Transporters involved in pH and K+ homeostasis affect pollen wall formation, male fertility, and embryo development.
ABSTRACT: Flowering plant genomes encode multiple cation/H+ exchangers (CHXs) whose functions are largely unknown. AtCHX17, AtCHX18, and AtCHX19 are membrane transporters that modulate K+ and pH homeostasis and are localized in the dynamic endomembrane system. Loss of function reduced seed set, but the particular phase(s) of reproduction affected was not determined. Pollen tube growth and ovule targeting of chx17chx18chx19 mutant pollen appeared normal, but reciprocal cross experiments indicate a largely male defect. Although triple mutant pollen tubes reach ovules of a wild-type pistil and a synergid cell degenerated, half of those ovules were unfertilized or showed fertilization of the egg or central cell, but not both female gametes. Fertility could be partially compromised by impaired pollen tube and/or sperm function as CHX19 and CHX18 are expressed in the pollen tube and sperm cell, respectively. When fertilization was successful in self-pollinated mutants, early embryo formation was retarded compared with embryos from wild-type ovules receiving mutant pollen. Thus CHX17 and CHX18 proteins may promote embryo development possibly through the endosperm where these genes are expressed. The reticulate pattern of the pollen wall was disorganized in triple mutants, indicating perturbation of wall formation during male gametophyte development. As pH and cation homeostasis mediated by AtCHX17 affect membrane trafficking and cargo delivery, these results suggest that male fertility, sperm function, and embryo development are dependent on proper cargo sorting and secretion that remodel cell walls, plasma membranes, and extracellular factors.
Project description:Key message: New gametic homozygous mutants. In angiosperms, a haploid male gamete (sperm cell) fuses with a haploid female gamete (egg cell) during fertilization to form a zygote carrying paternally and maternally derived chromosomes. Several fertilization-defective mutants in Arabidopsis thaliana, including a generative cell-specific 1 (gcs1)/hapless 2 mutant, the sperm cells of which are unable to fuse with female gametes, can only be maintained as heterozygous lines due to the infertile male or female gametes. Here, we report successful generation of a gcs1 homozygous mutant by heat-inducible removal of the GCS1 transgene. Using the gcs1 homozygous mutant as male, the defect in gamete fusion was observed with great frequency; in our direct observation by semi-in vivo fertilization assay using ovules, 100 % of discharged sperm cells in culture failed to show gamete fusion. More than 70 % of ovules in the pistil received a second pollen tube as attempted fertilization recovery. Moreover, gcs1 mutant sperm cells could fertilize female gametes at a low frequency in the pistil. This strategy to generate homozygous fertilization-defective mutants will facilitate novel approaches in plant reproduction research.
Project description:In Trimenia moorei, an extant member of the ancient angiosperm clade Austrobaileyales, we found a remarkable pattern of female gametophyte (egg-producing structure) development that strikingly resembles that of pollen tubes and their intrasexual competition within the maternal pollen tube transmitting tissues of most flowers. In contrast with most other flowering plants, in Trimenia, multiple female gametophytes are initiated at the base (chalazal end) of each ovule. Female gametophytes grow from their tips and compete over hundreds of micrometers to reach the apex of the nucellus and the site of fertilization. Here, the successful female gametophyte will mate with a pollen tube to produce an embryo and an endosperm. Moreover, the central tissue within the ovules of Trimenia, through which the embryo sacs grow, contains starch and other carbohydrates similar to the pollen tube transmitting tissues in the styles of most flowers. The pattern of female gametophyte development found in Trimenia is rare but by no means unique in angiosperms. Importantly, it seems that multiple female gametophytes are occasionally or frequently initiated in members of other ancient angiosperm lineages. The intensification of pollen tube (male gametophyte) competition and enhanced maternal selection among competing pollen tubes are considered to have been major contributors to the rise of angiosperms. Based on insights from Trimenia, we posit that prefertilization female gametophyte (egg) competition within individual ovules in addition to male gametophyte (sperm) competition and maternal mate choice may have been key features of the earliest angiosperms.
