Male Gametophyte Research Articles

Pollen development in cotton (Gossypium hirsutum) is highly sensitive to heat exposure during the tetrad stage.

The development of gametes in plants is acutely susceptible to heatwaves as brief as a few days, adversely affecting pollen maturation and reproductive success. Pollen in cotton (Gossypium hirsutum) was differentially affected when tetrad and binucleate stages were exposed to heat, revealing new insights into the interaction between heat and pollen development. Squares were tagged and exposed to 36/25°C (day/night, moderate heat) or 40/30°C (day/night, extreme heat) for 5 days. Mature pollen grains and leaves were collected for physiological and proteomic responses. While photosynthetic competence was not compromised even at 40°C, leaf tissues became leakier. In contrast, pollen grains were markedly smaller after the tetrad stage was exposed to 40°C and boll production was reduced by 65%. Sugar levels in pollen grains were elevated after exposure to heat, eliminating carbohydrate deficits as a likely cause of poor reproductive capacity. Proteomic analysis of pure pollen samples revealed a particularly high abundance of 70-kDa heat shock (Hsp70s) and cytoskeletal proteins. While short-term bursts of heat had a minor impact on leaves, male gametophyte development was profoundly damaged. Cotton acclimates to maxima of 36°C at both the vegetative and reproductive stages but 5-days exposure to 40°C significantly impairs reproductive development.

P.834Hyperactivated brain response to fear and disgust in schizophrenia

Pollination in flowering plants is the transfer of a pollen grain (male gametophyte) to a flower’s stigma (receptive surface of the female reproductive organ), where it may germinate, grow through the style, and fertilize an ovule to produce a seed.

The Importance of Cytokinins during Reproductive Development in Arabidopsis and Beyond.

Fertilization and seed formation are fundamental events in the life cycle of flowering plants. The seed is a functional unit whose main purpose is to propagate the plant. The first step in seed development is the formation of male and female gametophytes and subsequent steps culminate in successful fertilization. The detailed study of this process is highly relevant because it directly impacts human needs, such as protecting biodiversity and ensuring sustainable agriculture to feed the increasing world population. Cytokinins comprise a class of phytohormones that play many important roles during plant growth and development and in recent years, the role of this class of phytohormones during reproduction has become clear. Here, we review the role of cytokinins during ovule, pollen and seed formation at the genetic and molecular levels. The expansion of knowledge concerning the molecular mechanisms that control plant reproduction is extremely important to optimise seed production.

A method to establish an "immortalized F2 " sporophyte population in the economic brown alga Undaria pinnatifida (Laminariales: Alariaceae).

Studies of quantitative trait loci based on genetic linkage maps require the establishment of a mapping population. Permanent mapping populations are more ideal than temporary ones because they can be used repeatedly. However, there has been no reported permanent sporophyte population of economically important kelp species. Based on the characteristics of the kelp life cycle, we proposed a method to establish, and then constructed experimentally, an "immortalized F2 " (IF2 ) population of Undaria pinnatifida. Doubled-haploid "female" and "male" sporophytes were obtained through the parthenogenesis of a female gametophyte clone and the selfing of a "monoicous" gametophyte clone (originally male), respectively, and they were used as the parents. The F1 hybrid line was generated by crossing the female and male gametophytes derived from the respective female and male parents. Full-sibling F2 gametophyte clones, consisting of 260 females and 260 males, were established from an F1 hybrid sporophyte. Thirty-five females and 35 males were randomly selected and paired to give rise to an IF2 population containing 35 crossing lines. A parentage analysis using 10 microsatellite markers confirmed the accuracy of the IF2 population and indicated the feasibility of this method. This proposed method may be adapted for use in other kelp species, and thus, it will be useful for genetic studies of kelp.

The Arabidopsis R-SNARE protein YKT61 is essential for gametophyte development.

