Showing papers in "Sexual Plant Reproduction in 2004"

Primary structural features of the S haplotype-specific F-box protein, SFB, in Prunus

Abstract: The gene SFB encodes an F-box protein that has appropriate S-haplotype-specific variation to be the pollen determinant in the S-RNase-based gametophytic self-incompatibility (GSI) reaction in Prunus (Rosaceae). To further characterize Prunus SFB, we cloned and sequenced four additional alleles from sweet cherry (P. avium), SFB 1 , SFB 2 , SFB 4 , and SFB 5 . These four alleles showed haplotype-specific sequence diversity similar to the other nine SFB alleles that have been cloned. In an amino acid alignment of Prunus SFBs, including the four newly cloned alleles, 121 out of the 384 sites were conserved and an additional 65 sites had only conserva- tive replacements. Amino acid identity among the SFBs ranged from 66.0% to 82.5%. Based on normed variabil- ity indices (NVI), 34 of the non-conserved sites were considered to be highly variable. Most of the variable sites were located at the C-terminal region. A window- averaged plot of NVI indicated that there were two variable and two hypervariable regions. These variable and hypervariable regions appeared to be hydrophilic or at least not strongly hydrophobic, which suggests that these regions may be exposed on the surface and function in the allele specificity of the GSI reaction. Evidence of positive selection was detected using maximum likelihood meth- ods with sites under positive selection concentrated in the variable and hypervariable regions.

The local cytomechanical properties of growing pollen tubes correspond to the axial distribution of structural cellular elements

Abstract: Morphological studies of pollen tubes have shown that the configuration of structural cellular elements differs between the growing apex and the distal part of the cell. This polarized cellular organization reflects the highly anisotropic growth behavior of this tip growing cell. Accordingly, it has frequently been postulated that physical properties of pollen tubes such as cell wall plasticity should show anisotropic distribution, but no experimental evidence for this has been published hitherto. Using micro-indentation techniques, we quantify pollen tube resistance to lateral deformation forces and analyze its visco-elasticity as a function of distance from the growing apex. Our studies reveal that cellular stiffness is significantly higher at the distal portion of the cell. This part of the cell is also completely elastic, whereas the apex shows a visco-elastic component upon deformation. To relate these data to the architecture of the particular pollen tube investigated in this study, Papaver rhoeas, we analyzed the distribution of cell wall components such as pectin, callose, and cellulose as well as the actin cytoskeleton in this cell using fluorescence label. Our data revealed that, in particular, the degree of pectin methyl esterification and the configuration of the actin cytoskeleton correlate well with the distribution of the physical properties on the longitudinal axis of the cell. This suggests a role for these cellular components in the determination of the cytomechanics of pollen tubes.

Genetic and molecular analysis in Cristobalina sweet cherry, a spontaneous self-compatible mutant.

Abstract: Self-compatibility in a naturally self-incompatible species like sweet cherry is a highly interesting trait for breeding purposes and a powerful tool with which to investigate the basis of the self-incompatible reaction in gametophytic systems. However, natural self-compatibility in sweet cherry is a very rare phenomenon. Cristobalina is a local Spanish sweet cherry cultivar that has proven to be spontaneously self-compatible. In this work, the nature of the self-compatibility in Cristobalina has been studied using genetic and molecular approaches. Pollination studies and microscopic observations of pollen tube growth were carried out to confirm the self-compatible character and the results obtained indicate that self-compatibility is caused by a failure of the pollen and not the style factor. Polymerase chain reaction (PCR) analysis of progenies derived from Cristobalina revealed that self-compatibility in this genotype is not related uniquely to one of the two pollen S alleles, but that pollen grains carrying either of the two haplotypes can overcome the incompatibility barrier. Moreover, PCR analysis and microscopic observation of pollen tube growth in progeny derived from Cristobalina also confirmed that the self-compatible descendants can carry either of the two S haplotypes of their progenitor. Isolation and sequencing of the style S-RNases and pollen SFBs revealed that the DNA sequences of these factors are the same as those described in other self-incompatible sweet cherry cultivars with the same S alleles. Possible mechanisms to explain self-compatibility in Cristobalina are discussed.