Project description:In alders, where fertilization occurs approximately 8 weeks after pollination, the pollen tube (male gametophyte) grows intermittently in four steps in close association with the development of the ovary and its ovules. Pollen tubes stop growing in the style, at the ovarian locule, and at the chalaza (ovule), before reaching an embryo sac for fertilization. At the stage when the ovary develops an ovule primordium in each of the two locules, many pollen tubes germinate on the stigma, and a few of them reach the style, where they remain for approximately 7 weeks. Thereafter, a single tube resumes growing; with a short stop in the upper space of the ovarian locule, it reaches the older of the two ovules when it has developed a two-nucleate embryo sac. Except in the last step, where the tube grows from the chalaza to an embryo sac (female gametophyte), an eight-nucleate mature embryo sac is not necessary for pollen-tube guidance in the pistil. Although the intermittent pollen-tube growth appears to play an important role in the selection of a single pollen tube from many and one ovule from two, its detection provides insight into the study of the mechanism of pollen-tube guidance.
Project description:Plant seeds are essential for human beings, constituting 70% of carbohydrate resources worldwide; examples include rice, wheat, and corn. In angiosperms, fertilization of the egg by a sperm cell is required for seed formation; therefore, fertilization failure results in no seed formation, except in the special case of apomixis. Initially, plants produce many pollen grains inside the anthers; once the pollen grain is deposited onto the top of the pistil, the pollen tube elongates until it reaches the ovule. Generally, only one pollen tube is inserted into the ovule; however, we previously found that if fertilization by the first pollen tube fails, a second pollen tube could rescue fertilization via the so-called fertilization recovery system (FRS). Our previous reports also demonstrated that failed fertilization results in pollen tube-dependent ovule enlargement morphology (POEM), enlarged seeds, and partial seed coat formation if the pollen tube releases the pollen tube contents into the ovule. However, we have not determined whether all the ovules enlarge or produce seed coats if an ovule accepts the pollen tube contents. Therefore, we conducted a partial seed coat formation experiment taking into account both the FRS and POEM phenomena. Notably, the ratios of failed fertilization and the ovules with partial seed coats matched, indicating that all ovules initiate seed coat formation if the fertilization fails but the pollen tube contents enter the ovule. In addition, we confirmed that the agl62 mutant , defective in early endosperm formation, showed seed coat initiation with and without fertilization, indicating that for a normal seed coat initiation, fertilization is not required; however, for the completion of normal seed coat formation, both normal fertilization and endosperm formation are required. Further molecular evidence is required to understand these phenomena because very few factors related to FRS and POEM have been identified.
Project description:In double fertilization, a reproductive system unique to flowering plants, two immotile sperm are delivered to an ovule by a pollen tube. One sperm fuses with the egg to generate a zygote, the other with the central cell to produce endosperm. A mechanism preventing multiple pollen tubes from entering an ovule would ensure that only two sperm are delivered to female gametes. We use live-cell imaging and a novel mixed-pollination assay that can detect multiple pollen tubes and multiple sets of sperm within a single ovule to show that Arabidopsis efficiently prevents multiple pollen tubes from entering an ovule. However, when gamete-fusion defective hap2(gcs1) or duo1 sperm are delivered to ovules, as many as three additional pollen tubes are attracted. When gamete fusion fails, one of two pollen tube-attracting synergid cells persists, enabling the ovule to attract more pollen tubes for successful fertilization. This mechanism prevents the delivery of more than one pair of sperm to an ovule, provides a means of salvaging fertilization in ovules that have received defective sperm, and ensures maximum reproductive success by distributing pollen tubes to all ovules.
Project description:BET11 and 12 are required for pollen tube elongation. Pollen tubes are rapidly growing specialized structures that elongate in a polar manner. They play a crucial role in the delivery of sperm cells through the stylar tissues of the flower and into the embryo sac, where the sperm cells are released to fuse with the egg cell and the central cell to give rise to the embryo and the endosperm. Polar growth at the pollen tube tip is believed to result from secretion of materials by membrane trafficking mechanisms. In this study, we report the functional characterization of Arabidopsis BET11 and BET12, two genes that may code for Qc-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). Double mutants (bet11/bet12) in a homozygous/heterozygous background showed reduced transmission of the mutant alleles, reduced fertilization of seeds, defective embryo development, reduced pollen tube lengths and formation of secondary pollen tubes. Both BET11 and BET12 are required for fertility and development of pollen tubes in Arabidopsis. More experiments are required to dissect the mechanisms involved.