Gametophyte development is a pre-requisite for plant reproduction and seed yield; therefore, studies of gametophyte development help us understand fundamental biological questions and have potential applications in agriculture. The biogenesis and dynamics of endomembrane compartments are critical for cell survival, and their regulatory mechanisms are just beginning to be revealed. Here, we report that the Arabidopsis thaliana SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) protein YKT61 is essential for both male and female gametogenesis. By using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based genome editing, we demonstrated that male and female gametophytes carrying YKT61 loss-of-function alleles do not survive. Specifically, loss of YKT61 function resulted in the arrest of male gametophytic development at pollen mitosis I and the degeneration of female gametophytes. A three-base-pair deletion in YKT61 in the ykt61-3 mutant resulted in a single-amino acid deletion in the longin domain of YKT61; the resulting mutant protein does not interact with multiple SNAREs and showed substantially reduced membrane association, suggesting that the N-terminal longin domain of YKT61 plays multiple roles in its function. This study demonstrates that Arabidopsis YKT61 is essential for male and female gametogenesis and sets an example for functional characterization of essential genes with the combination of Cas9-mediated editing and expression from a Cas9-resistant transgene.

A mitochondrial prolyl aminopeptidase PAP2 releases N‐terminal proline and regulates proline homeostasis during stress response

Most mitochondrial proteins are synthesised in the cytosol and targeted into the organelle via N-terminal targeting peptides that are cleaved upon import. The free targeting peptide is subsequently processed in a stepwise manner, with single amino acids released as final products. Here, we have characterised a proline-cleaving aminopeptidase in Arabidopsis thaliana, prolyl aminopeptidase-2 (PAP2, At3g61540). Activity assays show that PAP2 has a preferred activity to hydrolyse N-terminal proline. Protein localisation studies revealed that PAP2 is exclusively targeted to mitochondria. Characterisation of pap2 mutants show defective pollen, enhanced dark-induced senescence and increased susceptibility to abiotic stresses, which are likely attributed to a reduced level of accumulated free proline. Taken together, these results demonstrate the role of PAP2 in proline cleavage from mitochondrial peptides and proline homeostasis, which is required for the development of male gametophyte, tolerance to abiotic stresses, and leaf senescence.

Transcriptomic and Proteomic Insights into Amborella trichopoda Male Gametophyte Functions.

Flowering plants (angiosperms) are characterized by pollen tubes (PTs; male gametophytes) carrying two immobile sperm cells that grow over long distances through the carpel toward the ovules, where double fertilization is executed. It is not understood how these reproductive structures evolved, which genes occur de novo in male gametophytes of angiosperms, and to which extent PT functions are conserved among angiosperms. To contribute to a deeper understanding of the evolution of gametophyte functions, we generated RNA sequencing data from seven reproductive and two vegetative control tissues of the basal angiosperm Amborella trichopoda and complemented these with proteomic data of pollen grains (PGs) and PTs. The eudicot model plant Arabidopsis (Arabidopsis thaliana) served as a reference organism for data analysis, as more than 200 genes have been associated with male gametophyte functions in this species. We describe methods to collect bicellular A. trichopoda PGs, to induce their germination in vitro, and to monitor PT growth and germ cell division. Transcriptomic and proteomic analyses indicate that A. trichopoda PGs are prepared for germination requiring lipids, energy, but likely also reactive oxygen species, while PTs are especially characterized by catabolic/biosynthetic and transport processes including cell wall biosynthesis and gene regulation. Notably, a number of pollen-specific genes were lacking in Arabidopsis, and the number of genes involved in pollen signaling is significantly reduced in A. trichopoda In conclusion, we provide insight into male gametophyte functions of the most basal angiosperm and establish a valuable resource for future studies on the evolution of flowering plants.

Ultrastructural characterization of microlipophagy induced by the interaction of vacuoles and lipid bodies around generative and sperm cells in Arabidopsis pollen

During pollen maturation, various organelles change their distribution and function during development as male gametophytes. We analyzed the behavior of lipid bodies and vacuoles involved in lipophagy in Arabidopsis pollen using serial section SEM and conventional TEM. At the bicellular pollen stage, lipid bodies in the vegetative cells lined up at the surface of the generative cell. Vacuoles then tightly attached, drew in, and degraded the lipid bodies and eventually occupied the space of the lipid bodies. Degradation of lipid began before transfer of the entire contents of the lipid body. At the tricellular stage, vacuoles instead of lipid bodies surrounded the sperm cells. The degradation of lipid bodies is morphologically considered microautophagy. The atg2-1 Arabidopsis mutant is deficient in one autophagy-related gene (ATG). In this mutant, the assembly of vacuoles around sperm cells was sparser than that in wild-type pollen. The deficiency of ATG2 likely prevents or slows lipid degradation, although it does not prevent contact between organelles. These results demonstrate the involvement of microlipophagy in the pollen development of Arabidopsis.

