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Ion-specific probes and fluorescent indicators have been key in establishing the role of ion signaling, namely calcium, protons, and anions, in plant development, providing a robust approach for monitoring spatiotemporal changes in intracellular ion dynamics. The integration of protons/pH in signaling mechanisms is especially important as reports of their biological functions continue to expand; however, attaining quantitative estimates with high spatiotemporal resolution in single cells poses a major research challenge. Here, we detail the use of the genetically encoded pH-sensitive pHluorin reporter expressed in Arabidopsis thaliana pollen tubes to assess cytosolic measurements with calibration to provide actual pH values. This technique enabled us to identify critical phenotypes and establish the importance of tip-focused pH gradient for pollen tube growth, although it can be adapted to other experimental systems.

The phytohormone ethylene has numerous effects on plant growth and development. Its immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), is a non-proteinogenic amino acid produced by ACC SYNTHASE (ACS). ACC is often used to induce ethylene responses. Here, we demonstrate that ACC exhibits ethylene-independent signaling in Arabidopsis thaliana reproduction. By analyzing an acs octuple mutant with reduced seed set, we find that ACC signaling in ovular sporophytic tissue is involved in pollen tube attraction, and promotes secretion of the pollen tube chemoattractant LURE1.2. ACC activates Ca2+-containing ion currents via GLUTAMATE RECEPTOR-LIKE (GLR) channels in root protoplasts. In COS-7 cells expressing moss PpGLR1, ACC induces the highest cytosolic Ca2+ elevation compared to all twenty proteinogenic amino acids. In ovules, ACC stimulates transient Ca2+ elevation, and Ca2+ influx in octuple mutant ovules rescues LURE1.2 secretion. These findings uncover a novel ACC function and provide insights for unraveling new physiological implications of ACC in plants., Ethylene is synthesized from the non-proteinogenic amino acid ACC. Here, Mou et al. show that ACC itself acts independently of ethylene to trigger secretion of a pollen tube attractant in the sporophytic tissue of Arabidopsis ovules and can activate Ca2+-currents via GLUTAMATE RECEPTOR-LIKE channels.

Pollen tubes are highly polarized tip-growing cells that depend on cytosolic pH gradients for signaling and growth. Autoinhibited plasma membrane proton (H+) ATPases (AHAs) have been proposed to energize pollen tube growth and underlie cell polarity, however, mechanistic evidence for this is lacking. Here we report that the combined loss of AHA6, AHA8, and AHA9 in Arabidopsis thaliana delays pollen germination and causes pollen tube growth defects, leading to drastically reduced fertility. Pollen tubes of aha mutants had reduced extracellular proton (H+) and anion fluxes, reduced cytosolic pH, reduced tip-to-shank proton gradients, and defects in actin organization. Furthermore, mutant pollen tubes had less negative membrane potentials, substantiating a mechanistic role for AHAs in pollen tube growth through plasma membrane hyperpolarization. Our findings define AHAs as energy transducers that sustain the ionic circuit defining the spatial and temporal profiles of cytosolic pH, thereby controlling downstream pH-dependent mechanisms essential for pollen tube elongation, and thus plant fertility.

Pollen tubes are highly polarized tip-growing cells that depend on cytosolic pH gradients for signaling and growth. Autoinhibited plasma membrane proton (H+) ATPases (AHAs) have been proposed to energize pollen tube growth and underlie cell polarity, however, mechanistic evidence for this is lacking. Here we report that the combined loss of AHA6, AHA8, and AHA9 in Arabidopsis thaliana delays pollen germination and causes pollen tube growth defects, leading to drastically reduced fertility. Pollen tubes of aha mutants had reduced extracellular proton (H+) and anion fluxes, reduced cytosolic pH, reduced tip-to-shank proton gradients, and defects in actin organization. Furthermore, mutant pollen tubes had less negative membrane potentials, substantiating a mechanistic role for AHAs in pollen tube growth through plasma membrane hyperpolarization. Our findings define AHAs as energy transducers that sustain the ionic circuit defining the spatial and temporal profiles of cytosolic pH, thereby controlling downstream pH-dependent mechanisms essential for pollen tube elongation, and thus plant fertility., Cytosolic ion gradients in growing pollen tubes are thought to be required for polar growth. Here the authors show that the Arabidopsis plasma membrane H+ ATPases, AHA6, AHA8, and AHA9, maintain tip-to-shank proton gradients, oscillations in cytosolic pH and actin organization to enable pollen tube elongation.

SummaryNitric oxide (NO) is a key signaling molecule that regulates diverse biological processes in both animals and plants. In animals, NO regulates vascular wall tone, neurotransmission and immune response while in plants, NO is essential for development and responses to biotic and abiotic stresses [1–3]. Interestingly, NO is involved in the sexual reproduction of both animals and plants mediating physiological events related to the male gamete [2, 4]. In animals, NO stimulates sperm motility [4] and binding to the plasma membrane of oocytes [5] while in plants, NO mediates pollen-stigma interactions and pollen tube guidance [6, 7]. NO generation in pollen tubes (PTs) has been demonstrated [8] and intracellular responses to NO include cytosolic Ca2+ elevation, actin organization, vesicle trafficking and cell wall deposition [7, 9]. However, the NO-responsive proteins that mediate these responses are still elusive. Here we show that PTs of Arabidopsis lacking the pollen-specific Diacylglycerol Kinase 4 (DGK4) grow slower and become insensitive to NO-dependent growth inhibition and re-orientation responses. Recombinant DGK4 protein yields NO-responsive spectral and catalytic changes in vitro which are compatible with a role in NO perception and signaling in PTs. NO is a fast, diffusible gas and, based on our results, we hypothesize it could serve in long range signaling and/or rapid cell-cell communication functions mediated by DGK4 downstream signaling during the progamic phase of angiosperm reproduction.

