-
Plant Reproduction Sep 2021Biolistic delivery into pollen. In recent years, genome editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding....
Biolistic delivery into pollen. In recent years, genome editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method has general limitations, such as the limited host range of Agrobacterium and difficulties in tissue culture, including callus induction and regeneration. To avoid these issues, we developed a method to genetically modify germ cells without the need for Agrobacterium-mediated transfection and tissue culture using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable promoter for driving the expression of the delivered gene in pollen tubes. We also evaluated the delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9-introduced pollen tubes, but were not detected in the negative control. Bombarded pollen germinated pollen tubes and delivered their contents into the ovules in vivo. Although it is necessary to improve biolistic delivery efficiency and establish a method for the screening of genome-modified seeds, our findings provide important insights for the detection and production of genome-modified seeds by pollen biolistic delivery.
Topics: Biolistics; CRISPR-Cas Systems; Gene Editing; Plant Breeding; Plants, Genetically Modified; Pollen Tube
PubMed: 34146158
DOI: 10.1007/s00497-021-00418-z -
Plant & Cell Physiology Nov 2021Many plant processes occur in the context of and in interaction with a surrounding matrix such as soil (e.g. root growth and root-microbe interactions) or surrounding... (Review)
Review
Many plant processes occur in the context of and in interaction with a surrounding matrix such as soil (e.g. root growth and root-microbe interactions) or surrounding tissues (e.g. pollen tube growth through the pistil), making it difficult to study them with high-resolution optical microscopy. Over the past decade, microfabrication techniques have been developed to produce experimental systems that allow researchers to examine cell behavior in microstructured environments that mimic geometrical, physical and/or chemical aspects of the natural growth matrices and that cannot be generated using traditional agar plate assays. These microfabricated environments offer considerable design flexibility as well as the transparency required for high-resolution, light-based microscopy. In addition, microfluidic platforms have been used for various types of bioassays, including cellular force assays, chemoattraction assays and electrotropism assays. Here, we review the recent use of microfluidic devices to study plant cells and organs, including plant roots, root hairs, moss protonemata and pollen tubes. The increasing adoption of microfabrication techniques by the plant science community may transform our approaches to investigating how individual plant cells sense and respond to changes in the physical and chemical environment.
Topics: Biological Assay; Bryophyta; Imaging, Three-Dimensional; Microfluidic Analytical Techniques; Plant Cells; Plant Roots; Pollen Tube; Protoplasts
PubMed: 34027549
DOI: 10.1093/pcp/pcab067 -
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 -
ELife Sep 2020The pollen tube in a flowering plant grows in a direction that is influenced by the mechanical properties of the stigma papillae and the organization of structures...
The pollen tube in a flowering plant grows in a direction that is influenced by the mechanical properties of the stigma papillae and the organization of structures called cortical microtubules inside these cells.
Topics: Arabidopsis; Cell Wall; Katanin; Microtubules; Pollen Tube
PubMed: 32867921
DOI: 10.7554/eLife.61647 -
Journal of Integrative Plant Biology Jul 2016In flowering plants, pollen tube growth is essential for delivery of male gametes into the female gametophyte or embryo sac for double fertilization. Although many genes...
In flowering plants, pollen tube growth is essential for delivery of male gametes into the female gametophyte or embryo sac for double fertilization. Although many genes have been identified as being involved in the process, the molecular mechanisms of pollen tube growth remains poorly understood. In this study, we identified that the Arabidopsis Transmembrane Protein 18 (AtTMEM18) gene played important roles in pollen tube growth. The AtTMEM18 shares a high similarity with the Transmembrane 18 proteins (TMEM18s) that are conserved in most eukaryotes and may play important roles in obesity in humans. Mutation in the AtTMEM18 by a Ds insertion caused abnormal callose deposition in the pollen grains and had a significant impact on pollen germination and pollen tube growth. AtTMEM18 is expressed in pollen grains, pollen tubes, root tips and other vegetative tissues. The pollen-rescued assays showed that the mutation in AtTMEM18 also caused defects in roots, stems, leaves and transmitting tracts. AtTMEM18-GFP was located around the nuclei. Genetic assays demonstrated that the localization of AtTMEM18 around the nuclei in the generative cells of pollen grains was essential for the male fertility. Furthermore, expression of the rice TMEM18-homologous protein (OsTMEM18) driven by LAT52 promoter could recover the fertility of the Arabidopsis attmem18 mutant. These results suggested that the TMEM18 is important for plant growth in Arabidopsis.
