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Nature Communications Jan 2023Pollen tube guidance within female tissues of flowering plants can be divided into preovular guidance, ovular guidance and a connecting stage called pollen tube...
Pollen tube guidance within female tissues of flowering plants can be divided into preovular guidance, ovular guidance and a connecting stage called pollen tube emergence. As yet, no female factor has been identified to positively regulate this transition process. In this study, we show that an ovary-expressed bHLH transcription factor Cucumis sativus ALCATRAZ (CsALC) functions in pollen tube emergence in cucumber. CsALC knockout mutants showed diminished pollen tube emergence, extremely reduced entry into ovules, and a 95% reduction in female fertility. Further examination showed two rapid alkalinization factors CsRALF4 and CsRALF19 were less expressed in Csalc ovaries compared to WT. Besides the loss of male fertility derived from precocious pollen tube rupture as in Arabidopsis, Csralf4 Csralf19 double mutants exhibited a 60% decrease in female fertility due to reduced pollen tube distribution and decreased ovule targeting efficiency. In brief, CsALC regulates female fertility and promotes CsRALF4/19 expression in the ovary during pollen tube guidance in cucumber.
Topics: Cucumis sativus; Ovary; Pollen Tube; Arabidopsis; Arabidopsis Proteins; Ovule
PubMed: 36650145
DOI: 10.1038/s41467-023-35936-z -
Methods in Molecular Biology (Clifton,... 2020Double-fertilization in angiosperms requires precise communication between the male gametophyte (pollen), the female tissues, and the associated female gametophyte...
Double-fertilization in angiosperms requires precise communication between the male gametophyte (pollen), the female tissues, and the associated female gametophyte (embryo sac) to facilitate efficient fertilization. Numerous small molecules, proteins, and peptides have been shown to impact double-fertilization through the disruption of pollen germination, pollen tube growth, pollen tube guidance, or pollen tube penetration of the female tissues. The genetic basis of signaling events that lead to successful double-fertilization has been greatly facilitated by studies in the model organism Arabidopsis thaliana, which possesses a relatively simple reproductive physiology and a widely available T-DNA mutant seed collection. In this chapter, we detail methods for determining the effects of single gene loss-of-function mutations on pollen behavior through the creation of an internally controlled fluorescent hemizygous complement line. By transforming a single copy of the disrupted gene back into the homozygous mutant background, a precise endogenous control is generated due to the fact that pollen containing equal ratios of mutant and complemented pollen can be collected from a single flower. Using this experimental design, we describe multiple assays that can be performed in series to assess mutant pollen defects in germination, pollen tube elongation rate, and pistil penetration, which can be easily quantified alongside a "near-wildtype" complemented counterpart.
Topics: Arabidopsis; Genetic Techniques; Loss of Function Mutation; Ovule; Plant Breeding; Plant Infertility; Pollen Tube
PubMed: 32529433
DOI: 10.1007/978-1-0716-0672-8_9 -
Current Opinion in Plant Biology Dec 2018Plant cells are enclosed in cell walls that weld them together, meaning that cells rarely change neighbours. Nonetheless, invasive growth events play critical roles in... (Review)
Review
Plant cells are enclosed in cell walls that weld them together, meaning that cells rarely change neighbours. Nonetheless, invasive growth events play critical roles in plant development and are often key hubs for the integration of environmental and/or developmental signalling. Here we review cellular processes involved in three such events: lateral root emergence, pollen tube growth through stigma and style tissues, and embryo expansion through the endosperm (Figures 1-3). We consider processes such as regulation of water fluxes and cell turgor (driving growth), cell wall modifications (e.g. cell separation) and cell death (for creating space) within these three contexts with the aim of identifying key mechanisms implicated in providing a chemical and biophysical environments permitting invasive growth events.
Topics: Cell Death; Cell Wall; Endosperm; Plant Cells; Plant Roots; Pollen Tube
PubMed: 29981931
DOI: 10.1016/j.pbi.2018.06.002 -
Biochemical and Biophysical Research... Jul 2020The conserved oligomeric Golgi (COG) complex, which consists of eight subunits named COG1-COG8, is highly conserved with homologous subunits present in most eukaryotic...
The conserved oligomeric Golgi (COG) complex, which consists of eight subunits named COG1-COG8, is highly conserved with homologous subunits present in most eukaryotic species. In yeast and mammalian, the COG complex has been implicated in the tethering of retrograde intra-Golgi vesicles. Although homologs of COG subunits have been identified in Arabidopsis, the functions of the complex and its subunits remain to be fully elucidated. In this study, we have utilized genetic and cytologic approaches to characterize the role of the COG6 subunit. We showed that a mutation in COG6 caused male transmission defect due to aberrant pollen tube growth. At the subcellular level, Golgi bodies exhibited altered morphology in cog6 pollen and cell wall components were incorrectly deposited in pollen tubes. COG6 fused to green fluorescent protein (GFP), which complemented the aberrant growth of cog6 pollen tubes, was localized to the Golgi apparatus. We propose that COG6, as a subunit of the COG complex, modulates Golgi morphology and vesicle trafficking homeostasis during pollen tube growth.
