Taxodiaceae) in Cupressus arizonica and Cupressus sempervirens (Cupressaceae) pollen grains

Abstract: Immunocytochemical localization of cross-reactive antigens to Cry j 1, the major allergen of Cryptomeria japonica, in the pollen grains of Cupressus arizonica and Cupressus sempervirens, was conducted using transmission electron microscopy. Mature and activated pollen grains were fixed for immunocytochemistry using conventional and freezing protocols. Sections containing the samples were incubated with anti-Cry j 1 monoclonal antibody (mAb) (KW-S91). Cross-reactive antigens to Cry j 1 were detected in the mature pollen grains of Cupressus arizonica and Cupressus sempervirens, and abundant gold particles were seen in the orbicules and wall, and in the Golgi, nucleus, and inclusions in reserve materials. After 5 min of activation, labelling noticeably decreased. From 15 min to 48 h, the cytoplasm exhibits a new labelling pattern, with gold markers being associated with protein storage vacuoles. The decrease in cross-reactive antigens during the first 5 min of activation could be due to a rapid release of proteins recognized by Cry j 1 mAb during pollen attachment in the pollination droplet. Our results show that the content of allergenic proteins is unstable, displaying variation relative to the progress of germination in Cupressus sempervirens and Cupressus arizonica pollen grains. The ability to do so may be viewed as an adaptive strategy of Cupressaceae pollen grains to maximize their biosynthetic efficiency.

Pollen tube growth and early ovule development following self- and cross-pollination in Eucalyptus nitens

Abstract: Controlled self- and cross-pollinations were conducted on flowers of five mature Eucalyptus nitens trees. Levels of self-sterility of the trees ranged from 25.8 to 93.6%. Pollen tube numbers in styles and ovule penetration by pollen tubes was investigated 2 weeks after pollination by fluorescence microscopy. There were no significant differences between treatments in the number of pollen tubes present in styles or in the percentage of ovules penetrated by pollen tubes. Embryology of material harvested 2 and 4 weeks after pollination was investigated by bright-field microscopy. Fertilisation had taken place by 2 weeks after pollination with nearly every ovule showing evidence of fertilisation. Cross-pollination resulted in a greater proportion of healthy, developing ovules, at both 2 and 4 weeks after pollination, compared with self-pollination. The proportion of degenerating ovules increased from 2 to 4 weeks after pollination. The reduced ability of E. nitens to set self-pollinated seed compared with cross-pollinated seed appears to be controlled by a post-zygotic mechanism. Differences in ovule size may potentially assist in the identification of trees incapable of setting self-pollinated seed.

Sexual polyembryony in almond

Abstract: Multiple embryos within the same seedcoat occur spontaneously in certain almond [P. dulcis (Mill.) D.A. Webb] cultivars including 'Nonpareil' and 'Mission'. Seedlings from the same polyembryonic seed are frequently viable, though one of the seedlings often shows weak growth and develops poorly. These dwarf seedlings have previously been reported as haploid. In this work, we have characterized several seedlings from 'Nonpareil' polyembryonic seed, including their germination and later growth. Isozyme and simple sequence repeat markers were used to analyze seedling genetic structure. In addition, individual mitotic karyotypes were determined following root-tip staining. The percentage of twin embryos showing aberrant growth was approximately 30%, with mortality rates of about 90%. The majority of these aberrant seedlings appear to be aneuploids. Most secondary embryos appear to be derived from the primary embryo following normal fertilization.

Expression of female sterility in alfalfa (Medicago sativa L.)

