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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 ion-selective vibrating probe has been used to detect and quantify the magnitude and direction of transmembrane fluxes of several ions in a wide range of biological systems. Inherently non-invasive, vibrating probes have been essential to access relevant electrophysiological parameters related to apical growth and morphogenesis in pollen tubes, a highly specialized cell where spatiotemporal tuning of ion dynamics is fundamental. Of relevance, crucial processes to the cell physiology of pollen tubes associated with protons and anions have been elucidated using vibrating probes, allowing the identification of diverse molecular players underlying and regulating their extracellular fluxes. The use of Arabidopsis thaliana as a genetic model system posed new challenges given their relatively small dimensions and difficult manipulation in vitro. Here, we describe protocol optimizations that made the use of the ion-selective vibrating probe in Arabidopsis pollen tubes feasible, ensuring consistent and reproducible data. Quantitative methods like this enabled characterizing phenotypes of ion transporter mutants, which are not directly detectable by evident morphological and reproductive defects, providing valuable insights into molecular and cellular mechanisms. The protocol for quantifying extracellular proton and anionic fluxes detailed here can be adjusted to other systems and species, while the sample preparation can be applied to correlated techniques, facilitating the research of pollen tube growth and development.

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.

Multiple, diverse, and complex Calcium currents characterize the developing pollen tube in the small mustard plant Arabidopsis and correlate with growth at the tip of the pollen tube. This system constitutes a practical model for screening for Ca 2+ -signaling mechanisms in plants. Wudick et al. analyzed multiple variants of glutamate receptor–like (GLR) channels and discovered that some work alone and others work in pairs or trios. Subcellular localization of GLRs is a complex response to CORNICHON sorting proteins, which leave some GLRs at the plasma membrane and ferry others to internal calcium reservoirs. The calcium current at the tip of the growing pollen tube apparently integrates multiple intracellular currents. Science , this issue p. 533

Glutamate receptor-like channels (GLRs) play vital roles in various physiological processes in plants, such as wound response, stomatal aperture control, seed germination, root development, innate immune response, pollen tube growth, and morphogenesis. Despite the importance of GLRs, knowledge about their molecular organization is limited. Here we employ X-ray crystallography and single-particle cryo-EM to solve structures of the Arabidopsis thaliana GLR3.4. Our structures reveal the tetrameric assembly of GLR3.4 subunits into a three-layer domain architecture, reminiscent of animal ionotropic glutamate receptors (iGluRs). However, the non-swapped arrangement between layers of GLR3.4 domains, binding of glutathione through S-glutathionylation of cysteine C205 inside the amino-terminal domain clamshell, unique symmetry, inter-domain interfaces, and ligand specificity distinguish GLR3.4 from representatives of the iGluR family and suggest distinct features of the GLR gating mechanism. Our work elaborates on the principles of GLR architecture and symmetry and provides a molecular template for deciphering GLR-dependent signaling mechanisms in plants.

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.

Glutamate receptor-like channels (GLRs) play important roles in numerous plant physiological processes. GLRs are homologous to ionotropic glutamate receptors (iGluRs) that mediate neurotransmission in vertebrates. Here we determine crystal structures of Arabidopsis thaliana GLR3.2 ligand-binding domain (LBD) in complex with glycine and methionine to 1.58- and 1.75-Å resolution, respectively. Our structures show a fold similar to that of iGluRs, but with several secondary structure elements either missing or different. The closed clamshell conformation of GLR3.2 LBD suggests that both glycine and methionine act as agonists. The mutation R133A strongly increases the constitutive activity of the channel, suggesting that the LBD mutated at the residue critical for agonist binding produces a more stable closed clamshell conformation. Furthermore, our structures explain the promiscuity of GLR activation by different amino acids, confirm evolutionary conservation of structure between GLRs and iGluRs, and predict common molecular principles of their gating mechanisms driven by bilobed clamshell-like LBDs.

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.

