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1.  Three MYB transcription factors control pollen tube differentiation required for sperm release 
Current biology : CB  2013;23(13):1209-1214.
In flowering plants, immotile sperm cells develop within the pollen grain and are delivered to female gametes by a pollen tube [1, 2]. Upon arrival at the female gametophyte, the pollen tube stops growing and releases sperm cells for successful fertilization [3]. Several female signaling components essential for pollen tube reception have been identified [4–11]); 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 [12–16] and degenerate upon pollen tube arrival [17, 18]. 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-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.
PMCID: PMC4009620  PMID: 23791732
2.  Exogenous γ-aminobutyric acid (GABA) affects pollen tube growth via modulating putative Ca2+-permeable membrane channels and is coupled to negative regulation on glutamate decarboxylase 
Journal of Experimental Botany  2014;65(12):3235-3248.
This work reveals how tobacco pistil and style may communicate with pollen tubes and regulate their growth during fertilization via the γ-aminobutyric acid–Ca2+-permeable channel–glutamate decarboxylase–calmodulin signalling pathway.
γ-Aminobutyric acid (GABA) is implicated in pollen tube growth, but the molecular and cellular mechanisms that it mediates are largely unknown. Here, it is shown that exogenous GABA modulates putative Ca2+-permeable channels on the plasma membranes of tobacco pollen grains and pollen tubes. Whole-cell voltage-clamp experiments and non-invasive micromeasurement technology (NMT) revealed that the influx of Ca2+ increases in pollen tubes in response to exogenous GABA. It is also demonstrated that glutamate decarboxylase (GAD), the rate-limiting enzyme of GABA biosynthesis, is involved in feedback controls of Ca2+-permeable channels to fluctuate intracellular GABA levels and thus modulate pollen tube growth. The findings suggest that GAD activity linked with Ca2+-permeable channels relays an extracellular GABA signal and integrates multiple signal pathways to modulate tobacco pollen tube growth. Thus, the data explain how GABA mediates the communication between the style and the growing pollen tubes.
PMCID: PMC4071839  PMID: 24799560
γ-Aminobutyric acid; Ca2+-permeable channel; cell–cell communication; glutamate decarboxylase; Nicotiana tabacum; pollen tube.
3.  Sulfinylated Azadecalins act as functional mimics of a pollen germination stimulant in Arabidopsis pistils 
Polarized cell elongation is triggered by small molecule cues during development of diverse organisms. During plant reproduction, pollen interactions with the stigma result in the polar outgrowth of a pollen tube, which delivers sperm cells to the female gametophyte to effect double fertilization. In many plants, pistils stimulate pollen germination. However, in Arabidopsis, the effect of pistils on pollen germination and the pistil factors that stimulate pollen germination remain poorly characterized. Here, we demonstrate that stigma, style, and ovules in Arabidopsis pistils stimulate pollen germination. We isolated an Arabidopsis pistil extract fraction that stimulates Arabidopsis pollen germination, and employed ultrahigh resolution ESI FT-ICR and MS/MS techniques to accurately determine the mass (202.126 daltons) of a compound that is specifically present in this pistil extract fraction. Using the molecular formula (C10H19NOS) and tandem mass spectral fragmentation patterns of the m/z (mass to charge ratio) 202.126 ion, we postulated chemical structures, devised protocols, synthesized N-Methanesulfinyl 1- and 2-azadecalins that are close structural mimics of the m/z 202.126 ion, and showed that they are sufficient to stimulate Arabidopsis pollen germination in vitro (30 µM stimulated ~50% germination) and elicit accession-specific response. Although N-Methanesulfinyl 2-azadecalin stimulated pollen germination in three species of Lineage I of Brassicaceae, it did not induce a germination response in Sisymbrium irio (Lineage II of Brassicaceae) and tobacco, indicating that activity of the compound is not random. Our results show that Arabidopsis pistils promote germination by producing azadecalin-like molecules to ensure rapid fertilization by the appropriate pollen.
PMCID: PMC3225508  PMID: 21801250
Pollen; pistil; germination; stimulant; chemical biology; functional mimic
4.  GABA Accumulation Causes Cell Elongation Defects and a Decrease in Expression of Genes Encoding Secreted and Cell Wall-Related Proteins in Arabidopsis thaliana 
Plant and Cell Physiology  2011;52(5):894-908.
GABA (γ-aminobutyric acid), a non-protein amino acid, is a signaling factor in many organisms. In plants, GABA is known to accumulate under a variety of stresses. However, the consequence of GABA accumulation, especially in vegetative tissues, remains poorly understood. Moreover, gene expression changes as a consequence of GABA accumulation in plants are largely unknown. The pop2 mutant, which is defective in GABA catabolism and accumulates GABA, is a good model to examine the effects of GABA accumulation on plant development. Here, we show that the pop2 mutants have pollen tube elongation defects in the transmitting tract of pistils. Additionally, we observed growth inhibition of primary root and dark-grown hypocotyl, at least in part due to cell elongation defects, upon exposure to exogenous GABA. Microarray analysis of pop2-1 seedlings grown in GABA-supplemented medium revealed that 60% of genes whose expression decreased encode secreted proteins. Besides, functional classification of genes with decreased expression in the pop2-1 mutant showed that cell wall-related genes were significantly enriched in the microarray data set, consistent with the cell elongation defects observed in pop2 mutants. Our study identifies cell elongation defects caused by GABA accumulation in both reproductive and vegetative tissues. Additionally, our results show that genes that encode secreted and cell wall-related proteins may mediate some of the effects of GABA accumulation. The potential function of GABA as a growth control factor under stressful conditions is discussed.
