Plasma membrane H+-ATPases sustain pollen tube growth and fertilization.
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ABSTRACT:
SUBMITTER: Hoffmann RD
PROVIDER: S-EPMC7224221 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Members of the P4 subfamily of P-type ATPases are thought to create and maintain lipid asymmetry in biological membranes by flipping specific lipids between membrane leaflets. In Arabidopsis, 7 of the 12 Aminophospholipid ATPase (ALA) family members are expressed in pollen. Here we show that double knockout of ALA6 and ALA7 (ala6/7) results in siliques with a ~2-fold reduction in seed set with a high frequency of empty seed positions near the bottom. Seed set was reduced to near zero when plants were grown under a hot/cold temperature stress. Reciprocal crosses indicate that the ala6/7 reproductive deficiencies are due to a defect related to pollen transmission. In-vitro growth assays provide evidence that ala6/7 pollen tubes are short and slow, with ~2-fold reductions in both maximal growth rate and overall length relative to wild-type. Outcrosses show that when ala6/7 pollen are in competition with wild-type pollen, they have a near 0% success rate in fertilizing ovules near the bottom of the pistil, consistent with ala6/7 pollen having short and slow growth defects. The ala6/7 phenotypes were rescued by the expression of either an ALA6-YFP or GFP-ALA6 fusion protein, which showed localization to both the plasma membrane and highly-mobile endomembrane structures. A mass spectrometry analysis of mature pollen grains revealed significant differences between ala6/7 and wild-type, both in the relative abundance of lipid classes and in the average number of double bonds present in acyl side chains. A change in the properties of the ala6/7 plasma membrane was also indicated by a ~10-fold reduction of labeling by lipophilic FM-dyes relative to wild-type. Together, these results indicate that ALA6 and ALA7 provide redundant activities that function to directly or indirectly change the distribution and abundance of lipids in pollen, and support a model in which ALA6 and ALA7 are critical for pollen fitness under normal and temperature-stress conditions.
| S-EPMC4404812 | biostudies-literature
Project description:BackgroundAs in animals, cell-cell communication plays a pivotal role in male-female recognition during plant sexual reproduction. Prelaid peptides secreted from the female reproductive tissues guide pollen tubes towards ovules for fertilization. However, the elaborate mechanisms for this dialogue have remained elusive, particularly from the male perspective.ResultsWe performed genome-wide quantitative liquid chromatography-tandem mass spectrometry analysis of a pistil-stimulated pollen tube secretome and identified 801 pollen tube-secreted proteins. Interestingly, in silico analysis reveals that the pollen tube secretome is dominated by proteins that are secreted unconventionally, representing 57 % of the total secretome. In support, we show that an unconventionally secreted protein, translationally controlled tumor protein, is secreted to the apoplast. Remarkably, we discovered that this protein could be secreted by infiltrating through the initial phases of the conventional secretory pathway and could reach the apoplast via exosomes, as demonstrated by co-localization with Oleisin1 exosome marker. We demonstrate that translationally controlled tumor protein-knockdown Arabidopsis thaliana plants produce pollen tubes that navigate poorly to the target ovule and that the mutant allele is poorly transmitted through the male. Further, we show that regulators of the endoplasmic reticulum-trans-Golgi network protein secretory pathway control secretion of Nicotiana tabacum Pollen tube-secreted cysteine-rich protein 2 and Lorelei-like GPI-anchor protein 3 and that a regulator of endoplasmic reticulum-trans-Golgi protein translocation is essential for pollen tube growth, pollen tube guidance and ovule-targeting competence.ConclusionsThis work, the first study on the pollen tube secretome, identifies novel genome-wide pollen tube-secreted proteins with potential functions in pollen tube guidance towards ovules for sexual reproduction. Functional analysis highlights a potential mechanism for unconventional secretion of pollen tube proteins and reveals likely regulators of conventional pollen tube protein secretion. The association of pollen tube-secreted proteins with marker proteins shown to be secreted via exosomes in other species suggests exosome secretion is a possible mechanism for cell-cell communication between the pollen tube and female reproductive cells.
