Pollen tube growth and guidance is regulated by POP2, an Arabidopsis gene that controls GABA levels

During angiosperm reproduction, pollen grains form a tube that navigates through female tissues to the micropyle, delivering sperm to the egg; the signals that mediate this process are poorly understood. Here, we describe a role for gamma-amino butyric acid (GABA) in pollen tube growth and guidance. In vitro, GABA stimulates pollen tube growth, although vast excesses are inhibitory. The Arabidopsis POP2 gene encodes a transaminase that degrades GABA and contributes to the formation of a gradient leading up to the micropyle. pop2 flowers accumulate GABA, and the growth of many pop2 pollen tubes is arrested, consistent with their in vitro GABA hypersensitivity. Some pop2 tubes continue to grow toward ovules, yet they are misguided, presumably because they target ectopic GABA on the ovule surface. Interestingly, wild-type tubes exhibit normal growth and guidance in pop2 pistils, perhaps by degrading excess GABA and sharpening the gradient leading to the micropyle.     

Deciphering the Possible Mechanism of GABA in Tobacco Pollen Tube Growth and Guidance

γ-Aminobutyric acid (GABA) is an inhibitory transmitter in animal central and peripheral nervous systems, and also plays an important role in pollen tube growth and guidance. However, the mechanisms underlying these effects in plants are poorly understood, mainly because the GABA receptor in plants has not been elucidated. To address this issue, we recently created quantum dot probes to identify possible GABA receptors on the membrane surfaces of pollen protoplasts. We found that GABA bound to cell membranes and regulated downstream Ca²⁺ oscillation in the cells. These results provide important clues to further specifying the nature of the binding sites and deciphering the role of GABA as a signal molecule in pollen tube growth and orientation.Key WordS: γ-aminobutyric acid, fertilization, GABA receptor, signal transduction and tobacco     

γ-Aminobutyric acid (GABA) homeostasis regulates pollen germination and polarized growth in Picea wilsonii

γ-Aminobutyric acid (GABA) is a four-carbon non-protein amino acid found in a wide range of organisms. Recently, GABA accumulation has been shown to play a role in the stress response and cell growth in angiosperms. However, the effect of GABA deficiency on pollen tube development remains unclear. Here, we demonstrated that specific concentrations of exogenous GABA stimulated pollen tube growth in Picea wilsonii, while an overdose suppressed pollen tube elongation. The germination percentage of pollen grains and morphological variations in pollen tubes responded in a dose-dependent manner to treatment with 3-mercaptopropionic acid (3-MP), a glutamate decarboxylase inhibitor, while the inhibitory effects could be recovered in calcium-containing medium supplemented with GABA. Using immunofluorescence labeling, we found that the actin cables were disorganized in 3-MP treated cells, followed by the transition of endo/exocytosis activating sites from the apex to the whole tube shank. In addition, variations in the deposition of cell wall components were detected upon labeling with JIM5, JIM7, and aniline blue. Our results demonstrated that calcium-dependent GABA signaling regulates pollen germination and polarized tube growth in P. wilsonii by affecting actin filament patterns, vesicle trafficking, and the configuration and distribution of cell wall components.     

Cell–cell communications and molecular mechanisms in plant sexual reproduction

Sexual reproduction is achieved by precise interactions between male and female reproductive organs. In plant fertilization, sperm cells are carried to ovules by pollen tubes. Signals from the pistil are involved in elongation and control of the direction of the pollen tube. Genetic, reverse genetic, and cell biological analyses using model plants have identified various factors related to the regulation of pollen tube growth and guidance. In this review, I summarize the mechanisms and molecules controlling pollen tube growth to the ovule, micropylar guidance, reception of the guidance signal in the pollen tube, rupture of the pollen tube to release sperm cells, and cessation of the tube guidance signal. I also briefly introduce various techniques used to analyze pollen tube guidance in vitro.     

花粉管生长和极性引导的孢子体和配子体控制研究进展

双受精是被子植物特有的生殖方式,精细胞只有通过花粉管穿过花柱才能到达子房、胚珠受精。花粉管在母本组织中的生长和引导包括孢子体控制(sporophytic control)和配子体控制(gametophytic control)两个连续的过程,现已克隆出不同阶段花粉管生长和引导的基因,通过分析其表达调控揭示出花粉管生长和引导的分子机制。该文就近年来国内外有关花粉管生长和极性引导的调控机制研究进展进行综述,并对禾本科(Poaceae)和十字花科(Brassicaceae)植物花粉管引导的异同点进行了比较分析。     

Attraction of tip-growing pollen tubes by the female gametophyte

Pollen tube guidance is the mechanism whereby the direction of pollen tube growth is controlled by female cells of the pistil. Some key genes and molecules have recently been identified as being involved in pollen tube guidance. In this review article, we discuss the molecular basis of pollen tube guidance, especially in Arabidopsis thaliana, by summarizing recent progress in various plant species. Attractant molecules and receptors for gametophytic pollen tube guidance are the focus of this article.     