Flower abscission and inflorescence carbohydrates in sensitive and non-sensitive cultivars of grapevine

Abstract: The Gewurztraminer (GW) and the Pinot noir (PN) cultivars of grapevine differ in their sensitivity to environmental factors that can cause flower abscission, cv. GW being the most sensitive. In order to further define the mechanisms leading to abscission, and owing to the importance of sugars in the achievement of sexual organ ontogenesis, we attempted to correlate the chronology of flower ontogenesis with the variations of carbohydrates in the inflorescence. In the vineyard, under optimal climatic conditions, fruit set of cv. GW and cv. PN was 82% and 65%, respectively. The sugar distribution was different in their inflorescences during the entire duration of flower development. Between stages 15 and 17, flowers of GW and PN reached the crucial meiosis stage. At that time, the inflorescences of cv. GW exhibited higher concentrations of starch and sucrose, whereas those of PN presented higher levels of glucose and fructose. Despite higher starch concentrations in GW inflorescences, starch reserves were present in the ovules and anthers of PN but not in those of GW. These results suggest that the higher content of reserve and transport carbohydrates in the inflorescences of GW favour flower development and fruit set under optimal environmental conditions. Furthermore, since meiosis represents a key step of female development, the different sugar concentrations in the inflorescences of the two cultivars at stages 15 and 17 could be related to the sensitivity to flower abscission under climatic stress. In particular, the presence of starch granules in PN ovules and anthers might explain the higher resistance of this cultivar to flower abscission.

Male and female gamete abortions, and reduced affinity between the uniting gametes as the causes for sterility in an indica/japonica hybrid in rice

Abstract: Hybrid sterility frequently occurs in crosses between indica and japonica subspecies of Asian cultivated rice. In this study, we investigated the cytological processes involved in formation and development of male and female gametes as well as their interactions in fertilization, using an indica/japonica hybrid in comparison with an indica/indica hybrid. It was found that more than 50% of the microspores generated in the indica/japonica hybrid could not develop into functional pollen. The abortion rate of microspores in the indica/japonica hybrid was much higher than that in the indica/indica hybrid. Abortive embryo sacs made up roughly 70% of the embryo sacs examined in the indica/japonica hybrid, which was also much higher than that detected in the indica/indica hybrid. Moreover, the amount of pollen adherence on stigmas of the indica variety upon hand-pollination with pollen from the japonica variety was much lower than the indica/indica pollination, and the number of pollen adhered on the stigmas by natural self-pollination was much greater in the indica/indica hybrid than in the indica/japonica hybrid. The indica/japonica hybrid also encountered difficulties in pollen tube growth after pollination, and the fertilization rate of the indica/japonica hybrid was much lower than that of the indica/indica hybrid. These results clearly illustrate the complexity of the mechanisms underlying inter-subspecific hybrid sterility in rice involving both pre- and post-zygotic reproductive isolation mechanisms.

The RHV region of S-RNase in the European pear ( Pyrus communis ) is not required for the determination of specific pollen rejection

Abstract: In the gametophytic self-incompatibility system, growth of self-pollen tubes in the style is inhibited in a haplotype-specific manner by S-RNase. The mechanism by which S-RNase confers its specificity is unknown. However, a hypervariable region (RHV in Rosaceae and HVa-HVb in Solanaceae) that differs among the many cloned S-RNase alleles has been proposed to be involved in conferring the S-haplotype specificity of the S-RNase. Region swapping experiments between S-RNases and crystallography of the enzyme support this assumption. However, the deduced amino acid sequences of Sn-RNase and Si-RNase alleles from the European pear (Pyrus communis) were recently found to have an identical RHV. In the present study it is shown that Sn-RNase does not prevent fertilization by Si-pollen haplotype, thus presenting a case in which RHV is not required for the determination of specific pollen rejection by S-RNase, and implying that other regions in the enzyme may be sufficient for this specificity.

Identification of a male-specific AFLP marker in a functionally dioecious fig, Ficus fulva Reinw. ex Bl. (Moraceae)

Abstract: A male-specific amplified fragment length polymorphism (AFLP) marker was identified in the functionally dioecious fig species, Ficus fulva A total of 89 polymorphic fragments from three primer combinations were produced, of which one (246 bp) was present in all males (n=23) and absent in all females (n=24) of two populations This strong association suggests a tight chromosomal linkage between the AFLP marker and the sex-controlling locus Further analysis indicated that the marker segregated in open-pollinated progenies from natural populations in a 1:1 ratio (n=156), implying that males are the heterogametic sex Chromosome preparations showed no evidence for morphologically distinct sex chromosomes The low frequencies of associated markers argue against a morphologically cryptic non-recombining sex chromosome The sex-locus is therefore likely to be autosomal The male-specific AFLP marker was sequenced and converted into a sequence characterised amplified region (SCAR) marker This SCAR marker produced a fragment of equal size in males and females, suggesting that sequence divergence between male- and female-specific chromosomal regions is low