We investigated the molecular basis and physiological implications of anion transport during pollen tube (PT) growth in Arabidopsis thaliana (Col-0). Patch-clamp whole-cell configuration analysis of pollen grain protoplasts revealed three subpopulations of anionic currents differentially regulated by cytoplasmic calcium ([Ca2+ ]cyt ). We investigated the pollen-expressed proteins AtSLAH3, AtALMT12, AtTMEM16 and AtCCC as the putative anion transporters responsible for these currents. AtCCC-GFP was observed at the shank and AtSLAH3-GFP at the tip and shank of the PT plasma membrane. Both are likely to carry the majority of anion current at negative potentials, as extracellular anionic fluxes measured at the tip of PTs with an anion vibrating probe were significantly lower in slah3-/- and ccc-/- mutants, but unaffected in almt12-/- and tmem16-/- . We further characterised the effect of pH and GABA by patch clamp. Strong regulation by extracellular pH was observed in the wild-type, but not in tmem16-/- . Our results are compatible with AtTMEM16 functioning as an anion/H+ cotransporter and therefore, as a putative pH sensor. GABA presence: (1) inhibited the overall currents, an effect that is abrogated in the almt12-/- and (2) reduced the current in AtALMT12 transfected COS-7 cells, strongly suggesting the direct interaction of GABA with AtALMT12. Our data show that AtSLAH3 and AtCCC activity is sufficient to explain the major component of extracellular anion fluxes, and unveils a possible regulatory system linking PT growth modulation by pH, GABA, and [Ca2+ ]cyt through anionic transporters. info:eu-repo/semantics/publishedVersion

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

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.

Mycorrhization is a typical example of a host-pathogen symbiotic interaction where the pathogen cell biology and the host immune response coevolved several functional links. Here, the role played by ion fluxes across the root concerning nutrient uptake, osmoregulation, growth and signaling events is addressed. An ion-selective vibrating probe system was used to determine the net fluxes of protons (H(+)), calcium (Ca(2+)) and anions (A(-)) along nonmycorrhizal and ectomycorrhizal (ECM) roots of Eucalyptus globulus colonized by Pisolithus sp. These data show that, from five root zones analyzed, the main effect of fungal colonization was localized to the elongation zone. Here, strong changes in ion dynamics and rhizosphere acidification capacity were observed. Additionally, ion fluxes exhibited periodic fluctuations. To verify whether these fluctuations corresponded to sustained oscillations, continuous wavelet time spectrum analysis was applied and it was determined that H(+) and A(-) fluxes from ECM roots had longer periods than nonmycorrhizal roots. By contrast, Ca(2+) oscillations were completely abolished following fungal interaction. These results are interpreted in the light of a working model in which nutrient uptake and stimulation of growth are mediated by ECM fungi and may be pH-dependent. Furthermore, the variations detected in ECM roots for H(+) and A(-) fluxes suggest a main contribution from the plant, while the results obtained for Ca(2+) point to a significant involvement of the fungus.

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.

Higher plants have evolved to be one of the predominant life forms on this planet. A great deal of this evolutionary success relies in a very short gametophytic phase which underlies the sexual reproduction cycle. Sexual plant reproduction takes place in special organs of the flower. In most species the processes of gametogenesis, pollination, syngamy and embryogenesis are sequentially coordinated to give rise to a functional seed in a matter of few weeks. Any of these processes is so intricately complex and precisely regulated that it becomes no wonder that each involves more specific genes and cellular processes than any other function in the plant life cycle. While variability generation - the evolutionary output of the sexual cycle - is the same as in any other Kingdom, plants do it using a completely original set of mechanisms, many of which are not yet comprehended. In this paper, we cover the fundamental features of male and female gametogenesis. While the physiological and cellular bases of these processes have been continuously described since the early nineteen century, recent usage of Arabidopsis and other species as central models has brought about a great deal of specific information regarding their genetic regulation. Transcriptomics has recently enlarged the repertoire and pollen became the first gametophyte to have a fully described transcriptome in plants. We thus place special emphasis on the way this newly accumulated genetic and transcriptional information impacts our current understanding of the mechanisms of gametogenesis.

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.

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.

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).

Patricio A. da Rocha*, Jose A. Feijo, Jefferson S. Mikalauskas and Stephen F. Ferrari 1 Postgraduate Programme in Zoology, Universidade Federal da Paraiba, Campus I, Departamento de Sistematica e Ecologia, 58.059-900 Joao Pessoa, Paraiba, Brazil, e-mail: parocha2@yahoo.com.br 2 Mammalian Zoology Laboratory, Institute of Biology, Universidade Federal Rural do Rio de Janeiro, Seropedica, Rio de Janeiro, Brazil 3 Department of Biology, Universidade Federal de Sergipe, Sao Cristovao, Sergipe, Brazil

