Plant Sexual Reproduction Research Articles

Signaling of cell fate determination by the TPD1 small protein and EMS1 receptor kinase

Sexual reproduction requires the specification of cells with distinct fates in plants and animals. The EMS1 (also known as EXS) leucine-rich repeat receptor-like kinase (LRR-RLK) and TPD1 small protein play key roles in regulating somatic and reproductive cell fate determination in Arabidopsis anthers. Here, we show that ectopic expression of TPD1 causes abnormal differentiation of somatic and reproductive cells in anthers. In addition, ectopic TPD1 activity requires functional EMS1. Yeast two-hybrid, pull-down, and coimmunoprecipitation analyses further demonstrate that TPD1 interacts with EMS1 in vitro and in vivo. Moreover, TPD1 induces EMS1 phosphorylation in planta. Thus, our results suggest that TPD1 serves as a ligand for the EMS1 receptor kinase to signal cell fate determination during plant sexual reproduction.

Localization of arabinogalactan-proteins in different stages of embryos and their role in cotyledon formation of Nicotiana tabacum L.

Arabinogalactan proteins (AGPs) have been implicated in plant development including sexual plant reproduction. In this paper, the expression of AGPs and the effects of β-glucosyl Yariv reagent (βGlcY, which binds arabinogalactan proteins) in embryo development and cotyledon formation were investigated. Immunofluorescence assay displayed that the expression of AGPs labeled with antibody JIM13 was developmentally regulated. In early stages, AGPs were evenly distributed in the whole embryo, except for a short polar expression in the basal suspensor cell. In the globular stage of embryo, AGPs were condensed in the embryo proper (EP), apex of the EP, and at the juncture of the EP and suspensor. In heart-shaped embryo, APGs were only present at the juncture of the EP and suspensor. Immunogold labeling assay showed that the strong expression of AGPs at the juncture of the EP and suspensor was localized in the cell wall. Provision of βGlcY to the in vitro ovule culture medium caused delayed growth of embryos, cotyledon defect and abnormal venation pattern. Consequently, βGlcY induced the death of defective seedlings with the characteristics of deformed or irregular single cotyledon. Our results suggested that AGPs play functional roles in embryo development, cotyledon formation and seedling morphology establishment in Nicotiana tabacum L.

Application of Arabidopsis AGAMOUS second intron for the engineered ablation of flower development in transgenic tobacco

To explore a new approach to generating reproductive sterility in transgenic plants, the barnase gene from Bacillus amyloliquefaciens was placed under the control of an 1853-bp nucleotide sequence from the 3'end of the second intron of Arabidopsis AGAMOUS and CaMV 35S (-60) minimal promoter [AG-I-35S (-60)::Barnase], and was introduced into tobacco through transformation mediated by Agrobacterium tumefaciens. All AG-I-35S (-60)::Barnase transgenic plants showed normal vegetative growth and 28% of the transgenic lines displayed complete ablation of flowering. Two transgenic lines, Bar-5 and Bar-15, were 98.1 and 98.4% sterile, respectively, as determined by seed production and germination. When controlled by AG-I-35S (-60) chimeric promoter, barnase mRNA was detected in the reproductive tissues of transgenic tobacco plants, but not in vegetative parts. This study presents the first application of an AG intron sequence in the engineered ablation of sexual reproduction in plants. The AG-I-35S (-60)::Barnase construct can be useful in diminishing pollen and seed formation in plants, providing a novel bisexual sterility strategy for interception of transgene escape and has other potentially commercial use for transgenic engineering.

Self-incompatibility inPapaver: signalling to trigger PCD in incompatible pollen

Sexual reproduction in higher plants uses pollination, involving interactions between pollen and pistil. Self-incompatibility (SI) prevents self-fertilization, providing an important mechanism to promote outbreeding. SI is controlled by the S-locus; discrimination occurs between incompatible pollen, which is rejected, while compatible pollen can achieve fertilization. In Papaver rhoeas, S proteins encoded by the pistil part of the S-locus interact with incompatible pollen to effect rapid inhibition of tip growth. This self-incompatible interaction triggers a Ca(2+)-dependent signalling cascade. SI-specific events triggered in incompatible pollen include rapid depolymerization of the actin cytoskeleton; phosphorylation of soluble inorganic pyrophosphatases, and activation of a MAPK. It has recently been shown that programmed cell death (PCD) is triggered by SI. This provides a precise mechanism for the specific destruction of 'self' pollen. Recent data providing evidence for SI-induced caspase-3-like protease activity, and the involvement of actin depolymerization and MAPK activation in SI-mediated PCD will be discussed. These studies not only significantly advance our understanding of the mechanisms involved in SI, but also contribute to our understanding of functional links between signalling components and initiation of PCD in a plant cell. Recent data demonstrating SI-mediated modification of soluble inorganic pyrophosphatases are also described.

