The Arabidopsis GPR1 Gene Negatively Affects Pollen Germination, Pollen Tube Growth, and Gametophyte Senescence.

Genes essential for gametophyte development and fertilization have been identified and studied in detail; however, genes that fine-tune these processes are largely unknown. Here, we characterized an unknown Arabidopsis gene, GTP-BINDING PROTEIN RELATED1 (GPR1). GPR1 is specifically expressed in ovule, pollen, and pollen tube. Enhanced green fluorescent protein-tagged GPR1 localizes to both nucleus and cytoplasm, and it also presents in punctate and ring-like structures. gpr1 mutants exhibit no defect in gametogenesis and seed setting, except that their pollen grains are pale in color. Scanning electron microscopy analyses revealed a normal patterned but thinner exine on gpr1 pollen surface. This may explain why gpr1 pollen grains are pale. We next examined whether GPR1 mutation affects post gametogenesis processes including pollen germination, pollen tube growth, and ovule senescence. We found that gpr1 pollen grains germinated earlier, and their pollen tubes elongated faster. Emasculation assay revealed that unfertilized gpr1 pistil expressed the senescence marker PBFN1:GUS (GUS: a reporter gene that encodes ß-glucuronidase) one-day earlier than the wild type pistil. Consistently, ovules and pollen grains of gpr1 mutants showed lower viability than those of the wild type at 4 to 5 days post anthesis. Together, these data suggest that GPR1 functions as a negative regulator of pollen germination, pollen tube growth, and gametophyte senescence to fine-tune the fertilization process.

Cytological, molecular mechanisms and temperature stress regulating production of diploid male gametes in Dianthus caryophyllus L.

In plant evolution, because of its key role in sexual polyploidization or whole genome duplication events, diploid gamete formation is considered as an important component in diversification and speciation. Environmental stress often triggers unreduced gamete production. However, the molecular, cellular mechanisms and adverse temperature regulating diplogamete production in carnation remain poorly understood. Here, we investigate the cytological basis for 2n male gamete formation and describe the isolation and characterization of the first gene, DcPS1 (Dianthus Caryophyllus Parallel Spindle 1). In addition, we analyze influence of temperature stress on diploid gamete formation and transcript levels of DcPS1. Cytological evidence indicated that 2n male gamete formation is attributable to abnormal spindle orientation at male meiosis II. DcPS1 protein is conserved throughout the plant kingdom and carries domains suggestive of a regulatory function. DcPS1 expression analysis show DcPS1 gene probably have a role in 2n pollen formation. Unreduced pollen formation in various cultivation was sensitive to high or low temperature which was probably regulated by the level of DcPS1 transcripts. In a broader perspective, these findings can have potential applications in fundamental polyploidization research and plant breeding programs.

Imaging sexual reproduction in Arabidopsis using fluorescent markers.

Sexual reproduction in higher plants is a stealth process as most events occur within tissues protected by multiple surrounding cell layers. Female gametes are produced inside the embryo sac surrounded by layers of ovule integument cells. Upon double fertilization, two male gametes are released at one end of the embryo sac and migrate towards their respective female partner to generate the embryo and its feeding tissue, the endosperm, within a seed. Since the early discovery of plant reproduction, advances in microscopy have contributed enormously to our understanding of this process (Faure and Dumas, Plant Physiol 125:102-104, 2001). Recently, live imaging of double fertilization has been possible using a set of fluorescent markers for gametes in Arabidopsis. The following chapter will detail protocols to study male and female gametogenesis and double fertilization in living tissues using fluorescent markers.

Identification of proteins enriched in rice egg or sperm cells by single-cell proteomics.

In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is specialized, fuses with the sperm cell, and converts into an active zygote for early embryogenesis remains unclear. This lack of knowledge is partly attributable to the difficulty of direct analyses of gametes in angiosperms. In the present study, proteins from egg and sperm cells obtained from rice flowers were separated by one-dimensional polyacrylamide gel electrophoresis and globally identified by highly sensitive liquid chromatography coupled with tandem mass spectroscopy. Proteome analyses were also conducted for seedlings, callus, and pollen grains to compare their protein expression profiles to those of gametes. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000265. A total of 2,138 and 2,179 expressed proteins were detected in egg and sperm cells, respectively, and 102 and 77 proteins were identified as preferentially expressed in egg and sperm cells, respectively. Moreover, several rice or Arabidopsis lines with mutations in genes encoding the putative gamete-enriched proteins showed clear phenotypic defects in seed set or seed development. These results suggested that the proteomic data presented in this study are foundational information toward understanding the mechanisms of reproduction and early development in angiosperms.