Premise of the study Upon pollination, dehydrated pollen grains take water out of the stigma surface, an event that constitutes the first functional checkpoint of sexual reproduction in higher plants. Little is known about possible functional connections between rehydration speed and further steps of fertilization. Here we addressed the mechanisms of water uptake control by dehydrated pollen grains. Because dehydrated cells have no energy-driven active mechanism such as membrane-based osmoregulation for controlling water movement, we tested the hypothesis that another mechanism might exist, namely, the use of hydrogel-behaving molecules. Methods We developed an imaging protocol to visualize and quantify the rate of water entry into pollen grains of Eucalyptus globulus and tested the influence of different treatments linked to hydrogel-behaving molecules. We complemented these analyses by immunostaining pectins in the pollen grain with monoclonal antibodies JIM5 and JIM7. Key results Water entry seemed to proceed exclusively through the germination apertures of the pollen grain, and the changes observed in different hydration media are compatible with hydrogel behavior. When JIM5 and JIM7 were used to characterize pectins on the germination apertures during hydration, pectin localization and esterification changed during hydration and were affected by the hydration solutions. These results suggest that chemical modification of the pectins may change their hydrogel behavior, thus modifying the hydration speed. Conclusions The hydrogel behavior of pectins and pectin localization on apertures strongly suggest that pectins act like "valves" for water entry, enabling a regulated process of water uptake into the dehydrated pollen grain. We propose that this regulation evolved in terms of achieving the correct self-organization of molecules and cellular components to resume metabolism and pollen tube growth, especially in species that are subject to demanding environmental water stress.

Post-print version of the article. Submitted by Pedro Gomes (psgomes@igc.gulbenkian.pt) on 2016-11-16T15:55:39Z No. of bitstreams: 8 Ortiz-Ramirez et. al. Molecular Plant 2016.pdf: 3618336 bytes, checksum: 6c7e8d70dfd297fbe551cc8540a1d162 (MD5) S1_Table.xlsx: 43094395 bytes, checksum: 88902e5fa186ab8873ebf00f48cca527 (MD5) S2_Table.xlsx: 30584 bytes, checksum: b7dad2287fd94b61d414b4f62f1be1c5 (MD5) S3_Table.xlsx: 64760 bytes, checksum: 91387f7977117c2080e38311999d0f1e (MD5) S4_Table.xlsx: 12018 bytes, checksum: 939b698d7a077a18ea22c38408ecf45d (MD5) S5_Table.xlsx: 23614 bytes, checksum: 27827ce836d39d91c00632f51ce36810 (MD5) S6_Table.xlsx: 399591 bytes, checksum: 881c815499f9f345efb7fb36460ebc04 (MD5) Supplemental figures 1-9.pdf: 1500308 bytes, checksum: 22bd0b0ad6f285cd49cff2434ccd2136 (MD5) Made available in DSpace on 2016-11-16T15:55:39Z (GMT). No. of bitstreams: 8 Ortiz-Ramirez et. al. Molecular Plant 2016.pdf: 3618336 bytes, checksum: 6c7e8d70dfd297fbe551cc8540a1d162 (MD5) S1_Table.xlsx: 43094395 bytes, checksum: 88902e5fa186ab8873ebf00f48cca527 (MD5) S2_Table.xlsx: 30584 bytes, checksum: b7dad2287fd94b61d414b4f62f1be1c5 (MD5) S3_Table.xlsx: 64760 bytes, checksum: 91387f7977117c2080e38311999d0f1e (MD5) S4_Table.xlsx: 12018 bytes, checksum: 939b698d7a077a18ea22c38408ecf45d (MD5) S5_Table.xlsx: 23614 bytes, checksum: 27827ce836d39d91c00632f51ce36810 (MD5) S6_Table.xlsx: 399591 bytes, checksum: 881c815499f9f345efb7fb36460ebc04 (MD5) Supplemental figures 1-9.pdf: 1500308 bytes, checksum: 22bd0b0ad6f285cd49cff2434ccd2136 (MD5) Previous issue date: 2016-02-01 ERA-NET: (2nd call ERA-NET for Coordinating Plant Sciences); Fundação para a Ciência e a Tecnologia.

Nitric oxide (NO) is a gaseous reactive oxygen species (ROS) that has evolved as a signaling hormone in many physiological processes in animals. In plants it has been demonstrated to be a crucial regulator of development, acting as a signaling molecule present at each step of the plant life cycle. NO has also been implicated as a signal in biotic and abiotic responses of plants to the environment. Remarkably, despite this plethora of effects and functional relationships, the fundamental knowledge of NO production, sensing, and transduction in plants remains largely unknown or inadequately characterized. In this review we cover the current understanding of NO production, perception, and action in different physiological scenarios. We especially address the issues of enzymatic and chemical generation of NO in plants, NO sensing and downstream signaling, namely the putative cGMP and Ca(2+) pathways, ion-channel activity modulation, gene expression regulation, and the interface with other ROS, which can have a profound effect on both NO accumulation and function. We also focus on the importance of NO in cell-cell communication during developmental processes and sexual reproduction, namely in pollen tube guidance and embryo sac fertilization, pathogen defense, and responses to abiotic stress.

The deposited article version is a "Accelerated Article Preview" provided by Nature Publishing Group, and it contains attached the supplementary materials within the pdf.». This publication hasn't any creative commons license associated. Glutamate receptors are well characterized channels that mediate cell-to-cell communication during neurotransmission in animals. Nevertheless, information regarding their functional role in organisms without nervous systems is still limited. In plants, Glutamate Receptor-like (GLR) genes have been implicated in defence against pathogens, reproduction, control of stomata aperture and light signal transduction(1-5). However, the numerous GLR genes present in angiosperm genomes (20 to 70)(6) has prevented the observation of strong phenotypes in loss-of-function mutants. Here, we show that in the moss Physcomitrella patens, a basal land plant, mutation of GLR genes cause sperm failure in targeting the female reproductive organs. In addition, we show that GLR genes encode non-selective Ca(2+) permeable channels that can regulate cytoplasmic Ca(2+) and are needed to induce the expression of a BELL1-like transcription factor essential for zygote development. Our work reveals novel functions for GLRs in sperm chemotaxis and transcriptional regulation. Sperm chemotaxis is essential for fertilization in both animals and early land plants like bryophytes and pteridophytes. Therefore, our results are suggestive that ionotropic glutamate receptors may have been conserved throughout plant evolution to mediate cell-to-cell communication during sexual reproduction. Phillips University; Oxford University; University of Marburg; University of Muenster; MarieCurie ITN-Plant Origins grant: (FP7-PEOPLE-ITN-2008); FCT grants: (BEX-BCM/0376/2012; PTDC/BIA-PLA/4018/2012); NSF-US grant: (MCB 1616437/2016). info:eu-repo/semantics/acceptedVersion