Characterization of a novel gene, BcMF7, that is expressed preferentially in pollen of Brassica campestris L. ssp. chinensis Makino

Pollen formation is important for plant sexual reproduction. To identify the genes that are involved in pollen formation, we performed the genome-wide transcriptional profiling in the flower buds of both male meiotic cytokinesis (mmc) mutant and its wild-type plants of Brassica campestris L. ssp. chinensis, syn. B. rapa L. ssp. chinensis. cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis showed that the mmc mutation resulted in changes in expression of a variety of genes. BcMF7, a transcript-derived fragment (TDF) accumulated in the wild-type flower buds was further characterized. The BcMF7 gene has 1161 bp in length with two introns. The full-length BcMF7 cDNA has 609 bp in length and encodes a protein of 129 amino acids. The deduced amino acid sequence of BcMF7 protein shares no similarity to any function-known protein in Swiss-Prot database, but has 8 protein kinase C phosphorylation sites, 2 caselin kinase II phosphorylation sites, 2 tyrosine kinase phosphorylation sites, 2 N-glycosylation sites and 2 N-myristolyation sites. Spatial and temporal expression patterns analysis showed that BcMF7 was expressed exclusively in pollen. The expression signal of BcMF7 was first detected at the tetrad stage of microspore development, reached a peak level at the uninucleate stage, and decreased to a slightly low level at the mature pollen stage. All these results show that BcMF7 may play a certain role in the signal transduction during pollen development.

Apomixis in Eulaliopsis binata: characterization of reproductive mode and endosperm development

Apomixis represents an alteration of classical sexual plant reproduction to produce seeds with essentially clonal embryos, stimulating wide interest from biologists and plant breeders for its ability to fix heterosis. Eulaliopsis binata (Poaceae), is identified here as a new apomictic species. Embryological investigation indicates that the developmental pattern of embryo sac formation in E. binata represents gametophytic apospory, the embryo originating from an unreduced cell, without fertilization and the mode of endosperm development was autonomous. Sexual embryo sacs were found with a frequency of 1–4% depending on the biotype. The DNA content of nuclei (C-value) in mature seeds was screened by flow cytometry (FCSS) and demonstrated that the endosperm was derived autonomously without fertilization and the three biotypes of E. binata showed varying degrees of apomixis. The Wide-leaf type showed obligate apomixis whereas the Slender-leaf and the Red-haulm type displayed facultative apomixis. In addition, adventitious embryos were observed on the wall of ovary, integument and nucellus cells, indicating that E. binata produces embryos via a mixture of apospory and adventitious embryony.

The function of actin-binding proteins in pollen tube growth

Pollen tube growth is a key step in sexual reproduction of higher plants. The pollen tube is a typical example of tip-growing cells and shows a polarized cytoplasm. To develop and maintain polarized growth, pollen tubes need a carefully regulated actin cytoskeleton. It is well known that actin-binding proteins are responsible for the direct control of dynamic actin filaments and serve as a link between signal transduction pathways and dynamic actin changes in determining cellular architecture. Several of these classes have been identified in pollen tubes and their detailed characterisation is progressing rapidly. Here, we aim to survey what is known about the major actin-binding proteins that affect actin assembly and dynamics, and their higher-order organisation in pollen tube growth.

Roles of Pectin Methylesterases in Pollen‐Tube Growth

AbstractElongation of the pollen tube in pistil is essential for delivering sperms into the female gametophyte in sexual plant reproduction. Recently, a group of cell wall enzymes, pectin methylesterases (PMEs), have been identified as playing an important role in this process. This article reviews the new understanding of the roles of PMEs in regulating pollen tube growth.