Gametophytic pollen tube guidance

The concept of a pollen tube attractant was proposed in the late nineteenth century when pollen tubes were found to grow toward excised pistil tissues on medium. Since then, for about 140 years, plant biologists have tried to identify the pollen tube attractants. However, no molecule has been convincingly demonstrated to be the true attractant that actually controls the navigation of pollen tubes in the pistil. The past decade has seen substantial progress in this field in terms of our understanding of the various mechanisms of pollen tube guidance. It was suggested that diffusible pollen tube attractants might provide localized signals that affect the directional growth of the pollen tube, especially in the last phase of guidance by the target female gametophyte. Here, we review the mechanisms of pollen tube guidance, with special focus on the gametophytic guidance and the attractant. The necessary and appropriate conditions required by the true attractant will be discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Plant reproduction: GABA gradient, guidance and growth

How a pollen tube manages to navigate through the female tissues during plant reproduction has been a mystery. A new analysis of an Arabidopsis mutant has provided the strongest evidence yet that a GABA gradient may be a critical signal for correct targeting of the pollen tube.     

花粉管引导机制的研究进展

花粉管引导是指显花植物在受精过程中,雌蕊组织与花粉管相互作用使花粉管定向生长并最终到达胚囊的过程,其机制颇为复杂。该文基于调控花粉管生长的孢子体引导和配子体细胞引导两个主要过程,阐述雌蕊中不同蛋白分子和其它小分子物质的浓度梯度在花粉管的孢子体组织引导中的作用,以及胚囊中不同类型的细胞及其相关基因与蛋白在花粉管的配子体细胞引导中的作用。同时,该文也对精细胞在花粉管引导中的作用进行了阐述。     

POD1 regulates pollen tube guidance in response to micropylar female signaling and acts in early embryo patterning in Arabidopsis

The pollen tube germinates from pollen and, during its migration, it perceives and responds to guidance cues from maternal tissue and from the female gametophyte. The putative female cues have recently been identified, but how the pollen tube responds to these signals remains to be unveiled. In a genetic screen for male determinants of the pollen tube response, we identified the pollen defective in guidance1 (pod1) mutant, in which the pollen tubes fail to target the female gametophyte. POD1 encodes a conserved protein of unknown function and is essential for positioning and orienting the cell division plane during early embryo development. Here, we demonstrate that POD1 is an endoplasmic reticulum (ER) luminal protein involved in ER protein retention. Further analysis shows that POD1 interacts with the Ca(2+) binding ER chaperone CALRETICULIN3 (CRT3), a protein in charge of folding of membrane receptors. We propose that POD1 modulates the activity of CRT3 or other ER resident factors to control the folding of proteins, such as membrane proteins in the ER. By this mechanism, POD1 may regulate the pollen tube response to signals from the female tissues during pollen tube guidance and early embryo patterning in Arabidopsis thaliana.     

Genetic Evidence for a Long-Range Activity That Directs Pollen Tube Guidance in Arabidopsis

The fertilization process of plants is governed by different kinds of cell-cell interactions. In higher plants, these interactions are required both for recognition of the pollen grain by the female reproductive system and to direct the growth of the pollen tube inside the ovary. Despite many years of study, the signaling mechanisms that guide the pollen tube toward its target, the ovule, are largely unknown. Two distinct types of principles, mechanical and chemotropic, have been suggested to account for the directed growth of the pollen tube. The first of these two types of models implies that the guidance of the pollen tube depends on the architecture and chemical properties of the female reproductive tissues, whereas the latter suggests that the ovule provides a signal for the target-directed growth of the pollen tube. To examine such a role for the ovules, we analyzed the growth path of pollen tubes in mutants defective in ovule development in Arabidopsis. The results presented here provide unique in vivo evidence for an ovule-derived, long-range activity controlling pollen tube guidance. A morphological comparison of the ovule mutants used in this study indicates that within the ovule, the haploid embryo sac plays an important role in this long-range signaling process.     