Pollen is the male gametophyte of higher plants and is responsible for the successful transport of the sperm cells to the ovules. After landing on a stigma, pollen grains will germinate and grow a pollen tube through the stigma and style tissue towards the ovules, where fertilization takes place. In terms of cell biology, the elongation of the pollen tube is characterized by a dramatically polarized growth process, tip growth, which is common to root hairs, fungal hyphae and some developing neurites. Due to their simple morphology and function, growing pollen tubes became the most well-established model system to study tip growth. Both the editors of this book have been enthusiastic paladins of this trend, even since they first met in 1994 during the 13th International Congress on Sexual Plant Reproduction in Vienna. Our intellectual enthusiasm was first materialized in an essay in which a naive and simple yet forward theoretical model was set forth, implying a set of electrochemistry rules to be at the core of a minimal set of mechanism underlying cell polarity establishment and maintenance during pollen tube growth (Feijo et al. ). In short, subcellular biophysical processes, like ion transport, endogenous electrical fields and a tip-focussed Ca2+ gradient, would regulate the shape and growth rate in an essentially self-organizing process. Ever since then, the multitude of nuts and bolts and genes and pathways and their biological consequences have amounted to a vast literature in practically every aspect of the biology of pollen. And yet, some of the essential parts of this naive biophysical view of the pollen tube remained elusive, namely the existence and features of the channels responsible for the unique ion biology of pollen (Michard et al.).

The male germ cells of angiosperm plants are neither free-living nor flagellated and therefore are dependent on the unique structure of the pollen grain for fertilization. During angiosperm male gametogenesis, an asymmetric mitotic division produces the generative cell, which is completely enclosed within the cytoplasm of the larger pollen grain vegetative cell. Mitotic division of the generative cell generates two sperm cells that remain connected by a common extracellular matrix with potential intercellular connections. In addition, one sperm cell has a cytoplasmic projection in contact with the vegetative cell nucleus. The shared extracellular matrix of the two sperm cells and the physical association of one sperm cell to the vegetative cell nucleus forms a linkage of all the genetic material in the pollen grain, termed the male germ unit. Found in species representing both the monocot and eudicot lineages, the cytoplasmic projection is formed by vesicle formation and microtubule elongation shortly after the formation of the generative cell and tethers the male germ unit until just prior to fertilization. The cytoplasmic projection plays a structural role in linking the male germ unit, but potentially plays other important roles. Recently, it has been speculated that the cytoplasmic projection and the male germ unit may facilitate communication between the somatic vegetative cell nucleus and the germinal sperm cells, via RNA and/or protein transport. This review focuses on the nature of the sperm cell cytoplasmic projection and the potential communicative function of the male germ unit.

The process of pollen germination and tube growth in the pistil involves a series of cell-cell interactions, some facilitating fertilization while others prohibiting pollen tube access to the female gametophyte, either because of incompatibility or as a result of mechanisms to avert polyspermy and to ensure reproductive success. Understanding pollen tube growth and guidance to the female gametophyte has long been a pursuit among plant biologists, and observations indicate that diverse strategies may be adopted by different plant species. Recent studies in Arabidopsis, maize, and Torenia fournieri suggest that low molecular weight secretory molecules probably play major roles in the short-range attraction of pollen tubes to the female gametophyte. The process of pollen tube growth in the pistil occurs beneath several cell layers so much of the information that conveys the intimate partnership between penetrating pollen tubes and the female tissues has come from fixed samples and observations of in vitro pollen tube growth responses to female factors. A unique glimpse of the in vivo pollen germination and tube growth process is provided here by intra-vital two-photon excitation (TPE) microscopy of pollinated Arabidopsis pistils that remained on intact plants. Further discoveries of critical factors of male or female origins and how they control the pollen tube growth and fertilization process will broaden our understanding of the common themes and diverse strategies that plants have evolved to ensure reproductive success. The advancement of imaging technology to monitor pollination and fertilization and the development of probes to monitor various aspects of the pollen tube growth process, including pollen intracellular dynamics, will allow us to superimpose details obtained from studying pollen tube growth in culture conditions to interpret and understand the in vivo events.

The guidance signals that drive pollen tube navigation inside the pistil and micropyle targeting are still, to a great extent, unknown. Previous studies in vitro showed that nitric oxide (NO) works as a negative chemotropic cue for pollen tube growth in lily ( Lilium longiflorum ). Furthermore, Arabidopsis thaliana Atnos1 mutant plants, which show defective NO production, have reduced fertility. Here, we focus in the role of NO in the process of pollen–pistil communication, using Arabidopsis in-vivo and lily semi-vivo assays. Cross-pollination between wild-type and Atnos1 plants shows that the mutation affects the pistil tissues in a way that is compatible with abnormal pollen tube guidance. Moreover, DAF-2DA staining for NO in kanadi floral mutants showed the presence of NO in an asymmetric restricted area around the micropyle. The pollen–pistil interaction transcriptome indicates a time-course-specific modulation of transcripts of AtNOS1 and two Nitrate Reductases ( nr1 and nr2 ), which collectively are thought to trigger a putative NO signaling pathway. Semi-vivo assays with isolated ovules and lily pollen further showed that NO is necessary for micropyle targeting to occur. This evidence is supported by CPTIO treatment with subsequent formation of balloon tips in pollen tubes facing ovules. Activation of calcium influx in pollen tubes partially rescued normal pollen tube morphology, suggesting that this pathway is also dependent on Ca 2+ signaling. A role of NO in modulating Ca 2+ signaling was further substantiated by direct imaging the cytosolic free Ca 2+ concentration during NO-induced re-orientation, where two peaks of Ca 2+ occur—one during the slowdown/stop response, the second during re-orientation and growth resumption. Taken together, these results provide evidence for the participation of NO signaling events during pollen–pistil interaction. Of special relevance, NO seems to directly affect the targeting of pollen tubes to the ovule's micropyle by modulating the action of its diffusible factors.