Topics: Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Cell Nucleus; Cell Proliferation; Gene Expression Regulation, Plant; Genetic Complementation Test; Germination; Green Fluorescent Proteins; Membrane Proteins; Mutation; Oryza; Phenotype; Plants, Genetically Modified; Pollen Tube; Subcellular Fractions
PubMed: 26699939
DOI: 10.1111/jipb.12459 -
The New Phytologist Nov 2019The causative link between phenotypic divergence and reproductive isolation is an important but poorly understood part of ecological speciation. We studied the effects...
The causative link between phenotypic divergence and reproductive isolation is an important but poorly understood part of ecological speciation. We studied the effects of floral-tube length variation on pollen placement/receipt positions and reproductive isolation. In a population of Lapeirousia anceps (Iridaceae) with bimodal floral-tube lengths, we labelled pollen of short- and long-tubed flowers with different colour fluorescent nanoparticles (quantum dots). This enabled us to map pollen placement by long- and short-tubed flowers on the only floral visitor, a long-proboscid fly. Furthermore, it allowed us to quantify pollen movement within and between short- and long-tubed flowers. Short- and long-tubed flowers placed pollen on different parts of the pollinator, and long-tubed flowers placed more pollen per visit than short-tubed flowers. This resulted in assortative pollen receipt (most pollen received comes from the same phenotype) and strong but asymmetric reproductive isolation, where short-tubed plants are more reproductively isolated than long-tubed plants. These results suggest that floral-tube length divergence can promote mechanical isolation in plants through divergence in pollen placement sites on pollinators. Consequently, in concert with other reproductive isolation mechanisms, selection for differences in floral-tube length can play an important role in ecological speciation of plants.
Topics: Animals; Biodiversity; Diptera; Iridaceae; Movement; Pollen Tube; Pollination; Reproductive Isolation; Species Specificity
PubMed: 31148172
DOI: 10.1111/nph.15971 -
Current Opinion in Plant Biology Oct 2019In flowering plants, extensive male-female interactions during pollen germination on the stigma, pollen tube growth and guidance in the transmitting tract, and pollen... (Review)
Review
In flowering plants, extensive male-female interactions during pollen germination on the stigma, pollen tube growth and guidance in the transmitting tract, and pollen tube reception by the female gametophyte are required for successful double fertilization in which various signaling cascades are involved. Peptide/receptor-like kinase-mediated signaling has been found playing important roles in these male-female interactions. Here, we mainly summarized the progress made on the regulatory roles of peptide/receptor-like kinase-mediated signaling pathways in four critical stages during reproduction in higher plants.
Topics: Female; Male; Ovule; Phosphotransferases; Pollen Tube; Pollination; Signal Transduction
PubMed: 30999163
DOI: 10.1016/j.pbi.2019.03.004 -
Arabidopsis SKU5 Similar 11 and 12 play crucial roles in pollen tube integrity, growth and guidance.The Plant Journal : For Cell and... Feb 2022Pollen tube integrity, growth and guidance are crucial factors in plant sexual reproduction. Members of the plant Skewed5 (SKU5) Similar (SKS) family show strong...
Pollen tube integrity, growth and guidance are crucial factors in plant sexual reproduction. Members of the plant Skewed5 (SKU5) Similar (SKS) family show strong similarity to multicopper oxidases (MCOs), but they lack conserved histidines in MCO active sites. The functions of most SKS family members are unknown. Here, we show that Arabidopsis pollen-expressed SKS11 and SKS12 play important roles in pollen tube integrity, growth and guidance. The sks11sks12 mutant exhibited significantly reduced male fertility. Most of the pollen from sks11sks12 plants burst when germinated, and the pollen tubes grew slowly and exhibited defective growth along the funiculus and micropyle. SKS11-GFP and SKS12-mCherry were detected at the cell wall in pollen tubes. The contents of several cell wall polysaccharides and arabinogalactans were decreased in the pollen tube cell walls of sks11sks12 plants. Staining with a reactive oxygen species (ROS)-sensitive dye and use of the H O sensor HyPer revealed that the ROS content in the pollen tubes of sks11sks12 plants was remarkably reduced. SKS11 , in which the last conserved histidine was mutated, could restore the mutant phenotypes of sks11sks12. Thus, SKS11/12 are required for pollen tube integrity, growth and guidance possibly by regulating the ROS level and cell wall polysaccharide deposition or remodeling in pollen tubes.