Evidence for a role of Arabidopsis CDT1 proteins in gametophyte development and maintenance of genome integrity.

Meristems retain the ability to divide throughout the life cycle of plants, which can last for over 1000 years in some species. Furthermore, the germline is not laid down early during embryogenesis but originates from the meristematic cells relatively late during development. Thus, accurate cell cycle regulation is of utmost importance to avoid the accumulation of mutations during vegetative growth and reproduction. The Arabidopsis thaliana genome encodes two homologs of the replication licensing factor CDC10 Target1 (CDT1), and overexpression of CDT1a stimulates DNA replication. Here, we have investigated the respective functions of Arabidopsis CDT1a and CDT1b. We show that CDT1 proteins have partially redundant functions during gametophyte development and are required for the maintenance of genome integrity. Furthermore, CDT1-RNAi plants show endogenous DNA stress, are more tolerant than the wild type to DNA-damaging agents, and show constitutive induction of genes involved in DNA repair. This DNA stress response may be a direct consequence of reduced CDT1 accumulation on DNA repair or may relate to the ability of CDT1 proteins to form complexes with DNA polymerase ε, which functions in DNA replication and in DNA stress checkpoint activation. Taken together, our results provide evidence for a crucial role of Arabidopsis CDT1 proteins in genome stability.

Sporogenesis and development of gametophytes in an endangered plant, Tetracentron sinense Oliv.

The sporogenesis and development of gametophytes in Tetracentron sinense Oliv. were studied with light microscopy. The anther has four microsporangia; its primary anther wall consists of an epidermis, an endothecium, one or two middle layers and one glandular tapetum. Simultaneous cytokinesis follows meiosis, forming a tetrahedral tetrad. Mature pollen grains are two-celled at the time of anther dehiscence. Its ovule is anatropous, bitegmic and crassinucellate; the development of the female gametophyte is of the monosporic 8-nucleate Polygonum type. Significantly, some striking features were first found in T. sinense: (1) anther dehiscence occurs soon after the endothecium fibrously thickens and the intersporangial septum degenerates; (2) tapetum degeneration is retarded, persisting up to the stage of two-celled pollen grain; (3) a few cellular events such as the vacuolization and the contraction and deformation of the pollen mother cell (PMC) and microspore are not normal at the PMC, dyad and tetrad stages. The abnormalities during male reproduction might be one of important factors resulting in the poor natural regeneration of T. sinense.

Non-additive effects of pollen limitation and self-incompatibility reduce plant reproductive success and population viability.

BACKGROUND AND AIMS: Mating system is a primary determinant of the ecological and evolutionary dynamics of wild plant populations. Pollen limitation and loss of self-incompatibility genotypes can both act independently to reduce seed set and these effects are commonly observed in fragmented landscapes. This study used a simulation modelling approach to assess the interacting effects of these two processes on plant reproductive performance and population viability for a range of pollination likelihood, self-incompatibility systems and S-allele richness conditions. METHODS: A spatially explicit, individual-based, genetic and demographic simulation model parameterized to represent a generic self-incompatible, short-lived perennial herb was used to conduct simulation experiments in which pollination probability, self-incompatibility type (gametophytic and sporophytic) and S-allele richness were systematically varied in combination to assess their independent and interacting effects on the demographic response variables of mate availability, seed set, population size and population persistence. KEY RESULTS: Joint effects of reduced pollination probability and low S-allele richness were greater than independent effects for all demographic response variables except population persistence under high pollinator service (>50 %). At intermediate values of 15-25 % pollination probability, non-linear interactions with S-allele richness generated significant reductions in population performance beyond those expected by the simple additive effect of each independently. This was due to the impacts of reduced effective population size on the ability of populations to retain S alleles and maintain mate availability. Across a limited set of pollination and S-allele conditions (P = 0·15 and S = 20) populations with gametophytic SI showed reduced S-allele erosion relative to those with sporophytic SI, but this had limited effects on individual fecundity and translated into only modest increases in population persistence. CONCLUSIONS: Interactions between pollen limitation and loss of S alleles have the potential to significantly reduce the viability of populations of a few hundred plants. Population decline may occur more rapidly than expected when pollination probabilities drop below 25 % and S alleles are fewer than 20 due to non-additive interactions. These are likely to be common conditions experienced by plants in small populations in fragmented landscapes and are also those under which differences in response between gameptophytic and sporophtyic systems are observed.