Summary Hydroxyproline O‐arabinosyltransferases (HPATs) are members of a small, deeply conserved family of plant‐specific glycosyltransferases that add arabinose sugars to diverse proteins including cell wall‐associated extensins and small signaling peptides. Recent genetic studies in flowering plants suggest that different HPAT homologs have been co‐opted to function in diverse species‐specific developmental contexts. However, nothing is known about the roles of HPATs in basal plants. We show that complete loss of HPAT function in Arabidopsis thaliana and the moss Physcomitrella patens results in a shared defect in gametophytic tip cell growth. Arabidopsis hpat1/2/3 triple knockout mutants suffer from a strong male sterility defect as a consequence of pollen tubes that fail to fully elongate following pollination. Knocking out the two HPAT genes of Physcomitrella results in larger multicellular filamentous networks due to increased elongation of protonemal tip cells. Physcomitrella hpat mutants lack cell‐wall associated hydroxyproline arabinosides and can be rescued with exogenous cellulose, while global expression profiling shows that cell wall‐associated genes are severely misexpressed, implicating a defect in cell wall formation during tip growth. Our findings point to a major role for HPATs in influencing cell elongation during tip growth in plants., Significance Statement Functionally important genes are evolutionarily conserved, but can be co‐opted for new functions in lineage‐specific contexts. Here we show that the phenotypes of mutants blocked in Hydroyproline O‐arabinosylation in Arabidopsis and in moss are opposite in tip‐growing cells, with shorter pollen tubes but longer protonemal cells, than in their WT counterparts.

The non-protein amino acid, gamma-aminobutyric acid (GABA) rapidly accumulates in plant tissues in response to biotic and abiotic stress, and regulates plant growth. Until now it was not known whether GABA exerts its effects in plants through the regulation of carbon metabolism or via an unidentified signalling pathway. Here, we demonstrate that anion flux through plant aluminium-activated malate transporter (ALMT) proteins is activated by anions and negatively regulated by GABA. Site-directed mutagenesis of selected amino acids within ALMT proteins abolishes GABA efficacy but does not alter other transport properties. GABA modulation of ALMT activity results in altered root growth and altered root tolerance to alkaline pH, acid pH and aluminium ions. We propose that GABA exerts its multiple physiological effects in plants via ALMT, including the regulation of pollen tube and root growth, and that GABA can finally be considered a legitimate signalling molecule in both the plant and animal kingdoms., GABA is an important neurotransmitter in animals, and while it accumulates in plants under stress, its potential role in plant signalling was poorly understood. Here, Ramesh et al. show that GABA rapidly alters the activity of plant ALMT anion channels modifying root growth and stress tolerance.

A multi-approach was used to study different aspects of self-incompatibility (SI) in almond (Prunus dulcis). First, a population of almond cultivars was characterised as to their individual S-allele combination using separation of stylar protein extracts (non-equilibrium pH gradient electrofocusing) followed by staining for RNase activity, which led to the identification of one putative new allele and several new S-allele combinations. Second, a field pollination scheme was designed to study pollen tube progression and to obtain a spatial and temporal characterisation of this reproductive stage in both incompatible and compatible crosses. In addition, an anti-serum was raised against a synthetic peptide designed from an almond S-protein (S8) and used for immunological in situ detection in pistil cryosections. S-RNases were found to accumulate intercellularly in the stylar transmitting tissue as previously reported for other rosaceous species. The results are discussed in view of the evolution of the gametophytic SI system and the models proposed for its mechanism. Gametophyte selection is also proposed as an important intraspecific barrier to fertilisation in this species.

Intraspecific barriers promote outcrossing while interspecific mechanisms may contribute to the isolation of species; both control the exchange of genes between plants. In this paper we establish that post-pollination mechanisms promote outcross and act at different temporal and spatial levels to control seed set and quality in Quercus species. Controlled pollinations were performed to investigate intraspecific crossing barriers in Q. suber and pollen-pistil interactions following interspecific pollinations with some simpatric Quercus species. Cytological data of intraspecific crosses in Q. suber and Q. ilex have shown different kinetics on pollen tube growth after self and outcross pollination, but pollen tubes were able to reach the base of the styles in both species and seed set was successful. Although some pre-zygotic interaction is occurring at the style, the most important interaction takes place at ovary. The cross between Q. ilex and Q. suber is possible only in one direction, revealing a case of unilateral incongruity. We show that the lack of seed set observed in the cross Q. suber×Q. ilex is due to the inability of pollen tubes to penetrate the transmitting tissue after germination. With Q. suber mainly as female parent, pollinations with other simpatric Quercus species have shown different levels of constraint on pollen tube progression at stigma and style levels. Eventual hybridisation between Quercus species will depend on the compatibility of pollen-pistil interactions, on the competitive ability of pollen genotypes, and, most important, on the overlapping of geographical, phenological and ecological factors. Differences in seed set and seed allocation was evident in all crosses, and was particularly outstanding in interspecific and in self intraspecific crosses, determining the ultimate level of seed production and quality in Quercus species.