Distribution of similar self-incompatibility (S) haplotypes in different genera, Raphanus and Brassica

Abstract: The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.

gemini pollen 2, a male and female gametophytic cytokinesis defective mutation

Abstract: Gametophytic cytokinesis is essential for the development and function of the male and female gametophytes. We have previously described the isolation and characterisation of gemini pollen 1 (gem1) that acts gametophytically to disturb asymmetric division and cytokinesis at pollen mitosis I (PMI) in Arabidopsis. Here we describe the genetic and cytological analysis of an independent gametophytic mutant, gem2, with similar characteristics to gem1, but which maps to a different genetic locus. gem2 shows reduced genetic transmission through both male and female gametes and leads to the production of divided or twin-celled pollen. Developmental analysis revealed that gem2 does not affect karyokinesis at PMI, but leads to repositioning of the cell plate, and partial or complete failure of cytokinesis, resulting in symmetrical divisions or binucleate pollen grains, respectively. Symmetrical divisions lead to altered pollen cell fate with both sister cells displaying vegetative cell fate. Moreover, we demonstrate that the predominant female defect in gem2 is a lack of cellularisation of the embryo sac during megagametogenesis. GEM2 therefore defines an independent genetic locus that is involved in the correct specification of both male and female gametophytic cytokinesis.

Isolation of S-RNase binding proteins from Solanum chacoense: identification of an SBP1 (RING finger protein) orthologue

Abstract: Currently, the most attractive working model of gametophytic self-incompatibility (SI) involving S-RNases postulates the presence of an inhibitor protein or complex expressed in pollen tubes that would counteract the cytotoxic effect of the ribonuclease activity of the S-RNase Since it has been previously shown that allele-specific recognition is mediated through the hypervariable domain sequence of the S-RNase, we have targeted this region to isolate pollen-expressed interacting proteins in the yeast two-hybrid system One of the isolated proteins corresponds to a RING finger protein highly similar to the previously isolated SBP1 protein from Petunia hybrida This protein is postulated to be part of the RING finger E3 ligase family The ScSBP1 gene is expressed in almost all tissues tested, suggesting a more general role than only being involved in SI Although the ScSBP1 gene is polymorphic, linkage analysis showed that it was unlinked to the S-locus The isolation of this S-RNase-binding protein in two different species and with four different S-RNase sequences as bait, strengthens its putative involvement in the SI response Furthermore, comparison of the bait sequences used suggests that the SBP1 protein interacts with conserved sequences located between the HVa and HVb domains

Fluctuations in the pollen tube tip-focused calcium gradient are not reflected in nuclear calcium level: a comparative analysis using recombinant yellow cameleon calcium reporter

Abstract: For the first time in pollen tubes, both cytoplasmic and nuclear calcium have been imaged to allow comparative analysis of calcium dynamics in these two compartments with high spatial and temporal dynamics. An improved cameleon (YC2.1) calcium reporter was expressed cytoplasmically in both Lilium longiflorum and Nicotiana tabacum pollen tubes and the periodically fluctuating tip-focused calcium gradient typical of normal growth was recorded by ratio image analysis. The nucleoplasmin targeting sequence was then used to localise expressed YC2.1 to the vegetative nucleus of N. tabacum pollen tubes to permit imaging of nuclear location, shape and calcium dynamics. Nuclear-targeted YC2.1 (NupYC2.1) showed an absence of any obvious regular fluctuations in nuclear calcium levels during tube extension in vitro with typical growth rate fluctuation. The use of targeted cameleons to study subcellular calcium dynamics in pollen tubes is discussed.

Studies on nuclear degeneration during programmed cell death of synergid and antipodal cells in Triticum aestivum

Abstract: Morphological changes in the nuclear degeneration of the synergid (mainly the synergid that receives the pollen tube) and antipodal cells in Triticum aestivum were studied. Although located in the same embryo sac, and derived from the same megaspore, nuclear degeneration of the synergid and antipodal cells differs greatly. Nuclear degeneration in the synergid is characterized by pycnosis, i.e., total chromatin condensation, nuclear deformation and distinct shrinkage in volume, followed by the formation of an irregular and densely stained mass—the degenerated nucleus—while the nucleolus disappears prior to the degradation of chromatin. In contrast, in the nuclear degeneration of antipodal cells, chromatin is only partly condensed and the nuclear volume changes only slightly after the distinct chromatin condensation. Chromatolysis then occurs, i.e., stainable contents disappear while the nuclear envelope is retained. The nucleoli persist after the disappearance of the chromatin. The possible functions of nuclear degeneration of synergid and antipodal cells are discussed, especially with respect to the guidance of pollen tube growth and the proliferation of free-nuclear endosperm. The degeneration of synergids and antipodal cells in T. aestivum are distinct forms of programmed cell death, regarded as cytoplasmic cell death and nuclear degradation in advance of cell death, respectively.