PMCID: PMC3093128  PMID: 21471118
Arabidopsis thaliana; Cell elongation; Cell wall; GABA; Microarray; Secretory pathway
5.  Loss of LORELEI function in the pistil delays initiation but does not affect embryo development in Arabidopsis thaliana 
Plant Signaling & Behavior  2010;5(11):1487-1490.
Double fertilization, uniquely observed in plants, requires successful sperm cell delivery by the pollen tube to the female gametophyte, followed by migration, recognition and fusion of the two sperm cells with two female gametic cells. The female gametophyte not only regulates these steps but also controls the subsequent initiation of seed development. Previously, we reported that loss of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein, in the female reproductive tissues causes a delay in initiation of seed development. From these studies, however, it was unclear if embryos derived from fertilization of lre-5 gametophytes continued to lag behind wild-type during seed development. Additionally, it was not determined if the delay in initiation of seed development had any lingering effects during seed germination. Finally, it was not known if loss of LORELEI function affects seedling development given that LORELEI is expressed in eight-day-old seedlings. Here, we showed that despite a delay in initiation, lre-5/lre-5 embryos recover, becoming equivalent to the developing wild-type embryos beginning at 72 hours after pollination. Additionally, lre-5/lre-5 seed germination, and seedling and root development are indistinguishable from wild-type indicating that loss of LORELEI is tolerated, at least under standard growth conditions, in vegetative tissues.
PMCID: PMC3115263  PMID: 21051955
LORELEI; glycosylphosphatidylinositol (GPI)-anchored protein; embryogenesis; DD45; seed germination; primary and lateral root growth; seedling development
6.  Penetration of the Stigma and Style Elicits a Novel Transcriptome in Pollen Tubes, Pointing to Genes Critical for Growth in a Pistil 
PLoS Genetics  2009;5(8):e1000621.
Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. Using microarray analysis in Arabidopsis, we show that pollen tubes that have grown through stigma and style tissues of a pistil have a distinct gene expression profile and express a substantially larger fraction of the Arabidopsis genome than pollen grains or pollen tubes grown in vitro. Genes involved in signal transduction, transcription, and pollen tube growth are overrepresented in the subset of the Arabidopsis genome that is enriched in pistil-interacted pollen tubes, suggesting the possibility of a regulatory network that orchestrates gene expression as pollen tubes migrate through the pistil. Reverse genetic analysis of genes induced during pollen tube growth identified seven that had not previously been implicated in pollen tube growth. Two genes are required for pollen tube navigation through the pistil, and five genes are required for optimal pollen tube elongation in vitro. Our studies form the foundation for functional genomic analysis of the interactions between the pollen tube and the pistil, which is an excellent system for elucidation of novel modes of cell–cell interaction.
Author Summary
For successful reproduction in flowering plants, a single-celled pollen tube must rapidly extend through female pistil tissue, locate female gametes, and deliver sperm. Pollen tubes undergo a dramatic transformation while growing in the pistil; they grow faster compared to tubes grown in vitro and become competent to perceive and respond to navigation cues secreted by the pistil. The genes expressed by pollen tubes in response to growth in the pistil have not been characterized. We used a surgical procedure to obtain large quantities of uncontaminated pollen tubes that grew through the pistil and defined their transcriptome by microarray analysis. Importantly, we identify a set of genes that are specifically expressed in pollen tubes in response to their growth in the pistil and are not expressed during other stages of pollen or plant development. We analyzed mutants in 33 pollen tube–expressed genes using a sensitive series of pollen function assays and demonstrate that seven of these genes are critical for pollen tube growth; two specifically disrupt growth in the pistil. By identifying pollen tube genes induced by the pistil and describing a mutant analysis scheme to understand their function, we lay the foundation for functional genomic analysis of pollen–pistil interactions.
PMCID: PMC2726614  PMID: 19714218
7.  Distinct short-range ovule signals attract or repel Arabidopsis thaliana pollen tubes in vitro 
BMC Plant Biology  2006;6:7.
Pollen tubes deliver sperm after navigating through flower tissues in response to attractive and repulsive cues. Genetic analyses in maize and Arabidopsis thaliana and cell ablation studies in Torenia fournieri have shown that the female gametophyte (the 7-celled haploid embryo sac within an ovule) and surrounding diploid tissues are essential for guiding pollen tubes to ovules. The variety and inaccessibility of these cells and tissues has made it challenging to characterize the sources of guidance signals and the dynamic responses they elicit in the pollen tubes.
Here we developed an in vitro assay to study pollen tube guidance to excised A. thaliana ovules. Using this assay we discerned the temporal and spatial regulation and species-specificity of late stage guidance signals and characterized the dynamics of pollen tube responses. We established that unfertilized A. thaliana ovules emit diffusible, developmentally regulated, species-specific attractants, and demonstrated that ovules penetrated by pollen tubes rapidly release diffusible pollen tube repellents.
These results demonstrate that in vitro pollen tube guidance to excised A. thaliana ovules efficiently recapitulates much of in vivo pollen tube behaviour during the final stages of pollen tube growth. This assay will aid in confirming the roles of candidate guidance molecules, exploring the phenotypes of A. thaliana pollen tube guidance mutants and characterizing interspecies pollination interactions.
PMCID: PMC1489931  PMID: 16595022

Results 1-7 (7)