To minimize the number of visits required to achieve seed-set from hand pollination whilst maintaining full control of the reproductive process in Eucalyptus globulus, we investigated the importance of style maturation on the progamic phase of reproduction. Controlled pollinations were performed in: (1) fully receptive styles; (2) immature cut styles at or after flower dehiscence; and (3) immature cut styles before flower dehiscence. We show here that pollen germination and tube growth to the ovule occurred in all three treatments, and all produced viable seeds. Remarkably, tubes growing on immature sliced styles of non-dehisced flowers reached the ovules by day 7, 1–2 d faster than tubes growing on normal receptive stigmas. Moreover, more tubes entered the ovary in this kind of pollination and, consequently, more viable seeds were produced. We established histochemically that tube growth in immature sliced styles is concomitant with the presence of lipid secretion. These results fully support the efficiency of the so-called ‘one-step pollination’ method in Eucalyptus globulus, which we now prove can be applied more successfully to immature non-dehisced flowers. General application of this procedure is likely to have beneficial impacts on the planning and efficiency of large-scale genetic improvement programs.

Cork oak (Quercus suber L.) is a monoecious wind-pollinated species with a protandrous system to ensure cross-pollination. To the best of our knowledge, this report provides the first insight into the sexual reproduction cycle in this species. The cork oak flowering season extends from April until the end of May. Our results show that, at anthesis, the pistillate flower is not completely formed and ovules are just starting to develop. Pollen reaching the dry stigmatic surface adheres to the receptive cells, germinates and penetrates the epidermis in aproximately 24 h, and grows through the intercellular spaces of a solid transmitting tissue. In cross-pollination, a sequential arrest of pollen tubes was observed along the style, providing preliminary evidence for a pollen tube competition mechanism. As a consequence, few pollen tubes reach the basal portion of the style. Furthermore, pollen tube growth is a discontinuous process since tubes are arrested in the basal portion of the style about 10–12 days after pollination. While tubes are latent, the ovarian loculus starts to develop from an emerging mass of sporogeneous cells which later will differentiate into the placenta and ovules. One and a half months after pollination ovules complete their differentiation, tubes resume growth and fertilisation occurs. Ovular abortion is frequent at this stage, and only one ovule will successfully mature during autumn into a monospermic seed.

Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients and fluxes, implying polar localization or regulation of the underlying transporters. The molecular identity and regulation of anion transporters in PTs is unknown. Here we report a negative gradient of cytosolic anion concentration focused on the tip, in negative correlation with the cytosolic Ca(2+) concentration. We hypothesized that a possible link between these two ions is based on the presence of Ca(2+)-dependent protein kinases (CPKs). We characterized anion channels and CPK transcripts in PTs and analyzed their localization. Yellow fluorescent protein (YFP) tagging of a homolog of SLOW ANION CHANNEL-ASSOCIATED1 (SLAH3:YFP) was widespread along PTs, but, in accordance with the anion efflux, CPK2/CPK20/CPK17/CPK34:YFP fluorescence was strictly localized at the tip plasma membrane. Expression of SLAH3 with either CPK2 or CPK20 (but not CPK17/CPK34) in Xenopus laevis oocytes elicited S-type anion channel currents. Interaction of SLAH3 with CPK2/CPK20 (but not CPK17/CPK34) was confirmed by Förster-resonance energy transfer fluorescence lifetime microscopy in Arabidopsis thaliana mesophyll protoplasts and bimolecular fluorescence complementation in living PTs. Compared with wild-type PTs, slah3-1 and slah3-2 as well as cpk2-1 cpk20-2 PTs had reduced anion currents. Double mutant cpk2-1 cpk20-2 and slah3-1 PTs had reduced extracellular anion fluxes at the tip. Our studies provide evidence for a Ca(2+)-dependent CPK2/CPK20 regulation of the anion channel SLAH3 to regulate PT growth.

One of the most interesting aspects of plant fertilization is the growth and orientation of the pollen tube from the stigma to the ovary. Considerable research has been carried out in this field but little is yet known about the mechanisms involved in the growth process. Recent research has been focused on the regulation of molecular events in order to discover the specific genes involved in tube growth. Important results in the biochemical and physiological aspects of molecular regulation have been reported. The following review attempts to cover these aspects. It is primarily based on talks presented by the authors at the 13th International Congress on Sexual Plant Reproduction and is mainly addressed to non-experts in the fields of electrophysiology and ion signalling. We aim to present a general overview of electrical currents, ion dynamics, and ion transporters in pollen, and their possible role during pollen tube germination and growth. Together with results on other tip-growing cells, a general model of pollen tube germination and growth as a self-organizing process is proposed.

Ionotropic glutamate receptors are ligand-gated, multimeric cation channels involved in neuronal transmission in vertebrates. In animals, ligand selectivity is highly specific, and depending on the subunit, channels open in response to glycine/D-serine or glutamate. The molecular evolution of the animal glutamate receptor family is an important element in the evolution of the central nervous system. Interestingly, glutamate receptor homologous channels (GLRs) can also be found in plants where they are, for instance, involved in calcium signaling during pollen tube growth or in long distance signaling. While the genome of the moss Physcomitrella patens codes for two GLRs, the GLR family largely diversified in flowering plants. The genome of Arabidopsis thaliana encodes 20 GLRs that subgroup in three clades, one of which is homologous to the moss GLRs, and two clades that are specific for higher plants.Our work consists of studying the molecular specificities of plant GLRs by addressing two main questions: (1) What is the evolutionary meaning of plant GLR diversification? (2) In which signaling pathways are GLRs from the different clades involved in? To address these questions we performed an electrophysiological characterization of GLRs by studying their activity in protoplasts from moss and Arabidopsis GLR knock-plants. To further characterize the function of GLRs, we also expressed and studied them in heterologous systems.Our work reveals unique gating properties of plant GLRs and gives novel insights into the specificity of plant GLRs compared to the animal ones.