Fertilization-induced changes in the microtubular architecture of the maize egg cell and zygote—an immunocytochemical approach adapted to single cells

Abstract: By using single cell micromanipulation techniques, we developed an immunocytochemical procedure to examine subcellular protein localization in isolated and cultured cells. Localization of microtubules was examined in isolated single egg cells and developing zygotes of maize with anti-α-tubulin antibodies. In egg cells, a few cortical microtubules were detected but well organized microtubules were rarely observed. In contrast, distinct cortical microtubules and strands of cytoplasmic microtubules radiating from the nucleus to the cell periphery were observed in developing zygotes. Solely cortical microtubules were observed in zygotes up to 7 h after in vitro fertilization. After this time, radiating microtubules additionally appeared, and persisted during zygote development. These results indicate early and pronounced fertilization-induced changes in microtubular organization in the fertilized egg cell of maize.

Defects in nucleolar migration and synapsis in male prophase I in the ask1-1 mutant of Arabidopsis

Abstract: The migration of nucleolus to the nuclear periphery and the onset of homologous chromosome synapsis are characteristics of the leptotene to zygotene transition in meiocytes Little is known about the genes regulating these processes in eukaryotes Here we report the characterization of morphological defects in prophase I in microsporocytes in the Arabidopsis skp1-like1 (ask1-1) mutant In ask1-1, the nucleolus of the microsporocyte failed to move to the nuclear periphery throughout prophase I, and the chromosomes failed to synapse, despite the formation of bivalents in late prophase I We also found that the transcription of the DMC1 gene and ASY1 gene, which were predominantly transcribed prior to zygotene in microsporocytes, was not affected in ask1-1 These findings, and the fact that ASK1 is a component of the SCF ubiquitin ligase, suggest that ASK1 regulates either the leptotene to zygotene transition or events associated with the developmental transition during male meiosis in Arabidopsis

Exploitation of genes affecting meiotic non-reduction and nuclear restitution: Arabidopsis as a model?

Abstract: Meiosis is a fascinating and complex phenomenon with intriguing practical potential. Some deviations in meiotic reduction are crucial for plant evolution and extremely important for plant breeding. In particular, the production of gametes with unreduced chromosome number and the diplosporic pathway of apomixis hold great promise for the genetic improvement of cultivated plants. The use of meiotic mutations is hampered by the fact that they do not occur widely in crop plants, and traditional breeding approaches to transfer these characters are difficult or impossible. For their exploitation, genetic engineering has been suggested as a promising and more direct way. Since molecular advances for isolating meiotic genes in flowering plants have been pursued mostly in Arabidopsis thaliana, mutants of this plant with alterations in meiotic reduction or meiotic nuclear restitution are the main focus of this review. Emphasis has been given to the cytological description of mutated phenotypes, their effect on male/female fertility, and to gene function. Perspectives for using meiotic genes of Arabidopsis to manipulate whole genomes through 2n gametes and for the exploitation of apomixis are also discussed.

Ploidy variation of progenies from intra- and inter-ploidy crosses with regard to trisomic production in asparagus (Asparagus officinalis L.)

Abstract: Ploidy variation within progenies from intra- and inter-ploidy crosses of asparagus were determined in this study. The DNA content of most reduced microspores was half that of somatic cells in diploid, triploid and tetraploid plants as determined by flow cytometry. Many viable seeds were obtained from various 2x × 2x and 2x × 3x intra- and inter-ploidy crosses. Nuclear DNA contents of these progenies from reciprocal crosses between diploids and triploids were similar to those expected of diploids, but with a wider range: hyper- and hypo-diploids including trisomics seem to be involved in the progenies. Indeed, the number of chromosomes in the progenies of 2x × 3x crosses ranged from 18 to 23. Based on frequencies of viable seeds and trisomic phenotypic appearance, it was concluded that the 2x × 3x cross was most efficient for obtaining trisomic plants in Asparagus officinalis.