Maize Pollen Coat Xylanase Facilitates Pollen Tube Penetration into Silk during Sexual Reproduction

Cell wall hydrolases are well documented to be present on pollen, but their roles on the stigma during sexual reproduction have not been previously demonstrated. We explored the function of the tapetum-synthesized xylanase, ZmXYN1, on maize (Zea mays L.) pollen. Transgenic lines (xyl-less) containing little or no xylanase in the pollen coat were generated with use of an antisense construct of the xylanase gene-coding region driven by the XYN1 gene promoter. Xyl-less and wild-type plants had similar vegetative growth. Electron microscopy revealed no appreciable morphological difference in anther cells and pollen between xyl-less lines and the wild type, whereas immunofluorescence microscopy and biochemical analyses indicated an absence of xylanase on xyl-less pollen. Xyl-less pollen germinated as efficiently as wild-type pollen in vitro in a liquid medium but less so on gel media of increasing solidity or on silk, which is indicative of partial impaired water uptake. Once germinated in vitro or on silk, the xyl-less and wild-type pollen tubes elongated at comparable rates. Tubes of germinated xyl-less pollen on silk did not penetrate into the silk as efficiently as tubes of wild-type pollen, and this lower efficiency could be overcome by the addition of xylanase to the silk. For wild-type pollen, coat xylanase activity on oat spelled xylan in vitro and tube penetration into silk were inhibited by xylose but not glucose. The overall findings indicate that maize pollen coat xylanase facilitates pollen tube penetration into silk via enzymatic xylan hydrolysis.

Isolation of 151 Mutants that Have Developmental Defects from T-DNA Tagging

In order to understand the mechanisms underlying plant development, a necessary first step involves the elucidation of the functions of the genes, via the analysis of mutants that exhibit developmental defects. In this study, an activation tagging mutant library harboring 80,650 independent Arabidopsis transformants was generated in order to screen for developmental mutants. A total of 129 mutants manifesting dominant developmental abnormalities were isolated, and their T-DNA insertion loci were mapped. The activation of one or more genes adjacent to a T-DNA insertion locus was confirmed in eight dominant mutants. A gene adjacent to the right border was usually activated by the 35S enhancers. Interestingly, the transcriptional activation of multiple genes within a broad range was observed in one of the mutants, which raises the possibility that activation by the 35S enhancers was not limited strictly to a single gene. In order to gain a better understanding of sexual reproduction in higher plants, we isolated 22 mutants exhibiting defects in female gametophyte development, and determined their T-DNA insertion loci. We propose that this mutant population may prove useful in the further determination of the functions of genes that play important roles in plant development.

The sweet stench of decay

The sweet stench of decay

In vitro fertilization as a tool for investigating sexual reproduction of angiosperms

In vitro fertilization (IVF) of isolated male and female gametes of flowering plants was first accomplished in the last decade. Successful isolation of male and female gametes, and culturing of in vitro zygotes to form new plants, is a prelude to the use of IVF for research into the cellular and molecular control of fertilization in higher plants and its application as a tool in biotechnology. Genes unique to male and female gametes and zygotes of higher plants, although currently incompletely characterized, are expected to permit direct molecular dissection of fertilization. By applying IVF and microculture to zygotes and endosperm obtained by both in vivo and in vitro methods, newly activated fusion products may be observed and manipulated in media where they are directly accessible to the techniques of molecular cell biology. IVF and zygote culture may also offer potential for creating new hybrid plants by fusing isolated gametes from different species to produce unique zygotes and ultimately plants that would be impossible to obtain using typical crossing techniques. Transformation and regeneration frequencies using IVF may also be high enough to avoid the necessity of adding controversial antibiotic and herbicide resistant genes to screen transformed products. This review describes advances using IVF in plant sexual reproduction and discusses its potential in the genetic improvement of flowering plants.

The ecological genetics of floral traits

Floral traits have fascinated evolutionary biologists since Darwin published 'The various contrivances by which orchids are fertilized by insects' in 1862. Because flowers are remarkably diverse and important for sexual reproduction in plants, floral traits are excellent subjects for studies of adaptive microevolution. In addition, floral traits are unique among plant traits because they can directly influence pollinator-mediated reproductive isolation between species. Consequently, floral traits can provide a direct link between patterns of evolution within species and rates of divergence between species.