| S-EPMC4853860 | biostudies-literature
Project description:In flowering plants, pollination, pollen tube growth, and fertilization are regarded as the first hierarchical processes of producing offspring. However, their independent contributions to fruit set and development remain unclear. In this study, we examined the effect of three different types of pollen, intact pollen (IP), soft X-ray-treated pollen (XP) and dead pollen (DP), on pollen tube growth, fruit development and gene expression in "Micro-Tom" tomato. Normal germination and pollen tube growth were observed in flowers pollinated with IP; pollen tubes started to penetrate the ovary at 9 h after pollination, and full penetration was achieved after 24 h (IP24h), resulting in ~94% fruit set. At earlier time points (3 and 6 h after pollination; IP3h and IP6h, respectively), pollen tubes were still in the style, and no fruit set was observed. Flowers pollinated with XP followed by style removal after 24 h (XP24h) also demonstrated regular pollen tubes and produced parthenocarpic fruits with ~78% fruit set. As expected, DP could not germinate and failed to activate fruit formation. Histological analysis of the ovary at 2 days after anthesis (DAA) revealed that IP and XP comparably increased cell layers and cell size; however, mature fruits derived from XP were significantly smaller than those derived from IP. Furthermore, there was a high correlation between seed number and fruit size in fruit derived from IP, illustrating the crucial role of fertilization in the latter stages of fruit development. RNA-Seq analysis was carried out in ovaries derived from IP6h, IP24h, XP24h and DP24h in comparison with emasculated and unpollinated ovaries (E) at 2 DAA. The results revealed that 65 genes were differentially expressed (DE) in IP6h ovaries; these genes were closely associated with cell cycle dormancy release pathways. Conversely, 5062 and 4383 DE genes were obtained in IP24h and XP24h ovaries, respectively; top enriched terms were mostly associated with cell division and expansion in addition to the 'plant hormone signal transduction' pathway. These findings indicate that full penetration of pollen tubes can initiate fruit set and development independently of fertilization, most likely by activating the expression of genes regulating cell division and expansion.
| S-EPMC10319911 | biostudies-literature
Project description:Ca2+ transport into synaptic vesicles (SVs) at the presynaptic terminals has been proposed to be an important process for regulating presynaptic [Ca2+] during stimulation as well as at rest. However, the molecular identity of the transport system remains elusive. Previous studies have demonstrated that isolated SVs exhibit two distinct Ca2+ transport systems depending on extra-vesicular (cytosolic) pH; one is mediated by a high affinity Ca2+ transporter which is active at neutral pH and the other is mediated by a low affinity Ca2+/H+ antiporter which is maximally active at alkaline pH of 8.5. In addition, synaptic vesicle glycoprotein 2 s (SV2s), a major SV component, have been proposed to contribute to Ca2+ clearance from the presynaptic cytoplasm. Here, we show that at physiological pH, the plasma membrane Ca2+ ATPases (PMCAs) are responsible for both the Ca2+/H+ exchange activity and Ca2+ uptake into SVs. The Ca2+/H+ exchange activity monitored by acidification assay exhibited high affinity for Ca2+ (Km ~ 400 nM) and characteristic divalent cation selectivity for the PMCAs. Both activities were remarkably reduced by PMCA blockers, but not by a blocker of the ATPase that transfers Ca2+ from the cytosol to the lumen of sarcoplasmic endoplasmic reticulum (SERCA) at physiological pH. Furthermore, we rule out the contribution of SV2s, putative Ca2+ transporters on SVs, since both Ca2+/H+ exchange activity and Ca2+ transport were unaffected in isolated vesicles derived from SV2-deficient brains. Finally, using a PMCA1-pHluorin construct that enabled us to monitor cellular distribution and recycling properties in living neurons, we demonstrated that PMCA1-pHluorin localized to intracellular acidic compartments and recycled at presynaptic terminals in an activity-dependent manner. Collectively, our results imply that vesicular PMCAs may play pivotal roles in both presynaptic Ca2+ homeostasis and the modulation of H+ gradient in SVs.