Effects of pollen-synthesized green fluorescent protein on pollen grain fitness

Abstract: Genetically engineered pollen with a visible marker gene could be useful to monitor the movement of transgenic pollen provided there are no negative physiological or fitness effects of expressing such a gene. In this study, we measured the fitness of Nicotiana tabacum cv. Xanthi pollen expressing the marker gene green fluorescent protein (GFP). Average pollen tube germination frequencies and pollen tube growth rates in vitro were measured in three different types of plants: (1) plants producing GFP in pollen cells only (LAT59-GFP), (2) plants synthesizing GFP under the control of a constitutive promoter (CaMV 35S) in which no GFP was produced in pollen, and (3) non-transgenic plants. Pollen synthesizing the GFP protein did not differ significantly in average pollen germination frequencies from pollen without GFP (P=0.65). Average pollen tube growth rates over a 5-h period did not differ significantly between transgenic and non-transgenic types (R 2=0.89, 0.98, and 0.95, respectively, for GFP-tagged, 35S-GFP, and wild type). Overall, GFP expression in pollen grains of tobacco was not found to have an effect on pollen fitness under the controlled experimental conditions of this study.

Characterization of two SEPALLATA MADS-box genes from the dioecious plant Silene latifolia

Abstract: Two SEPALLATA orthologs, SlSEP1 and SlSEP3, were isolated from male flower buds of the dioecious plant Silene latifolia. Both genes are located on autosomes and not on the sex chromosomes. SlSEP1 and SlSEP3 transcripts were detected specifically in male and female flower buds. Quantitative RT-PCR and in situ hybridization revealed that both genes were expressed in young flower meristems, developing petals, male anthers, and female ovules. However, neither transcript was detected in suppressed gynoecia of male flower buds or suppressed anthers of female flower buds. The genes were differentially expressed during anther development, with the SlSEP1 transcript accumulating in anther walls and tapetal cells, and the SlSEP3 transcript accumulating in tetrads as well as anther walls and tapeta. Transcripts of both genes were also detected in flower buds of mutants with deletions of some parts of the Y chromosome, suggesting that neither gene is directly involved in sex determination.

Calcium levels increase in the lily stylar transmitting tract after pollination

Abstract: Pollen tube growth in vitro requires calcium for most species but the in vivo source or reservoir of this calcium is not known. Using methods to localize calcium in situ, we confirm that low levels of calcium are detected in the transmitting tract extracellular matrix (ECM) in unpollinated lily styles. Pollination in lily induces an increase in the detectable levels of calcium in the transmitting tract ECM binding to the stylar cell and pollen tube walls. This calcium is detected in the cytoplasm and vesicles near the pollen tube tip.

Members of the S-receptor kinase multigene family in Senecio squalidus L. (Asteraceae), a species with sporophytic self-incompatibility.

Abstract: While the molecular basis of sporophytic self-incompatibility (SSI) has been investigated extensively in the Brassicaceae, almost nothing is known about the molecular regulation of SSI in other families, such as the Asteraceae. In species of Brassica and in Arabidopsis lyrata, a stigma-specific serine-threonine receptor kinase (SRK) and its cognate ligand, a pollen coating-borne cysteine-rich protein (SCR/SP11), determine the female and male sides of the SSI response, respectively. Here we have used RT-PCR with degenerate primers to conserved regions of SRK to amplify three SRK-like gene fragments expressed in stigmas of Senecio squalidus (Asteraceae). The Senecio S-receptor-like kinase (SSRLK) sequences share ~43% amino acid sequence identity with Brassica SRK3 but higher amino acid sequence identity (~50%) with two Solanum bulbocastanum receptor-like kinase genes of unknown function. Despite expression in stigmas, all three SSRLKs were expressed at varying levels in floral and vegetative tissues. No allelic polymorphism was detected for the three SSRLKs in two S homozygous lines of S. squalidus or three other lines of S. squalidus carrying different S alleles. A full-length cDNA clone was obtained for SSRLK1 and its predicted amino acid sequence revealed significant structural differences to Brassica SRKs, most notably a major N-terminal truncation of 169 amino acids and the presence of just 8 conserved cysteine residues within the putative receptor domain instead of 12. Together, the sequence characteristics and expression characteristics of SSRLKs suggest that they are unlikely to be directly involved in the SSI response of S. squalidus. These findings are discussed in terms of the evolution of the SRK multigene family and the molecular basis of SSI in S. squalidus and the Asteraceae.