Project description:In eukaryotes, fertilization relies on complex and specialized mechanisms that achieve the precise delivery of the male gamete to the female gamete and their subsequent union [1-4]. In flowering plants, the haploid male gametophyte or pollen tube (PT)  carries two nonmotile sperm cells to the female gametophyte (FG) or embryo sac  during a long assisted journey through the maternal tissues [7-10]. In Arabidopsis, typically one PT reaches one of the two synergids of the FG (Figure 1A), where it terminates its growth and delivers the sperm cells, a poorly understood process called pollen-tube reception. Here, we report the isolation and characterization of the Arabidopsis mutant abstinence by mutual consent (amc). Interestingly, pollen-tube reception is impaired only when an amc pollen tube reaches an amc female gametophyte, resulting in pollen-tube overgrowth and completely preventing sperm discharge and the development of homozygous mutants. Moreover, we show that AMC is strongly and transiently expressed in both male and female gametophytes during fertilization and that AMC functions in gametophytes as a peroxin essential for protein import into peroxisomes. These findings show that peroxisomes play an unexpected key role in gametophyte recognition and implicate a diffusible signal emanating from either gametophyte that is required for pollen-tube discharge.
Project description:In flowering plants, immotile sperm cells develop within the pollen grain and are delivered to female gametes by a pollen tube. Upon arrival at the female gametophyte, the pollen tube stops growing and releases sperm cells for successful fertilization. Several female signaling components essential for pollen tube reception have been identified; however, male components remain unknown. We show that the expression of three closely related MYB transcription factors is induced in pollen tubes by growth in the pistil. Pollen tubes lacking these three transcriptional regulators fail to stop growing in synergids, specialized cells flanking the egg cell that attract pollen tubes and degenerate upon pollen tube arrival. myb triple-mutant pollen tubes also fail to release their sperm cargo. We define a suite of pollen tube-expressed genes regulated by these critical MYBs and identify transporters, carbohydrate-active enzymes, and small peptides as candidate molecular mediators of pollen tube-female interactions necessary for flowering plant reproduction. Our data indicate that de novo transcription in the pollen tube nucleus during growth in the pistil leads to pollen tube differentiation required for release of sperm cells.
Project description:Flowering plants have immotile sperm that develop within pollen and must be carried to female gametes by a pollen tube. The pollen tube engages in molecular interactions with several cell types within the pistil and these interactions are essential for successful fertilization. We identified a group of three closely related pollen tube-expressed MYB transcription factors (MYB97, MYB101, MYB120), which are required for proper interaction of the pollen tube with the female gametophyte. These transcription factors are transcriptionally induced during growth in the pistil. They regulate a transcriptional network leading to proper differentiation and maturation of the pollen tube, promoting proper pollen tube-ovule interactions resulting in sperm release and double fertilization. We used microarrays to discover genes regulated by the transcription factors MYB97, MYB101 and MYB120 in pollen tubes growing through the pistil at 8 hours after pollination. Pistils were collected from ms1 (Male Sterile 1) pistils that were unpollinated, or pollinated with either wild type (Col-0) pollen or myb triple mutant (myb97-1, myb101-4, myb120-3) pollen for 8 hours. We sought to examine transcriptional changes that were taking place in pollen tubes before they reached ovules in wild type pollen tubes, and what portion of this transcriptional regulation was due to MYB97, MYB101 and MYB120. Analysis of growth in the pistil allows discovery of transcriptional changes taking place during pollen tube growth in its native environment, as opposed to mature pollen or in vitro grown pollen, which are essentially naive conditions, as neither have interacted with the pistil environment and any signalling factors found therein.
Project description:In double fertilization, the vegetative cell of the male gametophyte (pollen) germinates and forms a pollen tube that brings to the female gametophyte two sperm cells that fertilize the egg and central cell to form the embryo and endosperm, respectively. The 5-methylcytosine DNA glycosylase DEMETER (DME), expressed in the central cell, is required for maternal allele demethylation and gene imprinting in the endosperm. By contrast, little is known about the function of DME in the male gametophyte. Here we show that reduced transmission of the paternal mutant dme allele in certain ecotypes reflects, at least in part, defective pollen germination. DME RNA is detected in pollen, but not in isolated sperm cells, suggesting that DME is expressed in the vegetative cell. Bisulfite sequencing experiments show that imprinted genes (MEA and FWA) and a repetitive element (Mu1a) are hypomethylated in the vegetative cell genome compared with the sperm genome, which is a process that requires DME. Moreover, we show that MEA and FWA RNA are detectable in pollen, but not in isolated sperm cells, suggesting that their expression occurs primarily in the vegetative cell. These results suggest that DME is active and demethylates similar genes and transposons in the genomes of the vegetative and central cells in the male and female gametophytes, respectively. Although the genome of the vegetative cell does not participate in double fertilization, its DME-mediated demethylation is important for male fertility and may contribute to the reconfiguration of the methylation landscape that occurs in the vegetative cell genome.