Bridging the GAPs in plant reproduction: a comparison of plant and animal GPI-anchored proteins.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GAPs) are a unique type of membrane-associated proteins in eukaryotes. GPI and GAP biogenesis and function have been well studied in non-plant models and play an important role in the fertility of mouse sperm and egg. Although GPI and GAP biogenesis and function in plants are less known, they are critical for flowering plant reproduction because of their essential roles in the fertility of the male and female gametophytes. In Eukaryotes, GPI, a glycolipid molecule, can be post-translationally attached to proteins to serve as an anchor in the plasma membrane. GPI-anchoring, compared to other modes of membrane attachment and lipidation processes, localizes proteins to the extracellular portion of the plasma membrane and confers several unique attributes including specialized sorting during secretion, molecular painting onto membranes, and enzyme-mediated release of protein through anchor cleavage. While the biosynthesis, structure, and role of GPI are mostly studied in mammals, yeast and protists, the function of GPI and GAPs in plants is being discovered, particularly in gametophyte development and function. Here, we review GPI biosynthesis, protein attachment, and remodeling in plants with insights about this process in mammals. Additionally, we summarize the reproductive phenotypes of all loss of function mutations in Arabidopsis GPI biosynthesis and GAP genes and compare these to the reproductive phenotypes seen in mice to serve as a framework to identify gaps in our understanding of plant GPI and GAPs. In addition, we present an analysis on the gametophyte expression of all Arabidopsis GAPs to assist in further research on the role of GPI and GAPs in all aspects of the gametophyte generation in the life cycle of a plant.

Calcium dynamic in the developing microsporangiums of Chinese fir (Cunninghamia lanceolata)

Calcium dynamics in the developing pollen of Chinese fir (Cunninghamia lanceolata) were investigated using an antimonate calcium precipitation method. After microspores were released from the tetrad, abundant calcium precipitates formed on the surface of microspores. A characteristic event during microspore development is the formation of a large vacuole which displaces the nucleus to the periphery and produces polarity, which initiates male gametophyte development. During the formation of the large vacuole, calcium precipitates moved into the large vacuole via mitochondria, suggesting that calcium abundance is related to vacuole biogenesis and formation. After microspore division, the large vacuole reorganised and calcium became concentrated in smaller vacuoles. The pollen wall of Chinese fir consists mostly of the intine, composed of polysaccharose, and the surface of the pollen grains is covered with a thin indefinite structure, the exine, which is composed of sporopollenin. The function of calcium during pollen development and the characteristics of the pollen wall of Chinese fir are discussed.

The aquatic carnivorous plant Aldrovanda vesiculosa (Droseraceae) exhibits altered developmental stages in male gametophyte.

Aldrovanda vesiculosa (Droseraceae) is a rare aquatic carnivorous plant, distributed in Europe, Asia, Africa, and Australia. Aldrovanda populations can flower prolifically under favourable conditions, but seed set is very limited. We studied the structure of Aldrovanda pollen collected from flowers in different developmental stages (opened and non-opened anthers) from both European and Australian populations to elucidate pollination traits and the basis of poor seed set on the basis of microscopic observation of pollen and anther structure. Microscopic analyses of Aldrovanda pollen showed that this plant has pollen arranged in tetrads like other species in the Droseraceae family. In hydrated pollen, cytoplasmic protrusions originate from pores located along the equatorial wall of monads, and can develop into pollen tubes. Interestingly, pollen development from microspores occurs in open anthers, suggesting a delay of the developmental stages. In addition, pollen development displays altered sperm cell formation and precocious pollen germination. Precocious germination may characterize recalcitrant pollen, which naturally do not undergo dehydration before anthesis and remain partially hydrated, particularly in aquatic and wetland plants. These alterations of male gametophyte development could affect fertilization processes, and be the reason for the low reproductive capability of Aldrovanda observed both in the field and in cultures. Generally, reduced pollen longevity and very quick germination are considered an adaptation to aquatic or wet environments.