Abstract: Female sterility associated with the presence of callose in the nucellus at anthesis was studied in an F1 progeny of two alfalfa plants displaying 5 and 81% ovule sterility. Transgressive segregation was observed and 100% sterile plants were obtained. Two of the sterile plants were used for cytological analyses on sectioned and stain-cleared whole ovules, in comparison to a 100% fertile full sib plant. The first sign of sterility was callose deposition in the nucellus cell walls surrounding the sporogenous cells of the young ovules. At the same stage, no trace of callose was present in ovule primordia of the fertile plant. Megaspore mother cells differentiated in both fertile and sterile ovules and meiosis was initiated, as indicated by chromatin patterning typical of a zygotene stage. However, meiosis was never completed in the sterile plants. In the control, callose was deposited around the meiocyte and as sects between the cells of the dyads and tetrads during meiosis, and disappeared after the completion of meiosis; an embryo sac developed and female fertility was normal. In the sterile ovules, some nucellus cells enlarged and callose accumulation continued forming thick deposits. At anthesis, the sterile ovules lacked an embryo sac and showed massive callose accumulation in the nucellus. Male fertility was normal in female-sterile plants, thus a female-specific arrest of sporogenesis appears to be the cause of sterility. Pistil development was aberrant in some sterile genotypes, even with arrested pistil growth in early flower buds.

Immunocytochemical distribution of polygalacturonase and pectins in styles of distylous and homostylous Turneraceae

Abstract: Distyly is a plant breeding system in which two self-incompatible, but cross-compatible, floral morphs occur within populations. The morphs differ in having a reciprocal arrangement of styles and anthers. Little or nothing is known of the proteins involved in self-incompatibility for any distylous species. Here we show that a 35 kDa putative polygalacturonase is specific to the transmitting tissue of short-styled plants of five species in series Turnera. The polygalacturonase was not detected in styles of long-styled plants, or in styles of five homostylous self-compatible species in this series of the genus. It is also absent from two X-ray generated mutants and a spontaneous somatic homostylous mutant that arose on a short-styled plant and whose style does possess this polygalacturonase. Three more distantly related species in the Turneraceae were investigated. Turnera weddelliana (series Salicifoliae) does possess the polygalacturonase; however, T. diffusa (series Microphyllae), and Piriqueta caroliniana, showed no evidence of possessing this polygalacturonase using immunocytochemistry. Polygalacturonase assays revealed activity in styles of long- and short-styled plants, but showed no activity of the 35 kDa style polygalacturonase. The distribution of pectins in styles and pollen tubes revealed no difference between the long- and short-styled morphs. Methyl-esterified pectins occur throughout the style tissues, except in the transmitting tissue. The transmitting tissue possesses unesterified pectins that could provide a substrate for polygalacturonase activity. We propose that the style polygalacturonase might act in a complementary manner, allowing pollen of long-styled plants to grow through short styles or, alternatively, oligogalacturonide products of polygalacturonase activity might play a role in signalling compatible responses.

Transcript profiling on developing Petunia hybrida floral organs

Abstract: The cDNA-AFLP transcript profiling technique was used to analyse gene expression during flower development in Petunia hybrida. Reproductive and vegetative floral organs were sampled at five developmental stages and gene expression profiles were compared. This allowed us to assemble an inventory of genes expressed mainly in anthers during microspore development and in ovaries during macrosporogenesis. About 6,000 transcript tags were generated, 354 of which showed a modulated and/or organ-specific expression pattern. Stamen-specific transcripts exhibiting an upregulation in gene expression were well represented in our screening. Ovary-specific transcripts were less frequently observed and often displayed a constant level of gene expression. Of 194 fragments characterised further by sequencing, 35% showed homology with known genes in a database search. They belong to a wide range of gene classes, such as proteases, transcription factors and genes involved in metabolism, cell cycle and disease resistance. The usefulness of cDNA-AFLP transcript profiling as a tool to unravel complex developmental processes at the molecular level is discussed.