Topics: Arabidopsis; Arabidopsis Proteins; Genes, Plant; Golgi Apparatus; Microscopy, Electron, Transmission; Mutation; Plants, Genetically Modified; Pollen Tube
PubMed: 32499114
DOI: 10.1016/j.bbrc.2020.05.189 -
The New Phytologist Jul 2018The mitochondrial calcium uniporter complex (MCUc) was recently characterized in details in metazoans and consists of pore-forming units (MCUs) and regulatory factors...
The mitochondrial calcium uniporter complex (MCUc) was recently characterized in details in metazoans and consists of pore-forming units (MCUs) and regulatory factors that channel calcium (Ca ) ion into the mitochondria. MCUs participate in many stress and developmentally related processes involving Ca . Although multiple homologues of MCUs and one regulatory subunit are usually present in plants, the first functional characterization and contribution to Ca related processes of these proteins have been reported recently. Here, we focused on two predicted Arabidopsis MCUs and studied their role in the germination and the growth of pollen tube, a tip-growing cell type highly dependent on Ca homeostasis. Heterologous expression of MCU1 or MCU2 in yeast is sufficient to generate a mitochondrial Ca influx. MCU1 and MCU2 fluorescent reporters are co-expressed in the vegetative cell mitochondria of the pollen grain but are undetectable in the embryo sac. We demonstrate that MCU1 and MCU2 can form a heterotypic complex. Phenotypic analyses revealed an impaired pollen tube germination and growth in vitro only for the mcu2 mutants suggesting a predominant role of MCU2. Our results show that mitochondrial Ca controlled by MCUs is an additional player in Arabidopsis pollen tube germination and growth.
Topics: Arabidopsis; Arabidopsis Proteins; Calcium; Calcium Channels; Pollen; Pollen Tube; Pollination
PubMed: 29701876
DOI: 10.1111/nph.15189 -
Journal of Experimental Botany Dec 2017Brassicaceae pollen-stigma interactions have been extensively studied in Brassica and Arabidopsis species to identify cellular events triggered in the stigmatic papillae... (Review)
Review
Brassicaceae pollen-stigma interactions have been extensively studied in Brassica and Arabidopsis species to identify cellular events triggered in the stigmatic papillae by pollen contact. Compatible pollinations are linked to the activation of basal cellular responses in the stigmatic papillae, which include calcium gradients, actin networks, and polarized secretion. The occurrence of these cellular events in stigmatic papillae coincides with the stages of pollen hydration and pollen tube entry into the stigmatic papillar cell wall. However, the form of the vesicle trafficking appears to differ between species, with vesicle-like structures detected in Arabidopsis species while exosomes were found to be secreted in Brassica species. Around the same timeframe, self-incompatible pollen recognition leads altered cellular responses in the stigmatic papillae to interfere with basal compatible pollen responses and disrupt regulated secretion, causing self-pollen rejection. Here, the literature on the changing cellular dynamics in the stigmatic papillae following pollination is reviewed and discussed in the context of other well-characterized examples of polarized secretion in plants.
Topics: Arabidopsis; Autophagy; Brassica; Exosomes; Pollen Tube; Pollination; Protein Transport; Secretory Pathway; Vesicular Transport Proteins
PubMed: 29036428
DOI: 10.1093/jxb/erx340 -
Autophagy Mar 2023Macroautophagy/autophagy, a major catabolic pathway in eukaryotes, participates in plant sexual reproduction including the processes of male gametogenesis and the...
Macroautophagy/autophagy, a major catabolic pathway in eukaryotes, participates in plant sexual reproduction including the processes of male gametogenesis and the self-incompatibility response. Rapid pollen tube growth is another essential reproductive process that is metabolically highly demanding to drive the vigorous cell growth for delivery of male gametes for fertilization in angiosperms. Whether and how autophagy operates to maintain the homeostasis of pollen tubes remains unknown. Here, we provide evidence that autophagy is elevated in growing pollen tubes and critically required during pollen tube growth and male fertility in Arabidopsis. We demonstrate that SH3P2, a critical non-ATG regulator of plant autophagy, colocalizes with representative ATG proteins during autophagosome biogenesis in growing pollen tubes. Downregulation of expression significantly disrupts Arabidopsis pollen germination and pollen tube growth. Further analysis of organelle dynamics reveals crosstalk between autophagosomes and prevacuolar compartments following the inhibition of phosphatidylinositol 3-kinase. In addition, time-lapse imaging and tracking of ATG8e-labeled autophagosomes and depolarized mitochondria demonstrate that they interact specifically the ATG8-family interacting motif (AIM)-docking site to mediate mitophagy. Ultrastructural identification of mitophagosomes and two additional forms of autophagosomes imply that multiple types of autophagy are likely to function simultaneously within pollen tubes. Altogether, our results suggest that autophagy is functionally crucial for mediating mitochondrial quality control and canonical cytoplasm recycling during pollen tube growth. AIM: ATG8-family interacting motif; ATG8: autophagy related 8; ATG5: autophagy related 5; ATG7: autophagy related 7; BTH: acibenzolar-S-methyl; DEX: dexamethasone; DNP: 2,4-dinitrophenol; GFP: green fluorescent protein; YFP: yellow fluorescent protein; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PVC: prevacuolar compartment; SH3P2: SH3 domain-containing protein 2.