Plasma membrane anion transporters play fundamental roles in plant cell biology, especially in stomatal closure and nutrition. Notwithstanding, a lot is still unknown about the specific function of these transporters, their specific localization, or molecular nature. Here the fundamental roles of anionic transport in plant cells are reviewed. Special attention will be paid to them in the control of pollen tube growth. Pollen tubes are extreme examples of cellular polarity as they grow exclusively in their apical extremity. Their unique cell biology has been extensively exploited for fundamental understanding of cellular growth and morphogenesis. Non-invasive methods have demonstrated that tube growth is governed by different ion fluxes, with different properties and distribution. Not much is known about the nature of the membrane transporters responsible for anionic transport and their regulation in the pollen tube. Recent data indicate the importance of chloride (Cl(-)) transfer across the plasma membrane for pollen germination and pollen tube growth. A general overview is presented of the well-known accumulated data in terms of biophysical and functional characterization, transcriptomics, and genomic description of pollen ionic transport, and the various controversies around the role of anionic fluxes during pollen tube germination, growth, and development. It is concluded that, like all other plant cells so far analysed, pollen tubes depend on anion fluxes for a number of fundamental homeostatic properties.

International audience; Elevations in cytosolic free calcium concentration ([Ca(2+)](cyt)) constitute a fundamental signal transduction mechanism in eukaryotic cells, but the molecular identity of Ca(2+) channels initiating this signal in plants is still under debate. Here, we show by pharmacology and loss-of-function mutants that in tobacco and Arabidopsis, glutamate receptor-like channels (GLRs) facilitate Ca(2+) influx across the plasma membrane, modulate apical [Ca(2+)](cyt) gradient, and consequently affect pollen tube growth and morphogenesis. Additionally, wild-type pollen tubes grown in pistils of knock-out mutants for serine-racemase (SR1) displayed growth defects consistent with a decrease in GLR activity. Our findings reveal a novel plant signaling mechanism between male gametophyte and pistil tissue similar to amino acid-mediated communication commonly observed in animal nervous systems.

Plant reproduction depends on the concerted activation of many genes to ensure correct communication between pollen and pistil. Here, we queried the whole transcriptome of Arabidopsis (Arabidopsis thaliana) in order to identify genes with specific reproductive functions. We used the Affymetrix ATH1 whole genome array to profile wild-type unpollinated pistils and unfertilized ovules. By comparing the expression profile of pistils at 0.5, 3.5, and 8.0 h after pollination and applying a number of statistical and bioinformatics criteria, we found 1,373 genes differentially regulated during pollen-pistil interactions. Robust clustering analysis grouped these genes in 16 time-course clusters representing distinct patterns of regulation. Coregulation within each cluster suggests the presence of distinct genetic pathways, which might be under the control of specific transcriptional regulators. A total of 78% of the regulated genes were expressed initially in unpollinated pistil and/or ovules, 15% were initially detected in the pollen data sets as enriched or preferentially expressed, and 7% were induced upon pollination. Among those, we found a particular enrichment for unknown transcripts predicted to encode secreted proteins or representing signaling and cell wall-related proteins, which may function by remodeling the extracellular matrix or as extracellular signaling molecules. A strict regulatory control in various metabolic pathways suggests that fine-tuning of the biochemical and physiological cellular environment is crucial for reproductive success. Our study provides a unique and detailed temporal and spatial gene expression profile of in vivo pollen-pistil interactions, providing a framework to better understand the basis of the molecular mechanisms operating during the reproductive process in higher plants. Fundação para a Ciência e a Tecnologia grants: (FCT–PTDC/QUI/64339/06, SFRH/BD/1128/2000).

Using the tip-growing pollen tube of Arabidopsis thaliana and Nicotiana tabacum as a model to investigate endocytosis mechanisms, we show that phosphatidylinositol-4-phosphate 5-kinase 6 (PIP5K6) regulates clathrin-dependent endocytosis in pollen tubes. Green fluorescent protein–tagged PIP5K6 was preferentially localized to the subapical plasma membrane (PM) in pollen tubes where it apparently converts phosphatidylinositol 4-phosphate (PI4P) to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. RNA interference–induced suppression of PIP5K6 expression impaired tip growth and inhibited clathrin-dependent endocytosis in pollen tubes. By contrast, PIP5K6 overexpression induced massive aggregation of the PM in pollen tube tips. This PM abnormality was apparently due to excessive clathrin-dependent membrane invagination because this defect was suppressed by the expression of a dominant-negative mutant of clathrin heavy chain. These results support a role for PI(4,5)P2 in promoting early stages of clathrin-dependent endocytosis (i.e., membrane invagination). Interestingly, the PIP5K6 overexpression-induced PM abnormality was partially suppressed not only by the overexpression of PLC2, which breaks down PI(4,5)P2, but also by that of PI4Kβ1, which increases the pool of PI4P. Based on these observations, we propose that a proper balance between PI4P and PI(4,5)P2 is required for clathrin-dependent endocytosis in the tip of pollen tubes.

Pollen tubes elongate within the pistil to transport sperm cells to the embryo sac for fertilization. Growth occurs exclusively at the tube apex, rendering pollen tube elongation a most dramatic polar cell growth process. A hallmark pollen tube feature is its cytoskeleton, which comprises elaborately organized and dynamic actin microfilaments and microtubules. Pollen tube growth is dependent on the actin cytoskeleton; its organization and regulation have been examined extensively by various approaches, including fluorescent protein labeled actin-binding proteins in live cell studies. Using the previously described GFP-NtADF1 and GFP-LlADF1, and a new actin reporter protein NtPLIM2b-GFP, we re-affirm that the predominant actin structures in elongating tobacco and lily pollen tubes are long, streaming actin cables along the pollen tube shank, and a subapical structure comprising shorter actin cables. The subapical collection of actin microfilaments undergoes dynamic changes, giving rise to the appearance of structures that range from basket- or funnel-shaped, mesh-like to a subtle ring. NtPLIM2b-GFP is used in combination with a guanine nucleotide exchange factor for the Rho GTPases, AtROP-GEF1, to illustrate the use of these actin reporter proteins to explore the linkage between the polar cell growth process and its actin cytoskeleton. Contrary to the actin cytoskeleton, microtubules appear not to play a direct role in supporting the polar cell growth process in angiosperm pollen tubes. Using a microtubule reporter protein based on the microtubule end-binding protein from Arabidopsis AtEB1, GFP-AtEB1, we show that the extensive microtubule network in elongating pollen tubes displays varying degrees of dynamics. These reporter proteins provide versatile tools to explore the functional connection between major structural and signaling components of the polar pollen tube growth process.