AVAG2 is a putative D-class gene from an ornamental asparagus

Abstract: Members of the AGAMOUS (AG) family of MADS-box genes play important roles in regulating the development of reproductive organs in flowering plants. To elucidate the molecular mechanisms of floral development in Asparagus virgatus, we isolated and characterized an Asparagus AG-homologue, AVAG2. AVAG2 contains an open reading frame that encodes a deduced protein with 234 amino acid residues. Phylogenetic analysis indicated that AVAG2 belongs to the D-lineage of the AG gene family. AVAG2 mRNA was detected in the flower, but not in vegetative organs. Moreover, in in situ hybridization experiments, AVAG2 signals were observed in the stamens and carpels during early flower development, and appeared in the ovule only at later developmental stages. This suggests that the AVAG2 gene is involved in ovule formation. Thus, our expression data support the phylogenetic analysis indicating that AVAG2 belongs to the D-class gene family.

Male-sterile mutation alters Zea m 1 (β-expansin 1) accumulation in a maize mutant

Abstract: gaMS-2 is a gametophytic male-sterile mutant of maize, with sterile pollen grains developmentally blocked at the binucleate stage. To characterise differentially expressed proteins in gaMS-2 pollen, we compared protein profiles of anthers and mature pollen from heterozygous GaMS-2/gaMS-2 plants and wild type (wt) plants by two-dimensional electrophoresis (2-DE). A basic protein present at a greatly reduced level in GaMS-2/gaMS-2 anthers was subsequently identified by tandem mass spectrometry as Zea m 1 (a glycoprotein of 31 kDa), the major group-1 allergen of maize pollen and a member of the β-expansin 1 family. Moreover, we show that Zea m 1 can be deglycosylated by peptide N-glycosidase F. After deglycosylation, four major isoforms—Zea m 1a (more acetic), Zea m 1b, Zea m1c and Zea m 1d (more basic)—can be discriminated in wt anther in 2-DE immunoblots probed with a monoclonal antibody against the group-1 pollen allergen, whereas all the isoforms, especially Zea m 1a, exist at reduced levels in GaMS-2/gaMS-2 anthers. Furthermore, the reduced Zea m 1 accumulation in the mutant appears to occur in immature pollen but not in anther sporophytic tissues. Finally, we separated sterile pollen grains (at the mononucleate stage) from fertile ones using 42% Percoll solution, and found that Zea m 1 is barely detectable in sterile pollen grains. Together, our results indicate that a reduced Zea m 1 level is associated with the sterile phenotype of gaMS-2.

A mutant with constitutive sexual organ development in Marchantia polymorpha L.

Abstract: In lower land plants, genes controlling the transition from vegetative growth to sexual reproduction have not yet been identified. In the dioecious liverwort Marchantia polymorpha, the transition to sexual reproduction accompanied by the formation of sexual organs on the gametophytic thallus is initiated under long-day conditions. By particle bombardment-mediated mutagenesis, we generated a mutant of M. polymorpha that constitutively forms sexual organs. This mutant is fully fertile, showing that the mutation does not affect formation of male or female sexual organs per se. Genetic analysis reveals that this phenotype is caused by mutation of a single autosomal locus, suggesting that this mutation defines or controls a gene regulating the transition to sexual reproduction in M. polymorpha.

Transcript profiling of male-fertile and male-sterile sorghum indicates extensive alterations in gene expression during microgametogenesis

Abstract: Male-sterile sorghum carrying the IS1112C cytoplasm represents an unusual example of aberrant microgametogenesis wherein microspores develop into inviable pollen that remain physically intact until anther exsertion. These inviable pollen grains do not deposit starch, yet fluoresce with the vital stain fluoroscein diacetate. We sought to elucidate the extent of differential gene expression in this subtle example of defective microgametogenesis through cDNA-AFLP transcript profiling of near-isogenic male-fertile and male-sterile plants at an early stage representing early-mid microspores to early pollen, 7–11 days prior to anthesis, and a late stage representing young to nearly mature pollen, spanning the terminal 96 h of pollen development. The transition from early to late stages is characterized by changes in abundance of nearly 33% of shared transcripts, and early- or late-specific expression of about 10% of transcripts. Male-sterile plants exhibit extensive changes in regulatory patterns characteristic of fertile plants, including premature expression of late-specific, and prolonged expression of early-specific, transcripts. Genome-wide transcriptome patterns indicate the expression of an estimated 12,000 genes in early-mid microspores, and the abundance of at least 15% of these transcripts is altered in male-sterile plants. A near-isogenic line restored to male fertility is characterized by apparent normalized expression of most of these transcripts. The development of the microgametophyte is thus characterized by dynamic programmed changes in gene expression, and the expression of male sterility compounds these changes in a complex manner.