Analysis of four maize mutants arrested in early embryogenesis reveals an irregular pattern of cell division

Abstract: The process that leads to embryo formation appears to follow a defined pattern, whose sequential developmental steps—under strict genetic control—can be analysed through the study of mutants affecting embryogenesis. We present the analysis of four embryo-specific (emb) mutants of maize, characterised by abnormal development not overcoming the proembryo or early transition stage, that define three separate genes on the basis of their chromosomal location and complementation pattern. A common feature emerging from histological analysis is that suppression of morphogenesis is accompanied by an uncontrolled pattern of cell division. The block in embryo development is associated with abnormal suspensor proliferation, possibly due to the absence of a signal elaborated by the embryo proper and required for suspensor cell identity maintenance. Mutant endosperm morphogenesis is not impaired, as shown by the formation of the expected domains, i.e. aleurone, starchy endosperm, embryo-surrounding region and basal endosperm transfer layer. The program of cell death appears impaired in the mutants, as expected if this process is essential in determining the shape and morphology of the developing organs. An unexpected result is obtained when mutant embryo rescue is attempted. Immature embryos transferred to a basal medium germinated, yielding small but otherwise normal seedlings, an observation not consistent with the histological evidence of a complete absence of morphogenetic potential. The analysis of emb mutants appears a promising tool to elucidate crucial points of embryo development such as the coupling of cell division with morphogenesis, cell-to-cell interactions, the relationship between embryo and endosperm development, and the interaction between embryo proper and suspensor.

A defective pollen-pistil interaction contributes to hamper seed set in the parthenocarpic fruit tomato mutant

Abstract: In the parthenocarpic fruit (pat) tomato mutant, parthenocarpy is associated with partial aberrations of stamens (shortness and carpelloidy) and ovules (defective integument growth) that contribute to impair seed set. However, these do not seem to be the only reasons for seed infertility because hand-pollination fails to restore seed set in ovaries where a fraction of the ovules are still morphologically normal. Therefore, it is conceivable that other unreported defects occur during the reproductive process in the mutant. In this research, we show that the mutation does not affect pollen or embryo sac development and viability, but generates sporophytic effects that reduce seed production and seed size. While pollen germination and stylar growth were normal in mutant pistils, fertilization does not take place because of abnormalities in the pollen tube-ovary interaction in this genotype. Inside the ovary of pat plants, pollen tubes appeared to be disorientated; they wandered about in the ovarian cavity and often lost their adherence to the placental surface. Interestingly, in pat ovaries fertilization was strongly impaired even in those ovules that appeared normal. It may be that apparently 'normal' ovules cannot guide pollen tubes to their micropyle in the altered pat ovary because adhesion molecules are not properly arrayed on a placenta that is already preparing for cell division or, alternatively, chemotropic signals in the pat ovary may be altered by the presence of aberrant ovules, which are not simply devoid of attractivity, but disrupt pollen tube guidance overall.

halfman, an Arabidopsis male gametophytic mutant associated with a 150 kb chromosomal deletion adjacent to an introduced Ds transposable element

Abstract: To identify genes that play an important gametophytic role during pollen development, an Arabidopsis transposon (DsE) mutagenised population was screened for marker segregation ratio distortion. We report the characterisation of a male gametophytic mutant termed halfman (ham) that results in an aborted pollen phenotype in mature anthers. The genetic transmission efficiency of DsE was 6.6% through the male and 98.4% through the female, which suggested that HAM may encode essential male-specific component(s) required for pollen development. Molecular analysis of the insertion site revealed a single copy of the DsE element inserted into the second exon of the RLK5 gene that was adjacent to a large (~150 kb) genomic deletion. The deleted region is predicted to encode 38 genes and to include one or more genes with important function(s) during pollen maturation and seed development.