In order to cope with reproduction in a dry environment without any sort of motility, plants have developed a very specialized and unique sexual system. Of special notice, the two sperm cells that will perform the double fertilization typical of higher plants are carried by one of the fastest growing cells in nature, the pollen tube. This tube develops from the vegetative cell of the pollen grain upon germination on the female tissues. While it cannot be considered as a canonical excitable cell, pollen tubes depend for most of their fundamental functional features on a close regulation of ion dynamics, namely in terms of polarization of extracellular fluxes and formation of standing cytosolic free ion gradients, namely of calcium (Ca(2+)) and protons (H(+)). In turn, these imply that plasma membrane transporters are polarized, or polarly regulated, and that internal signaling cascades transduce this spatial information into the basic features of growth and morphogenesis needed for pollen tubes to target correctly the ovules and discharge the sperm cells. Because of the singularity of this organization, and the ease with which pollen tubes can be experimentally handled, recent years have witnessed an accumulation of data at many levels, from basic biophysical and cell biology characterization, to gene assignment and transcriptomic description of pollen development. In this review we aim to organize this information in terms of the basic biophysical features of membrane function and integrate it into conceptual testable hypotheses on how the dynamics of ion regulation may underlie fundamental properties of cell development.

International audience; The mutagenic activity of transposable elements (TEs) is suppressed by epigenetic silencing and small interfering RNAs (siRNAs), especially in gametes that could transmit transposed elements to the next generation. In pollen from the model plant Arabidopsis, we show that TEs are unexpectedly reactivated and transpose, but only in the pollen vegetative nucleus, which accompanies the sperm cells but does not provide DNA to the fertilized zygote. TE expression coincides with downregulation of the heterochromatin remodeler decrease in DNA methylation 1 and of many TE siRNAs. However, 21 nucleotide siRNAs from Athila retrotransposons are generated and accumulate in pollen and sperm, suggesting that siRNA from TEs activated in the vegetative nucleus can target silencing in gametes. We propose a conserved role for reprogramming in germline companion cells, such as nurse cells in insects and vegetative nuclei in plants, to reveal intact TEs in the genome and regulate their activity in gametes.

In flowering plants, the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part in fertilization are crucial goals in the study of plant reproduction. Studies of gene expression in male gametes of maize (Zea mays) and Plumbago and in lily (Lilium longiflorum) generative cells already showed that the previously held view of transcriptionally inert male gametes was not true, but genome-wide studies were lacking. Analyses in the model plant Arabidopsis (Arabidopsis thaliana) were hindered, because no method to isolate sperm cells was available. Here, we used fluorescence-activated cell sorting to isolate sperm cells from Arabidopsis, allowing GeneChip analysis of their transcriptome at a genome-wide level. Comparative analysis of the sperm cell transcriptome with those of representative sporophytic tissues and of pollen showed that sperm has a distinct and diverse transcriptional profile. Functional classifications of genes with enriched expression in sperm cells showed that DNA repair, ubiquitin-mediated proteolysis, and cell cycle progression are overrepresented Gene Ontology categories. Moreover, analysis of the small RNA and DNA methylation pathways suggests that distinct mechanisms might be involved in regulating the epigenetic state of the paternal genome. We identified numerous candidate genes whose involvement in sperm cell development and fertilization can now be directly tested in Arabidopsis. These results provide a roadmap to decipher the role of sperm-expressed proteins.

Polarized growth in pollen tubes results from exocytosis at the tip and is associated with conspicuous polarization of Ca2+, H+, K+, and Cl− -fluxes. Here, we show that cell polarity in Nicotiana tabacum pollen is associated with the exclusion of a novel pollen-specific H+-ATPase, Nt AHA, from the growing apex. Nt AHA colocalizes with extracellular H+ effluxes, which revert to influxes where Nt AHA is absent. Fluorescence recovery after photobleaching analysis showed that Nt AHA moves toward the apex of growing pollen tubes, suggesting that the major mechanism of insertion is not through apical exocytosis. Nt AHA mRNA is also excluded from the tip, suggesting a mechanism of polarization acting at the level of translation. Localized applications of the cation ionophore gramicidin A had no effect where Nt AHA was present but acidified the cytosol and induced reorientation of the pollen tube where Nt AHA was absent. Transgenic pollen overexpressing Nt AHA-GFP developed abnormal callose plugs accompanied by abnormal H+ flux profiles. Furthermore, there is no net flux of H+ in defined patches of membrane where callose plugs are to be formed. Taken together, our results suggest that proton dynamics may underlie basic mechanisms of polarity and spatial regulation in growing pollen tubes.

BACKGROUND: Pollen is the male gametophyte of higher plants. Upon pollination, it germinates and develops into a fast-growing cytoplasmic extension, the pollen tube, which ultimately delivers the sperm into the ovary. The biological relevance of its role, and the uniqueness of this kind of cellular organization, have made pollen the focus of many approaches, and it stands today as one of the best-known models in plant cell biology. In contrast, the genetic background of its development has been until recently largely unknown. Some genes involved have been described and a few functional mutants have been characterized, but only to a limited extent and allowing only a limited understanding of the regulatory mechanisms. Yet, being a relatively simple organ (2 or 3 cells), pollen stands as an excellent target for molecular-biology-based approaches. RECENT PROGRESS: Recent studies on Arabidopsis thaliana have characterized the transcriptional profile of pollen grains and microgametogenesis in comparison to sporophytic tissues. They underline the unique characteristics of pollen, not only in terms of a strongly reduced set of genes being expressed, but also in terms of the functions of the proteins encoded and the pathways they are involved in. These approaches have expanded the number of genes with known expression in pollen from a few hundred to nearly eight thousand. While for the first time allowing systems and/or gene-family approaches, this information also expands dramatically the possibility of hypothesis-driven experimentation based on specific gene function predictions. Recent studies reveal this to be the case in, for example, transcriptional regulation, cell-cycle progression and gene-silencing mechanisms in mature pollen.