Aspects of sexual failure in the reproductive processes of a rare bulbiliferous plant, Titanotrichum oldhamii (Gesneriaceae), in subtropical Asia

Abstract: Titanotrichum oldhamii produces both flowers and asexual bulbils on its inflorescences. However, field observations and herbarium collections indicate that seed set is infrequent and that most reproduction is from vegetative bulbils. We have investigated the failure of sexual reproduction and identified four major causes: (1) in the wild, the seed:ovule ratio for open pollination was only 1.9%, in contrast to 10.1% for artificial cross-pollination, implying poor pollinator services. (2) The overall reproductive success was 5–10 times greater in glasshouse pollination treatments compared to field treatments. This suggests suboptimal environmental conditions for seed set in natural habitats, which may eliminate natural seed set completely. (3) Pollination experiments showed that outcrossed populations set significantly more seed and had higher germination rates than intra-populational and self crosses, in both field and glasshouse experiments. T. oldhamii thus appears to benefit from wide outcrossing. Pollen transfer between populations, however, seems to be infrequent because of the rarity and scattered distribution of Titanotrichum populations. (4) Flowers near the apex of the inflorescence are less likely to set seed, especially late in the season when inflorescences convert to bulbil production. In these late flowers, pollen tubes showed poor guidance as they approach the micropyle of the ovules. Even under optimal glasshouse conditions, the average outcrossing success was only 0.229. Almost one-half of the ovules remained undeveloped and 13.5% of ovules aborted after pollination, indicating a strong shift of resource allocation toward vegetative bulbils and rhizome development. Efficient reproduction from asexual bulbils may thus have released Titanotrichum from strong selection for efficient sexual reproduction. However, occasional seed set observed in the wild may be very important for maintaining some genetic diversity in populations, and promoting overall fitness.

The PELPIII glycoproteins in Solanaceae: stylar expression and transfer into pollen tube walls

Abstract: The class III pistil-specific extensin-like proteins (PELPIII) of Nicotiana tabacum accumulate in the intercellular matrix (IM) of the style transmitting tissue (TT). After pollination, the 110–140 kDa PELPIII is translocated from the IM into the pollen tube walls. PELPIII-like sequences have been found in several solanaceous species. These sequences are expressed in mature non-pollinated styles at both RNA and protein level. Of the genus Nicotiana, the species N. alata, N. x sanderae and N. sylvestris (section Alatae), and N. tomentosiformis and N. otophora (section Tomentosae) showed an expression level of PELPIII homologues similar to that in mature styles of N. tabacum. PELPIII genes were absent in the most ancient species studied, namely N. trigonophylla (section Trigonophyllae). To study the species dependence of the translocation of PELPIII into the pollen tube wall in tobacco, interspecific pollinations on N. tabacum pistils were carried out with pollen from the incongruous species N. rustica, N. trigonophylla and Petunia hybrida, where PELPIII homologues are absent in the style. Immunocytological tests showed that the N. tabacum PELPIII is translocated into the pollen tube walls of all three species. Thus, the pollen tube walls of these species do not form a barrier for IM compounds such as the 110–140 kDa PELPIII and the absence of any possible effect of PELPIII on pollen tube growth cannot be due to failure of PELPIII transport through the wall. The importance of these findings is discussed with respect to the evolutionary origin of PELPIII, the pollen pistil interaction, the function of style TT-specific proteins and the physical properties of pollen tube walls.

Lily (Lilium longiflorum L.) pollen protoplast adhesion is increased in the presence of the peptide SCA