Characterization of the pollen growth transition in self-incompatible Petunia inflata

Abstract: In Petunia inflata, as in other species that shed bicellular pollen, early pollen tube growth in the pistil is slow, then increases 2- to 5-fold depending on the genotype of the female parent. We refer to the time point at which pollen tubes enter the accelerated phase of growth as the pollen growth transition (PGT). Here, we present evidence that pre-PGT and post-PGT growth are quantitatively and qualitatively different, and that the PGT is triggered when pollen tubes reach the transition zone (TZ) below the stigma. The capacity of various pistil zones to precipitate the PGT was tested through 'stump' pollinations: varying lengths of the pistil apex were excised, the cut surface of the remaining pistil (the stump) coated with stigmatic exudates then dusted with compatible pollen. Pollen applied to TZ tissues entered the PGT earlier than pollen growing in intact control pistils; the PGT was delayed in stylar stumps, largely because of delayed germination and reduced pre-PGT growth. In immature pistils, the PGT was delayed by several hours relative to its onset in mature pistils. The PGT fails to occur in pollen cultured in vitro. Collectively, the data suggest that pollen tubes become competent to enter the PGT when they reach a critical size, but the physicochemical environment of the transmitting tissue is necessary for triggering the cellular changes that result in accelerated growth. An analysis of the distribution of pollen tube tips before and after the PGT suggests that pollen competition is most intense during the pre-PGT phase.

Generation and analysis of expressed sequence tags from almond (Prunus dulcis Mill.) pistils

Abstract: Large-scale single-pass sequencing of expressed sequence tags (ESTs) can give a global picture of the genes involved in the development and function of organs and tissues. Almond (Prunus dulcis Mill. syn. Amygdalus communis L. var. dulcis) is a self-incompatible fruit tree of the family Rosaceae. As an attempt to examine the transcripts expressed during pistil development, we randomly selected and partially sequenced over 1,000 clones from a cDNA library of almond pistils. Analysis of the ESTs using the NCBI non-redundant protein database revealed that 456 (~45.4%) have significant similarity to protein coding sequences in the database. Among the 549 cDNAs with no similarity to known protein sequences, 56 clones have significant similarity to nucleotide sequences registered in the databases. Sequences were assembled using StackPACK and the estimated number of genes identified was 716: 121 contigs and 595 singletons. Only eight of the unique cDNA clones correspond to previously isolated gene sequences of P. dulcis. After functional classification of almond ESTs, "stress resistance and defense" and "protein synthesis and processing" ESTs were found to be the most numerous. Our EST analysis provides a first picture of the numerous almond genes potentially involved in pistil development. It also contributes to our knowledge about gene expression patterns in pistils while providing an extensive reservoir for gene cloning and genetic mapping in almond and other related fruit trees.

Involvement of the ubiquitin/proteasome pathway in the organisation and polarised growth of kiwifruit pollen tubes.

Abstract: We recently reported the involvement of the ubiquitin pathway in microgametophyte development, and a direct role for the 26S proteasome in regulating pollen tube emergence in kiwifruit. Here we show that the ubiquitin/proteasome proteolytic pathway is involved not only in early kiwifruit pollen tube organisation, but also in maintaining polarised growth of tubes. By immunofluorescence analysis we show that ubiquitin and ubiquitin-protein conjugates are distributed mainly at the apex of emerging tubes, in both untreated pollen grains and pollen grains treated with MG132, an inhibitor of proteasome function. In the latter case, polysiphonous germination occurred and all the emerging areas were highly fluorescent. By adding MG132 to pollen when normal tube growth had already been established, accumulation of ubiquitin-protein conjugates, as well as a drastic reduction in tube growth and dramatic modifications of tube tip morphology were observed. Significantly, differential interference contrast microscopy analysis demonstrated that the clear zone was largely reduced or absent, and the nuclei were disconnected in their movements, reaching, in some cases, the extreme apex of the tip. These findings provide evidence that the ubiquitin- and proteasome-dependent proteolytic system could modulate the abundance and/or activity of key regulatory proteins involved in pollen tube emergence and polarised growth.