Topics: Arabidopsis; Arabidopsis Proteins; Gene Expression Regulation, Plant; Genes, Plant; Membrane Glycoproteins; Phenotype; Pollen Tube
PubMed: 34775642
DOI: 10.1111/tpj.15580 -
The Plant Journal : For Cell and... Aug 2021Ethylene modulates plant developmental processes including flower development. Previous studies have suggested ethylene participates in pollen tube (PT) elongation, and...
Ethylene modulates plant developmental processes including flower development. Previous studies have suggested ethylene participates in pollen tube (PT) elongation, and both ethylene production and perception seem critical at the time of fertilization. The full gene set regulated by ethylene during PT growth is unknown. To study this, we used various EThylene Receptor (ETR) tomato (Solanum lycopersicum) mutants: etr3-ko, a loss-of-function (LOF) mutant; and NR (NEVER RIPE), a gain-of-function (GOF) mutant. The etr3-ko PTs grew faster than wild-type (WT) PTs. Oppositely, NR PT elongation was slower than in WT, and PTs displayed larger diameters. ETR mutations result in feedback control of ethylene production. Furthermore, ethylene treatment of germinating pollen grains increased PT length in etr-ko mutants and WT, but not in NR. Treatment with the ethylene perception inhibitor 1-methylcyclopropene decreased PT length in etr-ko mutants and WT, but had no effect on NR. This confirmed that ethylene regulates PT growth. The comparison of PT transcriptomes in LOF and GOF mutants, etr3-ko and NR, both harboring mutations of the ETR3 gene, revealed that ethylene perception has major impacts on cell wall- and calcium-related genes as confirmed by microscopic observations showing a modified distribution of the methylesterified homogalacturonan pectic motif and of calcium load. Our results establish links between PT growth, ethylene, calcium, and cell wall metabolism, and also constitute a transcriptomic resource.
Topics: Calcium; Cell Wall; Cyclopropanes; Ethylenes; Gene Expression Regulation, Plant; Solanum lycopersicum; Mutation; Plant Growth Regulators; Plant Proteins; Pollen Tube; Pollination; Signal Transduction; Transcriptome
PubMed: 34036648
DOI: 10.1111/tpj.15353 -
Journal of Experimental Botany Apr 2020Pollen tubes rapidly elongate, penetrate, and navigate through multiple female tissues to reach ovules for sperm delivery by utilizing a specialized form of polar growth... (Review)
Review
Pollen tubes rapidly elongate, penetrate, and navigate through multiple female tissues to reach ovules for sperm delivery by utilizing a specialized form of polar growth known as tip growth. This process requires a battery of cellular activities differentially occurring at the apical growing region of the plasma membrane (PM), such as the differential cellular signaling involving calcium (Ca2+), phospholipids, and ROP-type Rho GTPases, fluctuation of ions and pH, exocytosis and endocytosis, and cell wall construction and remodeling. There is an emerging understanding of how at least some of these activities are coordinated and/or interconnected. The apical active ROP modulates exocytosis to the cell apex for PM and cell wall expansion differentially occurring at the tip. The differentiation of the cell wall involves at least the preferential distribution of deformable pectin polymers to the apex and non-deformable pectin polymers to the shank of pollen tubes, facilitating the apical cell expansion driven by high internal turgor pressure. Recent studies have generated inroads into how the ROP GTPase-based intracellular signaling is coordinated spatiotemporally with the external wall mechanics to maintain the tubular cell shape and how the apical cell wall mechanics are regulated to allow rapid tip growth while maintaining the cell wall integrity under the turgor pressure. Evidence suggests that exocytosis and endocytosis play crucial but distinct roles in this spatiotemporal coordination. In this review, we summarize recent advances in the regulation and coordination of the differential pectin distribution and the apical domain of active ROP by exocytosis and endocytosis in pollen tubes.
Topics: Endocytosis; Exocytosis; Pectins; Plants; Pollen Tube; Signal Transduction; rho GTP-Binding Proteins
PubMed: 32173729
DOI: 10.1093/jxb/eraa134