Plant reproductive susceptibility to habitat fragmentation: review and synthesis through a meta‐analysis

The loss and fragmentation of natural habitats by human activities are pervasive phenomena in terrestrial ecosystems across the Earth and the main driving forces behind current biodiversity loss. Animal-mediated pollination is a key process for the sexual reproduction of most extant flowering plants, and the one most consistently studied in the context of habitat fragmentation. By means of a meta-analysis we quantitatively reviewed the results from independent fragmentation studies throughout the last two decades, with the aim of testing whether pollination and reproduction of plant species may be differentially susceptible to habitat fragmentation depending on certain reproductive traits that typify the relationship with and the degree of dependence on their pollinators. We found an overall large and negative effect of fragmentation on pollination and on plant reproduction. The compatibility system of plants, which reflects the degree of dependence on pollinator mutualism, was the only reproductive trait that explained the differences among the species' effect sizes. Furthermore, a highly significant correlation between the effect sizes of fragmentation on pollination and reproductive success suggests that the most proximate cause of reproductive impairment in fragmented habitats may be pollination limitation. We discuss the conservation implications of these findings and give some suggestions for future research into this area.

The relative importance of sexual and clonal reproduction for population growth in the long‐lived alpine plant Geum reptans

Summary Demographic studies in natural habitats are important for understanding the life history and mechanisms of population persistence of particular species. For plants living in high‐alpine habitats, it is of significant importance to what extent sexual or clonal reproduction contribute to population growth, but respective data are scarce. We studied the demography of Geum reptans, an alpine plant which reproduces by seeds and by vegetative rosettes formed at the ends of above‐ground stolons. In three consecutive years, growth, survival, and reproduction were measured in two populations with 579 and 301 individuals, respectively. The two resulting transition matrices per population were used to calculate population growth rates (λ) and elasticities, and to perform stochastic simulations to assess the relative importance of sexual vs. clonal reproduction for population growth. Population growth rates (λ) varied from 0.999 to 1.074 among years and populations. The frequency of seed and stolon production was relatively constant over the years. However, there was interannual variation in seed germination (0.7–2.4%) and in establishment of clonal offspring (53–74%). Elasticity analysis showed that changes in the survival of adults had the largest effect on λ, confirming the importance of longevity and persistence in the life history of G. reptans. Stochastic simulations showed that in G. reptans, independently of location and year, both sexual and clonal reproduction did not significantly differ in their contribution to the population growth rate λ. Establishment from seeds contributes to population growth particularly in favourable years, while the more regularly occurring reproduction by clonal offspring may ensure population growth in less favourable years. Sexual reproduction in clonal plants is frequently explained by the short‐ and long‐term benefits of genetic variation and the need for dispersal and colonization, for which seeds are better suited than vegetative organs. Our results, however, show the importance of sexual reproduction in a clonal plant for local dynamics as well, and prove the power of matrix models to evaluate the relative contributions of the two reproductive modes.

Pollen-specific pectin methylesterase involved in pollen tube growth

Pollen tube elongation in the pistil is a crucial step in the sexual reproduction of plants. Because the wall of the pollen tube tip is composed of a single layer of pectin and, unlike most other plant cell walls, does not contain cellulose or callose, pectin methylesterases (PMEs) likely play a central role in the pollen tube growth and determination of pollen tube morphology. Thus, the functional studies of pollen-specific PMEs, which are still in their infancy, are important for understanding the pollen development. We identified a new Arabidopsis pollen-specific PME, AtPPME1, characterized its native expression pattern, and used reverse genetics to demonstrate its involvement in determination of the shape of the pollen tube and the rate of its elongation.

Localization of arabinogalactan proteins in egg cells, zygotes, and two-celled proembryos and effects of -D-glucosyl Yariv reagent on egg cell fertilization and zygote division in Nicotiana tabacum L.