| S-EPMC6414521 | biostudies-literature
Project description:Sperm cells of seed plants have lost their motility and are transported by the vegetative pollen tube cell for fertilization, but the extent to which they regulate their own transportation is a long-standing debate. Here we show that Arabidopsis lacking two bHLH transcription factors produces pollen without sperm cells. This abnormal pollen mostly behaves like the wild type and demonstrates that sperm cells are dispensable for normal pollen tube development.
| S-EPMC5960590 | biostudies-literature
Project description:Background and aimsAn unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here.MethodsPollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM.ResultsAfter 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed.ConclusionsIt is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.
| S-EPMC4242329 | biostudies-literature
Project description:Proper growth of the pollen tube depends on an elaborate mechanism that integrates several molecular and cytological sub-processes and ensures a cell shape adapted to the transport of gametes. This growth mechanism is controlled by several molecules among which cytoplasmic and apoplastic polyamines. Spermine (Spm) has been correlated with various physiological processes in pollen, including structuring of the cell wall and modulation of protein (mainly cytoskeletal) assembly. In this work, the effects of Spm on the growth of pear pollen tubes were analyzed. When exogenous Spm (100 μM) was supplied to germinating pollen, it temporarily blocked tube growth, followed by the induction of apical swelling. This reshaping of the pollen tube was maintained also after growth recovery, leading to a 30-40% increase of tube diameter. Apical swelling was also accompanied by a transient increase in cytosolic calcium concentration and alteration of pH values, which were the likely cause for major reorganization of actin filaments and cytoplasmic organelle movement. Morphological alterations of the apical and subapical region also involved changes in the deposition of pectin, cellulose, and callose in the cell wall. Thus, results point to the involvement of Spm in cell wall construction as well as cytoskeleton organization during pear pollen tube growth.
| S-EPMC5627395 | biostudies-literature
Project description:Pollen germination (PG) and pollen tube growth (PTG) play crucial roles in sexual reproduction of flowering plants by sending sperm cells to the ovule. These two processes are regarded as ideal model system for the study of cell signaling and cell polarized growth. It has been considered for a long time that ion transports across the pollen tube membranes are essential for pollen tube navigation and growth. Previous transcriptome analyses for Arabidopsis have shown that the transcripts related to cellular transport are correspondingly overrepresented during the process of pollen tube growth. Here, we showed that 459 transporter genes expressed during PG and PTG in Arabidopsis. In addition, the gene expression profiles of ion (including Ca(2+), H(+), K(+), Cl(-)) channels and transporters were further analyzed. This analysis provides novel information for the potential candidate genes involving in ion fluxes across the pollen tube membranes and in regulation of pollen tube tip growth.
| S-EPMC2819455 | biostudies-literature
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.
| S-EPMC6468697 | biostudies-literature
Project description:In angiosperms, pollen tubes carry two sperm cells toward the egg and central cells to complete double fertilization. In animals, not only sperm but also seminal plasma is required for proper fertilization. However, little is known regarding the function of pollen tube content (PTC), which is analogous to seminal plasma. We report that the PTC plays a vital role in the prefertilization state and causes an enlargement of ovules without fertilization. We termed this phenomenon as pollen tube-dependent ovule enlargement morphology and placed it between pollen tube guidance and double fertilization. Additionally, PTC increases endosperm nuclei without fertilization when combined with autonomous endosperm mutants. This finding could be applied in agriculture, particularly in enhancing seed formation without fertilization in important crops.
| S-EPMC5091356 | biostudies-literature