Upon germination, pollen forms a tube that elongates dramatically through female tissues to reach and fertilize ovules. While essential for the life cycle of higher plants, the genetic basis underlying most of the process is not well understood. We previously used a combination of flow cytometry sorting of viable hydrated pollen grains and GeneChip array analysis of one-third of the Arabidopsis (Arabidopsis thaliana) genome to define a first overview of the pollen transcriptome. We now extend that study to approximately 80% of the genome of Arabidopsis by using Affymetrix Arabidopsis ATH1 arrays and perform comparative analysis of gene family and gene ontology representation in the transcriptome of pollen and vegetative tissues. Pollen grains have a smaller and overall unique transcriptome (6,587 genes expressed) with greater proportions of selectively expressed (11%) and enriched (26%) genes than any vegetative tissue. Relative gene ontology category representations in pollen and vegetative tissues reveal a functional skew of the pollen transcriptome toward signaling, vesicle transport, and the cytoskeleton, suggestive of a commitment to germination and tube growth. Cell cycle analysis reveals an accumulation of G2/M-associated factors that may play a role in the first mitotic division of the zygote. Despite the relative underrepresentation of transcription-associated transcripts, nonclassical MADS box genes emerge as a class with putative unique roles in pollen. The singularity of gene expression control in mature pollen grains is further highlighted by the apparent absence of small RNA pathway components.

The application of second harmonic generation (SHG) microscopy to plant materials has been neglected hitherto even though it would seem to have promise for identification and characterisation of biologically and commercially important plant polysaccharides. We have found that imaging of cellulose requires rather high laser powers which are above optimal values for live cell imaging. Starch, however is easily imaged by the technique at laser fluences compatible with extended cell viability. This also has useful applications in imaging plant-derived starchy food products. Lignin in plant cell walls shows a strong 3-photon excited fluorescence which may be enhanced by resonance effects.

Pollen tubes are a good model for the study of cell growth and morphogenesis because of their extreme elongation without cell division. Yet, knowledge about the genetic basis of pollen germination and tube growth is still lagging behind advances in pollen physiology and biochemistry. In an effort to reduce this gap, we have developed a new method to obtain highly purified, hydrated pollen grains of Arabidopsis through flowcytometric sorting, and we used GeneChips (Affymetrix, Santa Clara, CA; representing approximately 8,200 genes) to compare the transcriptional profile of sorted pollen with those of four vegetative tissues (seedlings, leaves, roots, and siliques). We present a new graphical tool allowing genomic scale visualization of the unique transcriptional profile of pollen. The 1,584 genes expressed in pollen showed a 90% overlap with genes expressed in these vegetative tissues, whereas one-third of the genes constitutively expressed in the vegetative tissues were not expressed in pollen. Among the 469 genes enriched in pollen, 162 were selectively expressed, and most of these had not been associated previously with pollen. Their functional classification reveals several new candidate genes, mainly in the categories of signal transduction and cell wall biosynthesis and regulation. Thus, the results presented improve our knowledge of the molecular mechanisms underlying pollen germination and tube growth and provide new directions for deciphering their genetic basis. Because pollen expresses about one-third of the number of genes expressed on average in other organs, it may constitute an ideal system to study fundamental mechanisms of cell biology and, by omission, of cell division.

The formation of nitrogen-fixing nodules in leguminous plants involves a subtle, two-way interaction between a bacterium and its host plant. The host plant produces signaling molecules, e.g. flavonoids, to which the bacterium responds, generating its own set of signaling molecules, the Nod factors.

Rhizobium Nod metabolites alone can trigger several plant responses. The dynamics of intracellular Ca2+ ions in Phaseolus vulgaris root hairs in the presence and absence of Nod factors have been studied by radiometric ion imaging, resulting in a rapid Ca2+ increase as well as changes around the vacuole and nuclear area in the presence of R. etli Nod factors (Cardenas et al. submitted).

Pollen tubes follow attractants secreted by the ovules. In a recent paper in BMC Plant Biology, Stewman and colleagues have quantified the parameters of this attraction and used them to calibrate a mathematical model that reproduces the process and enables predictions on the nature of the female attractant and the mechanisms of the male response. See research article: http://www.biomedcentral.com/1471-2229/10/32

Cytochemical detection of ATPase activity in the pollen grain (PG) and pollen tube (PT) of Agapanthus umbelatus showed that the enzymes concerned presented specific patterns of membrane distribution according to their ionic dependencies and to the timecourse of germination and tube growth. In the pollen tubes Ca2+-ATPases were mainly localized in mitochondria and ER membranes, while Mg2+-ATPases were found especially in the tonoplast and in the membrane of the P-particles. K+-ATPases showed a high activity at the plasma membrane. In the pollen grain similar patterns of ATPase activity were observed. The highest activity of all three types was observed at the plasma membrane of the grain and at the intine and inner exine layers of the cell wall. The activity observed in the pollen grain cell wall decreased with germination time. In vivo germination studies in the presence of specific inhibitors of the ATPases showed patterns of inhibition that could be correlated with the corresponding ATPase putative role.

The application of second-harmonic generation (SHG) microscopy to plant materials has been neglected hitherto even though it would seem to have promise for identification and characterization of biologically and commercially important plant polysaccharides. We find that imaging of cellulose requires rather high laser powers, which are above optimal values for live cell imaging. Starch, however, is easily imaged by the technique at laser fluences compatible with extended cell viability. This also has useful applications in imaging plant-derived starchy food products. Lignin in plant cell walls shows a strong three-photon excited fluorescence, which may be enhanced by resonance effects.