Arabinogalactan proteins (AGPs) have been implicated in a variety of plant development processes including sexual plant reproduction. As a crucial developmental event, plant sexual reproduction generally occurs inside an ovule embedded in an ovary. The inaccessibility of the egg cells, zygotes, and embryos has hindered our understanding of the importance of AGPs in the early events involving fertilization, zygotic division, and early embryogenesis. In this study, the well-established in vitro zygote and ovary culture systems, together with immunofluorescence and immunogold labelling techniques, were employed to investigate the role of AGPs in the early events of sexual reproduction in Nicotiana tabacum. Dramatic changes in AGP content during ovule development were evidenced by western blotting. Subcellular localization revealed that AGPs are localized in the plasma membrane, cell wall, and cytoplasm of pre- and post-fertilized egg cells, and cytoplasm and vacuoles of two-celled proembryos. Abundant AGPs were detected in unfertilized egg cells; however, the level of AGPs substantially decreased in fertilized egg cells. Polar distribution of AGPs in elongated zygotes was observed. The early two-celled proembryos just from zygote division displayed accumulation of AGPs at a low level, while in the elongated two-celled proembryos at the late stage, the AGP content clearly increased. Provision of betaGlcY, a synthetic phenylglycoside that specifically binds AGPs, to the in vitro cultures of isolated zygote and fertilized ovaries increased abnormal symmetrical division of zygotes. In the culture of pollinated but unfertilized ovaries, addition of betaGlcY resulted in arrest of fertilization of the egg cells, but had no effect on fertilization of the central cells. The possible roles of AGPs in fertilization, zygotic division, and proembryo development are discussed.

Effects of Environmental Salinity on Vertebrate Florivory and Wetland Communities

Iris hexagona is a perennial freshwater species indigenous to Louisiana wetlands. Coastal iris populations that are exposed to saline conditions often suffer heavy floral browsing. This paper is the first to photographically document florivory by white-tailed deer (Odocoileus virginianus Zimmerman), which consume iris flowers and immature seed capsules. Severe florivory can prevent sexual reproduction in plants and eliminate habitat for floral arthropods. In this three-year study, we monitored environmental salinity, assessed natural levels of florivory, and established deer exclosures to test the effects of florivory on arthropod abundance and diversity. In two out of three years, florivory rates were positively correlated with the level of salinity at individual I. hexagona populations. In all three years, florivory in the brackish marsh was an order of magnitude greater than florivory in intermediate and freshwater sites. In 2003, the number and diversity of arthropods was significantly greater on unbrowsed (caged) than browsed (open) irises. In October 2002, Hurricane Lili struck our main study site, causing extensive damage to the landscape and heavy deer mortality. Despite this, floral browsing was high (88%) in spring 2003, and arthropod numbers and diversity declined significantly. Our results suggest that salinity may facilitate floral browsing, which can have direct effects on plant fitness and indirect consequences for arthropod communities in wetland plant populations.

Differentiation of generative and vegetative cells by unequal cell division

Summary: Cellular differentiation of a generative and a vegetative cell during pollen development is requisite for sexual plant reproduction and is initiated by an unequal(asymmetric)cell division. The smaller, generative cell produces two sperm cells, or male gametes, whereas the larger, vegetative cell produces an elongated pollen tube, agametophytic cell, to deliver the male gametes to the embryo sac. The distinct differences between two kinds of cell are found in organelle composition and cytoskeletal organization in their cytoplasm. Recent transcriptome analysis also revealed the difference in gene expression profiles between both cells. We pay attention to the difference of theirchromatin configuration, that is assumed to be associated with the difference in gene activity between the two types of nucleus. Namely, the generative nucleus has highly condensed chromatin, as do the nuclei of animal sperm, whereas the vegetative nucleus contains diffuse chromatin. For this reason, by developing an efficient method to isolate the generative and vegetative nuclei from lily pollen protoplasts, their histone composition was investigated biochemically and immunocytochemically. Expression analysis of isolated histone genes was also done. As a result, we detected, in addition to 1)replication-dependent, somatic types of histones common to both nuclei, 2)replication-independent, generative and sperm nuclei-specific histone variants(gH 2 A, gH 2 B and gH 3)and 3)replication-independent, vegetative nucleus-specific histone variant(H 3. 3). The significance of this discovery will be discussed with respect to the function and developmental fates of both cells in the process of sexual plant reproduction.

A reference map of the Arabidopsis thaliana mature pollen proteome

The male gametophyte (or pollen) plays an obligatory role during sexual reproduction of higher plants. The extremely reduced complexity of this organ renders pollen a valuable experimental system for studying fundamental aspects of plant biology such as cell fate determination, cell–cell interactions, cell polarity, and tip-growth. Here, we present the first reference map of the mature pollen proteome of the dicotyledonous model plant species, Arabidopsis thaliana. Based on two-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization time-of-flight, and electrospray quadrupole time-of-flight mass spectrometry, we reproducibly identified 121 different proteins in 145 individual spots. The presence, subcellular localization, and functional classification of the identified proteins are discussed in relation to the pollen transcriptome and the full protein complement encoded by the nuclear Arabidopsis genome.