Arabidopsis HAP2 (GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization

In flowering plants, sperm cells develop in the pollen cytoplasm and are transported through floral tissues to an ovule by a pollen tube, a highly polarized cellular extension. After targeting an ovule, the pollen tube bursts, releasing two sperm that fertilize an egg and a central cell. Here, we identified the gene encoding Arabidopsis HAP2, demonstrating that it is allelic to GCS1. HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to ovules. We identified an insertion (hap2-1) that disrupts the C-terminal portion of the protein and tags mutant pollen grains with the beta-glucuronidase reporter. By monitoring reporter expression, we showed that hap2-1 does not diminish pollen tube length in vitro or in the pistil, but it reduces ovule targeting by twofold. In addition, we show that the hap2 sperm that are delivered to ovules fail to initiate fertilization. HAP2 is predicted to encode a protein with an N-terminal secretion signal, a single transmembrane domain and a C-terminal histidine-rich domain. These results point to a dual role for HAP2, functioning in both pollen tube guidance and in fertilization. Moreover, our findings suggest that sperm, long considered to be passive cargo, are involved in directing the pollen tube to its target.     

Exogenous free ubiquitin enhances lily pollen tube adhesion to an in vitro stylar matrix and may facilitate endocytosis of SCA

Pollen tube adhesion and guidance on extracellular matrices within the pistil are essential processes that convey the pollen tube cell and the sperm cells to the ovule. In this study, we purified an additional molecule from the pistil that enhances pollen tube adhesion when combined with the SCA (stigma/stylar cysteine-rich adhesin)/pectin matrix in our in vitro assay. The enhancer of adhesion was identified as free ubiquitin (Ub). This was confirmed by use of bovine Ub as a substitute for lily (Lilium longiflorum Thunb.) stigma Ub. To study the interaction of SCA and Ub with the lily pollen tube, we labeled both proteins with biotin. We observed uptake of biotin-labeled SCA and Ub into the pollen tube cells in vitro using confocal microscopy. For SCA, a strong signal occurred first at the tip of the pollen tube, suggestive of an endocytosis event, and then progressively throughout the tube cytoplasm. SCA was also localized inside the in vivo pollen tube using immunogold electron microscopy and found to be present in endosomes, multivesicular bodies, and vacuoles, all known to be endocytic compartments. It was also confirmed that SCA is endocytosed in the in vitro adhesion assay. Internalization of SCA was increased in pollen tubes treated with exogenous Ub compared to those without Ub, suggesting that Ub may facilitate SCA endocytosis. These results show that Ub can act as an enhancer of pollen tube adhesion in vitro and that it is taken up into the pollen tube as is SCA. The Ub machinery may play a role in pollen tube adhesion and guidance in lily.     

Two Mitogen-Activated Protein Kinases,MPK3 and MPK6, Are Required for Funicular Guidance of Pollen Tubes in Arabidopsis