Effects of low temperature in spring on fertility of pollen and formation of grain number in wheat

<p indent=0mm>Grain yield and the number of grains per ear of wheat decreased under the low temperature after jointing stage. In order to explore the physiological reasons of the decrease of grains number per ear by low temperature in spring, the spring wheat variety Yangmai 16 and the semi-winter wheat variety Xumai 30 were used as tested varieties to analyze the effects of low temperature in spring on the fertility of pollen and the formation of grain numbers at the appearance of the penultimate leaf stage (5℃/–3℃, day/night), booting stage (8℃/–1℃, day/night), and anthesis stage (12℃/4℃, day/night). The results showed that low temperature at the appearance of the penultimate leaf stage and booting stage resulted in abnormal metabolism of starch and protein in the anther both at the binuclear and tri-nuclear pollen stages and delayed degradation of the tapetum. The low temperature at the booting stage caused abnormal meiosis of pollen mother cells, abnormalities in chromosome pairing, which affected the formation of male gametophyte. The abortion rate of pollen was more significantly increased under low temperature at the appearance of the penultimate leaf stage than that at the booting stage, and it was the minimum at anthesis stage. The abortion rate of pollen in Xumai 30 was higher than that in Yangmai 16. The low temperature at three stages had significant effects on the number of spikelets, the number of fertile spikelets and the number of grains per spikelet. The effect of low temperature at the appearance of the penultimate leaf stage on grains number per spikelet was the most significant. There was a significant positive correlation between the number of grains per spike and pollen fertility, which was affected by abnormal meiosis, delayed degradation of the tapetum, and undersupply of nutriment for anther development. Therefore, the decline of pollen fertility caused by low temperature in spring is the main reason for the decrease of grains number per spike.

Identification of New Mutant Alleles of Augmin Subunits Broadens Spectrum of Augmin Function During Sexual Reproduction in Arabidopsis

In flowering plants, production of functional gametophytes is prerequisite for the double fertilization that leads to successful proliferation. Pollen grains as male gametophytes in Arabidopsis thaliana are uniquely patterned with two sperm cells inside a vegetative cell. To better understand the genetic regulation underlying pollen development, we morphologically screened DAPI-stained mature pollen in a mutant population generated using an activation tagging vector in this study. As a result, we identified two independent lines, AL318 and AL434, exhibiting similar mutant phenotypes regarding nucleus number and pollen pattern. Genetic analysis showed that the two mutants are maintained in heterozygotes but not in homozygotes, due to highly reduced genetic transmission from both sexes. Developmental analysis revealed that mutant microspores at the polarized stage either completely fail to enter pollen mitosis I, or abnormally divide with altered division asymmetry, resulting in mature pollen without the male germline. We found that these defects arise from genetic lesions in the AUG2 and AUG4 genes, members of the augmin complex that mediates microtubule (MT)-dependent MT nucleation in a broad range of species. Collectively, our study provides genetic evidence that the augmin complex plays a critical role in mitotic entry and proper execution for pollen mitosis I, broadening the spectrum of augmin function during sexual reproduction in Arabidopsis.

AtPIG-S, a predicted Glycosylphosphatidylinositol Transamidase subunit, is critical for pollen tube growth in Arabidopsis