Analysis of ZmAE3 upstream sequences in maize endosperm and androgenic embryos

Abstract: The single copy gene ZmAE3 (androgenic embryo) has been isolated from early androgenic embryos of maize. It codes for a small hydrophilic protein with partial similarity to basal layer antifungal proteins. During normal reproductive development ZmAE3 is specifically expressed in the embryo-surrounding region, a specialised region of the endosperm. Here we report the cloning, sequencing and functional analysis of upstream sequences of ZmAE3. The fusion of a 1.1 kb potential promoter fragment of ZmAE3 to a uidA reporter gene resulted in β-glucuronidase activity in transiently transformed androgenic embryos and in Black Mexican Sweet maize suspension cells. The AE box, an 18 bp sequence present in the ZmAE3 promoter and in other endosperm-specific promoters, was shown to bind proteins present specifically in nuclear extracts of the lower half of the maize kernel. The potential role of this and other cis elements in the binding of trans-acting factors will be discussed.

Histology of sterile male and female cones in Pinus monticola (western white pine)

Abstract: Two years of histological samples were collected from a Pinus monticola Dougl. (western white pine) tree identified as not producing mature pollen or seed cones. Anatomical information was collected to the ultrastructural level, to assess possible mechanisms for pollen and cone abortion resulting in sterility. Development of male and female gametophytes in the sterile western white pine tree was arrested after meiosis and before further cell divisions could take place. Sterile male gametophytes (pollen grains) had poorly developed pollen walls and sacci, reduced and degenerative cytoplasm, and no evidence of stored starch grains. The pollen cone aborted prior to pollen dehiscence. Meiosis of the megaspore mother cell in the ovule produced four megaspores, but development was stopped at the functional megaspore stage. The seed cone aborted in the first year of growth before winter dormancy. Tapetal tissue in sterile microsporangia appeared similar to that of fertile microsporangia, until the vacuolate, uninucleate microspore stage. Tapetal cells and thecal fluid surrounding the sterile microspores persisted well past the time when microsporangia on fertile trees started the process of maturation and desiccation. At pollen dehiscence, sterile pollen cones did not release any pollen and the microsporangia were filled with a sticky fluid. The behaviour of the tapetum in P. monticola sterile cones is compared with reports of tapetal function and malfunction reported in studies of angiosperm and other gymnosperm species. The occurrence and timing of gametophyte abortion in both cone sexes suggests a genetic rather than environmental basis for the sterility mechanism.

A functional study of stylar hydroxyproline-rich glycoproteins during pollen tube growth

Abstract: Class III pistil-specific extensin-like proteins (PELPIII) are chimeric hydroxyproline-rich glycoproteins with properties of both extensins and arabinogalactan proteins. The abundance and specific localization of PELPIII in the intercellular matrix (IM) of tobacco (Nicotiana tabacum) stylar transmitting tissue, and translocation of PELPIII from the IM into the pollen tube wall after pollination, presume the biological function of these glycoproteins to be related to plant reproduction. Here we show that in in vitro assays the translocation of PELPIII is specifically directed to the callose inner wall of the pollen tubes, indicating that protein transfer is not dependent on the physiological conditions of the transmitting tract. We designed a set of experiments to elucidate the biological function of PELPIII in the stylar IM. To study the function of the specific interaction between PELPIII proteins and the pollen tube wall, one of the PELPIII proteins (MG15) was ectopically expressed in pollen tubes and targeted to the tube wall. We also generated transgenic tobacco plants in which PELPIII proteins were silenced. In vitro bioassays were performed to test the influence of purified PELPIII on pollen tube growth, as compared to tobacco transmitting tissue-specific proteins (TTS) that were previously shown to stimulate pollen tube growth. The various tests described for activity of PELPIII proteins all gave consistent and mutually affirmative results: the biological function of PELPIII proteins is not directly related to pollen tube growth. These data show that similar stylar glycoproteins may act very differently on pollen tubes.