Double fertilization in flowering plants requires the delivery of two immotile sperm cells to the female gametes by a pollen tube, which perceives guidance cues, modifies its tip growth direction, and eventually enters the micropyle of the ovule. In spite of the recent progress, so far, little is known about the signaling events in pollen tubes in response to the guidance cues. Here, we show that MPK3 and MPK6, two Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinases, mediate the guidance response in pollen tubes. Genetic analysis revealed that mpk3 mpk6 double mutant pollen has reduced transmission. However, direct observation of mpk3 mpk6 mutant pollen phenotype was hampered by the embryo lethality of double homozygous mpk3^–/– mpk6^–/– plants. Utilizing a fluorescent reporter-tagged complementation method, we showed that the mpk3 mpk6 mutant pollen had normal pollen tube growth but impaired pollen tube guidance. In vivo pollination assays revealed that the mpk3 mpk6 mutant pollen tubes were defective in the funicular guidance phase. By contrast, semi-in vitro guidance assay showed that the micropylar guidance of the double mutant pollen tube was normal. Our results provide direct evidence to support that the funicular guidance phase of the pollen tube requires an in vivo signaling mechanism distinct from the micropyle guidance. Moreover, our finding opened up the possibility that the MPK3/MPK6 signaling pathway may link common signaling networks in plant stress response and pollen-pistil interaction.In flowering plants, successful fertilization is dependent on extensive cell-cell communication between male and female gametophytes. After landing on a compatible stigma surface, a mature pollen grain germinates to form a pollen tube, which penetrates the stigma, perceives guidance cues along the growth path, and modifies its tip growth direction toward the ovule (Hülskamp et al., 1995). In Arabidopsis (Arabidopsis thaliana), the pollen tube guidance can be divided into two phases: funicular guidance, in which the pollen tube emerges from the septum and proceeds to a funiculus, and micropylar guidance, in which the pollen tube grows toward and enters the micropyle of an ovule (Hülskamp et al., 1995).In pollen tube, it is believed that receptors on the tube tip perceive various guidance cues and regulate downstream signaling pathways to modify tip reorientation toward the ovule (Higashiyama, 2010; Takeuchi and Higashiyama, 2011). Two receptor-like kinase genes, Lost In Pollen tube guidance1 (LIP1) and LIP2, are involved in guidance control of pollen tubes. LIP1 and LIP2 were anchored to the membrane in the pollen tube tip region via palmitoylation, which was essential for their guidance control (Liu et al., 2013). Therefore, LIP1 and LIP2 are the essential components of the receptor complex in micropylar guidance. The Glu receptor-like channels facilitate Ca²⁺ influx across the plasma membrane and regulate pollen tube growth and morphogenesis (Michard et al., 2011). This interesting work revealed that there is a signaling mechanism between the male gametophyte and pistil tissue that is similar to the amino acid-mediated communication in animal nervous systems (Michard et al., 2011). Recent findings also highlight the importance of the endoplasmic reticulum (ER), ion homeostasis, and protein processing in pollen tube guidance (Li et al., 2011; Lu et al., 2011; Li and Yang, 2012). Two pollen-expressed cation proton exchangers (CHXs), CHX21 and CHX23, were reported to mediate K⁺ transport in ER and are essential for the pollen tube to respond to directional signals from the ovule in Arabidopsis (Lu et al., 2011). POLLEN DEFECTIVE IN GUIDANCE1 plays an important role in micropylar guidance in pollen tube (Li et al., 2011). It is an ER luminal protein involved in ER protein retention and interacts with a luminal chaperone involved in Ca²⁺ homeostasis and ER quality control (Li et al., 2011). Therefore, the ER quality control is likely an important mechanism in surveillance of signaling factors in pollen tube guidance (Li and Yang, 2012).In spite of the recent progresses, so far, little is known about the cytoplasmic signaling events in pollen tubes in response to the guidance cues. Mitogen-activated protein kinase (MAPK, or MPK) cascades are conserved signaling pathways that respond to extracellular stimuli and regulate various cellular activities. In Arabidopsis, MPK3 and MPK6 are induced by various biotic and abiotic stresses and collaboratively play important roles in defense response and plant development (Zhang, 2008). Here, we show that MPK3 and MPK6 are also critical to pollen tube guidance. Utilizing a fluorescent reporter-tagged complementation method, we demonstrated that mpk3 mpk6 pollen was defective in pollen tube guidance at the funicular guidance phase. Intriguingly, the micropylar guidance of mpk3 mpk6 pollen tube is not affected.     

Pollen tube targeting and axon guidance: parallels in tip growth mechanisms

The growth of pollen tubes to plant egg cells and the guidance of axons to neural synapses are classic examples of targeted cell growth. Despite the evolutionary time that separates animals and plants, axon and pollen tube guidance share remarkable mechanistic similarities. In both instances, extracellular cues are transduced by intracellular signal-transduction pathways that culminate in directed tip growth. Do the mechanistic similarities extend to the molecular level? Here, we address this question by a comprehensive review of the molecules and pathways involved in pollen tube targeting and axon guidance. The emerging scenario is that similar intracellular molecules are recruited to control tip growth, while different extracellular molecules mediate guidance through the distinct plant and animal extracellular matrices.     

Development and function of the synergid cell

The synergid cells are located in the female gametophyte and are essential for angiosperm reproduction. During the fertilization process, a pollen tube grows into one of the synergid cells, ceases growth, ruptures, and releases its two sperm cells into this cell. The synergid cells produce an attractant that guides the pollen tube to the female gametophyte and likely contain factors that control arrest of pollen tube growth, pollen tube discharge, and gamete fusion. The synergid cells contain an elaborated cell wall at their micropylar poles, the filiform apparatus that likely plays a role in pollen tube guidance and pollen tube reception. Recent genetic, molecular, and physiological studies in Arabidopsis, maize, and Torenia have provided insights into synergid cell development and the control of pollen tube growth by the synergid cell.