BackgroundGlycosylphosphatidylinositol (GPI) addition is one of the several post-translational modifications to proteins that increase their affinity for membranes. In eukaryotes, the GPI transamidase complex (GPI-T) catalyzes the attachment of pre-assembled GPI anchors to GPI-anchored proteins (GAPs) through a transamidation reaction. A mutation in AtGPI8 (gpi8–2), the putative catalytic subunit of GPI-T in Arabidopsis, is transmitted normally through the female gametophyte (FG), indicating the FG tolerates loss of GPI transamidation. In contrast, gpi8–2 almost completely abolishes male gametophyte (MG) function. Still, the unexpected finding that gpi8–2 FGs function normally requires further investigation. Additionally, specific developmental defects in the MG caused by loss of GPI transamidation remain poorly characterized.ResultsHere we investigated the effect of loss of AtPIG-S, another GPI-T subunit, in both gametophytes. Like gpi8–2, we showed that a mutation in AtPIG-S (pigs-1) disrupted synergid localization of LORELEI (LRE), a putative GAP critical for pollen tube reception by the FG. Still, pigs-1 is transmitted normally through the FG. Conversely, pigs-1 severely impaired male gametophyte (MG) function during pollen tube emergence and growth in the pistil. A pPIGS:GFP-PIGS transgene complemented these MG defects and enabled generation of pigs-1/pigs-1 seedlings. However, the pPIGS:GFP-PIGS transgene seemingly failed to rescue the function of AtPIG-S in the sporophyte, as pigs-1/pigs-1, pPIGS:GFP-PIGS seedlings died soon after germination.ConclusionsCharacterization of pigs-1 provided further evidence that the FG tolerates loss of GPI transamidation more than the MG and that the MG compared to the FG may be a better haploid system to study the role of GPI-anchoring. Pigs-1 pollen develops normally and thus represent a tool in which GPI anchor biosynthesis and transamidation of GAPs have been uncoupled, offering a potential way to study free GPI in plant development. While previously reported male fertility defects of GPI biosynthesis mutants could have been due either to loss of GPI or GAPs lacking the GPI anchor, our results clarified that the loss of mature GAPs underlie male fertility defects of GPI-deficient pollen grains, as pigs-1 is defective only in the downstream transamidation step.

Genome-Wide Identification and Expression Analysis of MIKC-Type MADS-Box Gene Family in Punica granatum L.

MADS-box is a critical transcription factor regulating the development of floral organs and plays essential roles in the growth and development of floral transformation, flower meristem determination, the development of male and female gametophytes, and fruit development. In this study, 36 MIKC-type MADS-box genes were identified in the ‘Taishanhong’ pomegranate genome. By utilizing phylogenetic analysis, 36 genes were divided into 14 subfamilies. Bioinformatics methods were used to analyze the gene structure, conserved motifs, cis-acting elements, and the protein interaction networks of the MIKC-type MADS-box family members in pomegranate, and their expressions pattern in different tissues of pomegranate were analyzed. Tissue-specific expression analysis revealed that the E-class genes (PgMADS03, PgMADS21, and PgMADS27) were highly expressed in floral tissues, while PgMADS29 was not expressed in all tissues, indicating that the functions of the E-class genes were differentiated. PgMADS15 of the C/D-class was the key gene in the development network of pomegranate flower organs, suggesting that PgMADS15 might play an essential role in the peel and inner seed coat development of pomegranate. The results in this study will provide a reference for the classification, cloning, and functional research of pomegranate MADS-box genes.

The integral spliceosomal component CWC15 is required for development in Arabidopsis

Efficient mRNA splicing is a prerequisite for protein biosynthesis and the eukaryotic splicing machinery is evolutionarily conserved among species of various phyla. At its catalytic core resides the activated splicing complex Bact consisting of the three small nuclear ribonucleoprotein complexes (snRNPs) U2, U5 and U6 and the so-called NineTeen complex (NTC) which is important for spliceosomal activation. CWC15 is an integral part of the NTC in humans and it is associated with the NTC in other species. Here we show the ubiquitous expression and developmental importance of the Arabidopsis ortholog of yeast CWC15. CWC15 associates with core components of the Arabidopsis NTC and its loss leads to inefficient splicing. Consistent with the central role of CWC15 in RNA splicing, cwc15 mutants are embryo lethal and additionally display strong defects in the female haploid phase. Interestingly, the haploid male gametophyte or pollen in Arabidopsis, on the other hand, can cope without functional CWC15, suggesting that developing pollen might be more tolerant to CWC15-mediated defects in splicing than either embryo or female gametophyte.

Reactive Oxygen Species as Mediators of Gametophyte Development and Double Fertilization in Flowering Plants.