Identification and evolutionary analysis of a relic S-RNase in Antirrhinum

Abstract: In several gametophytic self-incompatible species of the Solanaceae, a group of RNases named relic S-RNase has been identified that belong to the S-RNase lineage but are no longer involved in self-incompatibility. However, their function, evolution and presence in the Scrophulariaceae remained largely unknown. Here, we analyzed the expression of S-RNase and its related genes in Antirrhinum, a member of the Scrophulariacaeae, and identified a pistil-specific RNase gene; AhRNase29 encodes a predicted polypeptide of 235 amino acids with an estimated molecular weight of 26 kDa. Sequence and phylogenetic analyses indicated that AhRNase29 forms a monophyletic clade with Antirrhinum S-RNases, similar to that observed for other relic S-RNases. Possible evolution and function of relic S-RNases are discussed.

Identification of self-incompatibility groups in Hatiora and Schlumbergera (Cactaceae)

Abstract: The Cactaceae, a family of about 1,800 species of succulent perennials, contains numerous species that exhibit self-incompatibility (SI). The objective of the current study was to determine the number of incompatibility groups present among diploid (2n=2x=22) cultivars of the genera Schlumbergera Lem. (Christmas cacti) and Hatiora Britton & Rose (Easter cacti). Two partial diallel crosses were performed, one with 19 cultivars of Christmas cacti [= S. truncata (Haworth) Moran and S. × buckleyi (Buckley) Tjaden] and the other with 10 cultivars of Easter cacti [= H. gaertneri (Regel) Barthlott, H. rosea (Lagerheim) Barthlott, and H. × graeseri Barthlott ex D. Hunt]. The compatibility/incompatibility status of crosses was determined by percent fruit set and presence of seed in mature fruit. None of the cultivars set fruit when selfed or crossed with a cultivar in the same incompatibility group, but fruit set ranged from 35% to 100% following compatible crosses. For the Christmas cacti, eight intra-incompatible but reciprocally compatible groups were identified, with 13 of the 19 cultivars assigned to three incompatibility groups (68%). The ten cultivars of Easter cacti yielded nine intra-incompatible but reciprocally compatible groups, with two cultivars in one incompatibility group and the other eight cultivars each assigned to a unique group. One cultivar of Christmas cactus ('Abendroth 6') was incompatible when crossed as a male with cultivars in incompatibility group 2 but was compatible in reciprocal crosses, results that suggest that this cultivar is an S-allele homozygote. The crossing relationships are consistent with a one-locus, gametophytic SI system with multiple alleles. Allozyme locus Lap-1, shown previously to be linked with the S locus (recombination frequency 7%) in Schlumbergera, exhibited insufficient allelic diversity for determining the S genotypes of the 19 cultivars of Christmas cacti. Based on the number of incompatibility groups in each diallel, at least five S-alleles occur in the 19 Christmas cacti and the 10 Easter cacti.

Swimming behavior and ultrastructure of sperm of Lygodium japonicum (Pteridophyta)

Abstract: Swimming behavior of the sperm of Lygodium japonicum (Pteridophyta) and the associated ultrastructure of the flagellar apparatus were studied by video microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The sperm has approximately 70 flagella that emerge from a sinistrally-coiled flagellar apparatus, and swims forward by ciliary beat of these flagella. Backward swimming was not observed even after sperm collided with obstacles. Video microscopy showed that the flagella of the swimming sperm are oriented laterally and oblique-anteriorly. TEM and SEM observations revealed that the basal bodies of these flagella are arranged in at least two rows and oriented in the same directions as observed by video microscopy. These basal bodies (flagella) are categorized into two types according to their orientation: group I (laterally directed) and group II (oblique-anteriorly directed). The directionality of the basal bodies appears to be fixed by electron-dense material around their base. The outer dynein arms of the flagellar axoneme are entirely absent. These morphological characteristics of basal bodies (flagella) may relate to the lack of backward swimming behavior of the sperm. Based on these results, the evolution of swimming behavior in the archegoniates is discussed in connection with lack of backward swimming in a distantly related green alga, Mesostigma viride, and the Streptophyta.