Topics: Arabidopsis; Autophagy; Pollen Tube; Arabidopsis Proteins; Mitochondria; Phosphatidylinositol 3-Kinases; Fertility
PubMed: 35786359
DOI: 10.1080/15548627.2022.2095838 -
Trends in Plant Science May 2018In flowering plants, pollen tubes deliver and release the immotile sperms to the female gametes for fertilization, but mechanisms remain unclear. New results show that...
In flowering plants, pollen tubes deliver and release the immotile sperms to the female gametes for fertilization, but mechanisms remain unclear. New results show that the RALF family peptides control pollen-tube integrity and burst by binding to CrRLK1L family receptors and cell wall leucine-rich repeat extensins.
Topics: Arabidopsis; Cell Wall; Ligands; Magnoliopsida; Pollen Tube
PubMed: 29571836
DOI: 10.1016/j.tplants.2018.03.005 -
Methods in Molecular Biology (Clifton,... 2020Conspicuous intracellular gradients manifest and/or drive intracellular polarity in pollen tubes. However, quantifying these gradients raises multiple technical...
Conspicuous intracellular gradients manifest and/or drive intracellular polarity in pollen tubes. However, quantifying these gradients raises multiple technical challenges. Here we present a sensible computational protocol to analyze gradients in growing pollen tubes and to filter nonrepresentative time points. As an example, we use imaging data from pollen tubes expressing a genetically encoded ratiometric Ca probe, Yellow CaMeleon 3.6, from which a kymograph is extracted. The tip of the pollen tube is detected with CHUKNORRIS, our previously published methodology, allowing the reconstruction of the intracellular gradient through time. Statistically confounding time points, such as growth arrest where gradients are highly oscillatory, are filtered out and a mean spatial profile is estimated with a local polynomial regression method. Finally, we estimate the gradient slope by the linear portion of the decay in mean fluorescence, offering a quantitative method to detect phenotypes of gradient steepness, location, intensity, and variability. The data manipulation protocol proposed can be achieved in a simple and efficient manner using the statistical programming language R, opening paths to perform high-throughput spatiotemporal phenotyping of intracellular gradients in apically growing cells.
Topics: Arabidopsis; Calcium; Cell Polarity; Kymography; Metabolic Flux Analysis; Microscopy, Fluorescence; Pollen Tube; Software
PubMed: 32529438
DOI: 10.1007/978-1-0716-0672-8_14 -
Plant Physiology Jul 2016Root hair cells and pollen tubes, like fungal hyphae, possess a typical tip or polar cell expansion with growth limited to the apical dome. Cell expansion needs to be... (Review)
Review
Root hair cells and pollen tubes, like fungal hyphae, possess a typical tip or polar cell expansion with growth limited to the apical dome. Cell expansion needs to be carefully regulated to produce a correct shape and size. Polar cell growth is sustained by oscillatory feedback loops comprising three main components that together play an important role regulating this process. One of the main components are reactive oxygen species (ROS) that, together with calcium ions (Ca(2+)) and pH, sustain polar growth over time. Apoplastic ROS homeostasis controlled by NADPH oxidases as well as by secreted type III peroxidases has a great impact on cell wall properties during cell expansion. Polar growth needs to balance a focused secretion of new materials in an extending but still rigid cell wall in order to contain turgor pressure. In this review, we discuss the gaps in our understanding of how ROS impact on the oscillatory Ca(2+) and pH signatures that, coordinately, allow root hair cells and pollen tubes to expand in a controlled manner to several hundred times their original size toward specific signals.
Topics: Calcium; Cell Membrane; Cell Polarity; Cytoplasm; Homeostasis; Hydrogen-Ion Concentration; Peroxidases; Plant Cells; Plant Proteins; Plant Roots; Pollen Tube; Reactive Oxygen Species
PubMed: 27208283
DOI: 10.1104/pp.16.00191