Reactive oxygen species (ROS) are toxic by-products of aerobic metabolism. In plants, they also function as important signaling molecules that regulate biotic and abiotic stress responses as well as plant growth and development. Recent studies have implicated ROS in various aspects of plant reproduction. In male gametophytes, ROS are associated with germline development as well as the developmentally associated programmed cell death of tapetal cells necessary for microspore development. ROS have a role in regulation of female gametophyte patterning and maintenance of embryo sac polarity. During pollination, ROS play roles in the generation of self-incompatibility response during pollen-pistil interaction, pollen tube growth, pollen tube burst for sperm release and fertilization. In this mini review, we provide an overview of ROS production and signaling in the context of plant reproductive development, from female and male gametophyte development to fertilization.

Nanocarriers for Transgene Expression in Pollen as a Plant Biotechnology Tool

As the male gametophyte of flowering plants, pollen grains are ideal targets for the introduction of transgenes for plant genetic engineering and crop improvement. However, the difficulty of delive...

Cytological stability of wheat amphidiploids and cultivars in meiosis I

Introgression of genes controlling useful traits into a common wheat gene pool is still a highly actual task. Amphidiploids usually serve as a bridge for transferring alien genes into the wheat genome. One of the conditions of successful use of amphidiploids as sources of alien genes is their cytological stability and fertility. Cytological stability of the genome substitution amphidiploids Aurotica and Auroale was determined through characterization of different meiosis stages by studding cytological preparations. Amphidiploids were characterized by deviations from the bivalent chromosome conjugation, appearance of multivalent chromosome associations, and univalent chromosomes in metaphase I. In the anaphase, bridges were observed. Univalents and chromosome fragments resulted from the bridges breakage caused appearance of micronuclei in tetrads. Conjugation of chromosomes in common wheat cultivars, studied for comparison, was bivalent, with the absolute preference of closed bivalents in metaphase plates. The meiotic index calculated on quantitative characteristics of amphidiploids with micronuclei and polyads was similar for Aurotica and Auroale and varied between 64–69 %. In tetrads of Auroale no triads were found, whereas in Aurotica triads were usual. So, the meiotic index in itself is not an indicator for the meiotic stability of a plant sample. More exhaustive information can be received through structuring of the meiotic index for assessment of components which are used for its calculation. The received results enable to assume that meiotic abnormalities in Aurotica may be caused by disorder of synchrony in meiosis II processes, whereas for Auroale this disorder is not typical. Gametogenesis in amphidiploids sometimes demonstrated appearance of the third generative nucleus in the male gametophyte. Determined meiosis characteristics explain the decline in seed fertility of Aurotica, and in particular Auroale, compared to common wheat cultivars.Manuscript received 13.04.2020

The identification of differentially expressed genes in male and female gametophytes of simple thalloid liverwort Pellia endiviifolia sp. B using an RNA-seq approach

Main conclusionThis study shows differences in gene expression between male and female gametophytes of the simple thalloid liverwort with a distinction between the vegetative and reproductive phases of growth.Pellia endiviifolia is a simple thalloid liverwort that, together with hornworts and mosses, represents the oldest living land plants. The limited taxon sampling for genomic and functional studies hampers our understanding of processes governing evolution of these plants. RNA sequencing represents an attractive way to elucidate the molecular mechanisms of non-model species development. In the present study, RNA-seq was used to profile the differences in gene expression between P. endiviifolia male and female gametophytes, with a distinction between the vegetative and reproductive phases of growth. By comparison of the gene expression profiles from individuals producing sex organs with the remaining thalli types, we have determined a set of genes whose expression might be important for the development of P. endiviifolia reproductive organs. The selected differentially expressed genes (DEGs) were categorized into five main pathways: metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems. A comparison of the obtained data with the Marchantia polymorpha transcriptome resulted in the identification of genes exhibiting a similar expression pattern during the reproductive phase of growth between members of the two distinct liverwort classes. The common expression profile of 87 selected genes suggests a common mechanism governing sex organ development in both liverwort species. The obtained RNA-seq results were confirmed by RT-qPCR for the DEGs with the highest differences in expression level. Five Pellia-female-specific and two Pellia-male-specific DEGs showed enriched expression in archegonia and antheridia, respectively. The identified genes are promising candidates for functional studies of their involvement in liverwort sexual reproduction.