Abstract: The style of lily produces a specialized extracellular matrix (ECM) in the transmitting tract epidermis that functions to guide pollen tubes to the ovary. This adhesive ECM contains low esterified pectins and a peptide, SCA (stigma/stylar cysteine-rich adhesin). Together they form a matrix to which pollen tubes adhere as they grow through the style. Pollen tubes also adhere to each other but only when grown in vivo, not in vitro. Pollen does not produce detectable SCA, but when SCA is added to an in vitro growth medium, it binds to pollen tubes that have esterified and low-esterified pectins in their walls. Since adhesion of the pollen tube to the stylar matrix requires tip growth, we hypothesized that the pectin wall at the pollen tube tip interacted with the SCA protein to initiate adhesion with the stylar pectin [Lord (2000) Trends Plant Sci 5:368–373]. Here, we use a pollen protoplast system to examine the effect of SCA on protoplast adhesion when it is added to the growth medium in the absence of the stylar pectin. We found that SCA induces a 2-fold increase in protoplast adhesion when it is added at the start of protoplast culture. This effect is less when SCA is added to the medium after the cell wall on the protoplast has begun to regenerate. We show that among the first components deposited in the new wall are arabinogalactan proteins (AGPs) and highly esterified pectins. We see no labeling for low esterified pectins even after 3 days of culture. In the pollen protoplast culture, adhesion occurs in the absence of the low esterified pectin. The newly formed wall on the protoplast mirrors that of the pollen tube tip in lily, which is rich in AGPs and highly esterified pectins. Thus, the protoplast system may be useful for isolating the pollen partner for SCA in this adhesion event.

The AVAG1 gene is involved in development of reproductive organs in the ornamental asparagus, Asparagus virgatus

Abstract: The AGAMOUS (AG)-like gene has been reported to be involved in the formation of the stamens and carpels. The genus Asparagus contains hermaphrodite and dioecious species, and analysis of the AG-like genes in these species may reveal how these different reproductive systems have evolved in this genus. We isolated one AG-like gene, designated AVAG1, from the ornamental hermaphrodite species Asparagus virgatus. Phylogenetic analysis showed that the AVAG1 gene is closely related to HAG1 from Hyacinthus and PeMADS1 from Phalaenopsis. Northern blot analysis showed that AVAG1 transcripts were detected in flower buds, but not in roots, stems or phylloclades. In situ hybridization analyses revealed that the AVAG1 mRNA is expressed specifically in the floral meristem and the developing reproductive organs. Early in flower development, expression of AVAG1 was restricted mainly to the stamens and carpels, with AVAG1 expression in the stamen disappearing at later stages of flower development, although it remained strong in the ovule.

Changes of protein profiles during pollen development in Lilium longiflorum

Abstract: Pollen proteins of Lilium longiflorum were examined at different developmental stages (young, mature and cultured) using two-dimensional differential gel electrophoresis. Quantitative changes of six proteins (MP1–MP6) during pollen development were observed in the acidic and low molecular weight region. After water absorption on the culture medium, the quantities of all six proteins were drastically changed. Mass spectrometric analysis revealed that MP2, MP3, MP4 and MP6 are late embryogenesis abundant (LEA) (D-7) protein, profilin 3, profilin 1 and enolase, respectively. The remaining two proteins (MP1 and MP5) could not be identified by mass spectrometric analysis. Immunogold electron microscopic examination showed the presence of these proteins in different regions: MP1 around lipid bodies, suggesting possible involvement in lipid metabolism, MP4 near actin in the cytoplasm, indicating the possibility of its interaction with actin in the regulatory pathways of pollen, and MP2 and MP6 in the cytoplasm.

AOM3 and AOM4: two MADS box genes expressed in reproductive structures of Asparagus officinalis

Abstract: The MADS box genes participate in different steps of vegetative and reproductive plant development, including the most important phases of the reproductive process. Here we describe the isolation and characterisation of two Asparagus officinalis MADS box genes, AOM3 and AOM4. The deduced AOM3 protein shows the highest degree of similarity with ZAG3 and ZAG5 of maize, OsMADS6 of rice and AGL6 of Arabidopsis thaliana. The deduced AOM4 protein shows the highest degree of similarity with AOM1 of asparagus, the SEP proteins of Arabidopsis and the rice proteins OsMADS8, OsMADS45 and OsMADS7. The high level of identity between AOM1 and AOM4 made impossible the preparation of probes specific for one single gene, so the hybridisation signal previously described for AOM1 is probably due to the expression of both genes. The expression profile of AOM3 and AOM1/AOM4 during flower development is identical, and similar to that of the SEP genes. Asparagus genes, however, are expressed not only in flower organs, but also in the different meristem present on the apical region of the shoot during the flowering season: the apical meristem and the three lateral meristems emerging from the leaf axillary region that will give rise to flowers and lateral inflorescences during flowering season, and to phylloclades and branches during the subsequent vegetative phase. The expression of AOM3 and AOM1/AOM4 in these meristems appears to be correlated with the reproductive function of the apex as the hybridisation signal disappears when the apex switches to vegetative function.