Sporogenesis and development of gametophytes in an endangered plant, Tetracentron sinense Oliv

The sporogenesis and development of gametophytes in Tetracentron sinense Oliv. were studied with light microscopy. The anther has four microsporangia; its primary anther wall consists of an epidermis, an endothecium, one or two middle layers and one glandular tapetum. Simultaneous cytokinesis follows meiosis, forming a tetrahedral tetrad. Mature pollen grains are two-celled at the time of anther dehiscence. Its ovule is anatropous, bitegmic and crassinucellate; the development of the female gametophyte is of the monosporic 8-nucleate Polygonum type. Significantly, some striking features were first found in T. sinense: (1) anther dehiscence occurs soon after the endothecium fibrously thickens and the intersporangial septum degenerates; (2) tapetum degeneration is retarded, persisting up to the stage of two-celled pollen grain; (3) a few cellular events such as the vacuolization and the contraction and deformation of the pollen mother cell (PMC) and microspore are not normal at the PMC, dyad and tetrad stages. The abnormalities during male reproduction might be one of important factors resulting in the poor natural regeneration of T. sinense.

The RPT2 subunit of the 26S proteasome directs complex assembly, histone dynamics, and gametophyte and sporophyte development in Arabidopsis.

The regulatory particle (RP) of the 26S proteasome contains a heterohexameric ring of AAA-ATPases (RPT1-6) that unfolds and inserts substrates into the core protease (CP) for degradation. Through genetic analysis of the Arabidopsis thaliana gene pair encoding RPT2, we show that this subunit plays a critical role in 26S proteasome assembly, histone dynamics, and plant development. rpt2a rpt2b double null mutants are blocked in both male and female gamete transmission, demonstrating that the subunit is essential. Whereas rpt2b mutants are phenotypically normal, rpt2a mutants display a range of defects, including impaired leaf, root, trichome, and pollen development, delayed flowering, stem fasciation, hypersensitivity to mitomycin C and amino acid analogs, hyposensitivity to the proteasome inhibitor MG132, and decreased 26S complex stability. The rpt2a phenotype can be rescued by both RPT2a and RPT2b, indicative of functional redundancy, but not by RPT2a mutants altered in ATP binding/hydrolysis or missing the C-terminal hydrophobic sequence that docks the RPT ring onto the CP. Many rpt2a phenotypes are shared with mutants lacking the chromatin assembly factor complex CAF1. Like caf1 mutants, plants missing RPT2a or reduced in other RP subunits contain less histones, thus implicating RPT2 specifically, and the 26S proteasome generally, in plant nucleosome assembly.

[Allelopathic effects of companion species on spore germination and gametophyte development in Cibotium barometz].

OBJECTIVE: To study the relationship between Cibotium barometz and its companion species, Arachniodes falcate and Alpinia japonica, we used aqueous leachates of the two companions to deal with C. barometz spores. METHOD: Spores of C. barometz were translated on MS culture which contained different concentration of aqueous extracts of the two companions, the germination and gametophyte development were observed and recorded. RESULT: All extracts inhibited and delayed the C. barometz spores germination and rhizoid elongation was inhibited. It also had obvious inhibition to the prothallus formation and sexual differentiation. And the higher concentration, the more obvious inhibition of aqueous extracts of the two companion species. CONCLUSION: The two companion species have allelopathic effects on the spore germination and gametophyte development of C. barometz. And it may have an influence on sporogon ontogenesis and the population expansion.

Evidence for the involvement of the Arabidopsis SEC24A in male transmission.

Eukaryotic cells use COPII-coated carriers for endoplasmic reticulum (ER)-to-Golgi protein transport. Selective cargo capture into ER-derived carriers is largely driven by the SEC24 component of the COPII coat. The Arabidopsis genome encodes three AtSEC24 genes with overlapping expression profiles but it is yet to be established whether the AtSEC24 proteins have overlapping roles in plant growth and development. Taking advantage of Arabidopsis thaliana as a model plant system for studying gene function in vivo, through reciprocal crosses, pollen characterization, and complementation tests, evidence is provided for a role for AtSEC24A in the male gametophyte. It is established that an AtSEC24A loss-of-function mutation is tolerated in the female gametophyte but that it causes defects in pollen leading to failure of male transmission of the AtSEC24A mutation. These data provide a characterization of plant SEC24 family in planta showing incompletely overlapping functions of the AtSEC24 isoforms. The results also attribute a novel role to SEC24 proteins in a multicellular model system, specifically in male fertility.

[Cytogenetic peculiarities of cell genesis in apical meristems under gametophytic apomixis (using autonomous apomicts of the Asteraceae as an example)].

Cytogenetic peculiarities of cell genesis in apical meristems of apomicts has been analyzed using a series of the Asteraceae species as an example. The extent to which aneu- and mixoploids are spread among plants in the investigated populations of the Asteraceae species is so high (up to 30-60% of the studied plants and their offspring), that it seems reasonable to suppose that their rise is a natural phenomenon. It has been shown that in the aposporous facultative apomict Pilosella officinarum microgametophyte is a relatively stable element of the seed reproduction system from the point of view of caryotypical variation.

Extremes in rapid cellular morphogenesis: post-transcriptional regulation of spermatogenesis in Marsilea vestita.

The endosporic male gametophyte of the water fern, Marsilea vestita, provides a unique opportunity to study the mechanisms that control cell fate determination during a burst of rapid development. In this review, we show how the spatial and temporal control of development in this simple gametophyte involves several distinct modes of RNA processing that allow the translation of specific mRNAs at distinct stages during gametogenesis. During the early part of development, nine successive cell division cycles occur in precise planes within a closed volume to produce seven sterile cells and 32 spermatids. There is no cell movement in the gametophyte; so, cell position and size within the spore wall define cell fate. After the division cycles have been completed, the spermatids become sites for the de novo formation of basal bodies, for the assembly of a complex cytoskeleton, for nuclear and cell elongation, and for ciliogenesis. In contrast, the adjacent sterile cells exhibit none of these changes. The spermatids differentiate into multiciliated, corkscrew-shaped gametes that resemble no other cells in the entire plant. Development is controlled post-transcriptionally. The transcripts stored in the microspore are released (unmasked) in the gametophyte at different times during development. At the start of these studies, we identified several key mRNAs that undergo translation at specific stages of gametophyte development. We developed RNA silencing protocols that enabled us to block the translation of these proteins and thereby establish their necessity and sufficiency for the completion of specific stages of gametogenesis. In addition, RNAi enabled us to identify additional proteins that are essential for other phases of development. Since the distributions of mRNAs and the proteins they encode are not identical in the gametophyte, transcript processing is apparently important in allowing translation to occur under strict temporal and spatial control. Transcript polyadenylation occurs in the spermatogenous cells in ways that match the translation of specific mRNAs. We have found that the exon junction complex plays key roles in transcript regulation and modifications that underlie cell specification in the gametophyte. We have recently become interested in the mechanisms that control the unmasking of the stored transcripts and have linked the synthesis and redistribution of spermidine in the gametophyte to the control of mRNA release from storage during early development and later to basal body formation, cytoskeletal assembly, and nuclear and cell elongation in the differentiating spermatids.

Arabidopsis FIMBRIN5, an actin bundling factor, is required for pollen germination and pollen tube growth.

Actin cables in pollen tubes serve as molecular tracks for cytoplasmic streaming and organelle movement and are formed by actin bundling factors like villins and fimbrins. However, the precise mechanisms by which actin cables are generated and maintained remain largely unknown. Fimbrins comprise a family of five members in Arabidopsis thaliana. Here, we characterized a fimbrin isoform, Arabidopsis FIMBRIN5 (FIM5). Our results show that FIM5 is required for the organization of actin cytoskeleton in pollen grains and pollen tubes, and FIM5 loss-of-function associates with a delay of pollen germination and inhibition of pollen tube growth. FIM5 decorates actin filaments throughout pollen grains and tubes. Actin filaments become redistributed in fim5 pollen grains and disorganized in fim5 pollen tubes. Specifically, actin cables protrude into the extreme tips, and their longitudinal arrangement is disrupted in the shank of fim5 pollen tubes. Consequently, the pattern and velocity of cytoplasmic streaming were altered in fim5 pollen tubes. Additionally, loss of FIM5 function rendered pollen germination and tube growth hypersensitive to the actin-depolymerizing drug latrunculin B. In vitro biochemical analyses indicated that FIM5 exhibits actin bundling activity and stabilizes actin filaments. Thus, we propose that FIM5 regulates actin dynamics and organization during pollen germination and tube growth via stabilizing actin filaments and organizing them into higher-order structures.

Syncyte formation in the microsporangium of Chrysanthemum (Asteraceae): a pathway to infraspecific polyploidy.

Polyploidy, which is thought to have played an important role in plant evolution and speciation, is prevalent in Chrysanthemum (x = 9). In fact, polyploid series are known in C. zawadskii (2x, 4x, 6x, 8x, and 10x) and C. indicum (2x, 4x, and 6x), but the mechanism by which polyploidization occurs is unknown. Here we show that in diploid individuals of both C. zawadskii and C. indicum, the fusion between two adjacent pollen mother cells (PMCs) occurs at a frequency of 1.1-1.3% early in the first meiotic division. While possessing the chromosomes of both PMCs, the fused cell or syncyte undertakes subsequent meiotic division processes as a single large PMC, producing four 2n pollen grains that are able to germinate. Despite their low frequency, syncyte formation may have played a major role in the production of infraspecific polyploids in Chrysanthemum.