Arbuscular mycorrhiza (AM), a type of plant-fungal endosymbiosis, and nodulation, a bacterial-plant endosymbiosis, are the most ubiquitous symbioses on earth. Recent findings have established part of a shared genetic basis underlying these interactions. Here, we approach root endosymbioses through the lens of the homology and modularity concepts aiming at further clarifying the proximate and ultimate causes for the establishment of these biological systems. We review the genetics that underlie interspecific signaling and its concomitant shift in genetic programs for either partner. Also, through the comparative analysis of genetic modules shared by AM and nodulation symbioses, we identify fundamental nodes in these networks, suggesting the elemental steps that may have permitted symbiotic adaptation. Here, we show that this approach, allied to recent technical advances in the study of genetic systems architecture, can provide clear testable hypotheses for the advancement of our understanding on the evolution and development of symbiotic systems.

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.

Environmental and developmental signals can elicit differential activation of membrane proton (H+) fluxes as one of the primary responses of plant and fungal cells. In recent work,1 we could determine that during the presymbiotic growth of arbuscular mycorrhizal (AM) fungi specific domains of H+ flux are activated by clover root factors, namely host root exudates or whole root system. Consequently, activation on hyphal growth and branching were observed and the role of plasma membrane H+-ATPase was investigated. The specific inhibitors differentially abolished most of hyphal H+ effluxes and fungal growth. As this enzyme can act in signal transduction pathways, we believe that spatial and temporal oscillations of the hyphal H+ fluxes could represent a pH signature for both early events of the AM symbiosis and fungal ontogeny. Addendum to: Ramos AC, Facanha AR, Feijo JA. Proton (H+) flux signature for the presymbiotic development of the arbuscular mycorrhizal fungi. New Phytol 2008; 178:177-88.

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.

New molecules, including protein kinases, lipids and molecules that have neurotransmitter activities in animals have emerged as important players in tip-growing cells. Transcriptomics analysis reveals that the largest single class of genes expressed in pollen tubes encode signal transducers, reflecting the necessity to decode complex and diverse pathways that are associated with tip growth. Many of these pathways may use common intracellular second messengers, with ions and reactive oxygen species emerging as two major common denominators in many of the processes involved in tip growth. These second messengers might influence the actin cytoskeleton through known interactions with actin-binding proteins. In turn, changes in the dynamic properties of the cytoskeleton would define the basic polarity events needed to shape and modify tip-growing cells.

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.

Immobilized cultured tobacco cells become polarized upon the addition of naphthalene-1-acetic acid and start to elongate from an initial spherical shape. The question as to how a diffuse-growing cell forms a polar axis is addressed here with approaches successfully applied to the study of tip growth. With two kinds of vibrating probes the electric current flow and proton fluxes were mapped around such elongating cells. No consistent polar pattern of ion fluxes, which is typical for actively tip-growing cells, was detected. Therefore, other signals must provide the positional information needed for polar axis formation. Furthermore, neither a specific pattern of intracellular Ca(2+) concentration nor a polar distribution of putative ion-channel antagonist-binding sites were found in elongating tobacco cells. Auxin flux, on the other hand, was found to be important as TIBA, an inhibitor of polar auxin transport, clearly inhibited elongation in a concentration-dependent way. Cross-linking of arabinogalactan-proteins with the beta-Yariv reagent also resulted in inhibition of elongation. A model is proposed for the induction of polar growth where localized auxin efflux starts a signal cascade that triggers molecules that reorient microtubules. These then guide cellulose deposition in the cell wall, which in turn alters cell wall mechanics and leads to elongation. In this scheme, arabinogalactan-proteins are not causal agents but are probably important regulators of growth and survival of the cell.

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).

Research in sexual plant reproduction has been boosted by the concentration of means and minds around central issues, such as incompatibility, microspore development or, to a less extent, the progamic phase of fertilisation. These areas are now pivotal since they involve basic biological questions still unanswered and which became recently accessible due to powerful genetic and molecular techniques. While these combined efforts resulted in the outcome of major findings that projected the field to a place not easy to by-pass even in the general context of Biology, this was only possible because of the existence of a few model species, particularly adapted to the experimental needs of the molecular weaponry. Most notably, Arabidopsis now plays a central role, since it possesses most of the development paths that characterise the majority of Angiosperms (with the exception of secondary growth). While this approach is not questionable, since it permits a faster advance in models and concepts, the reductionism underlying the focus on a few model species may create a void of knowledge on the natural variability and diversity of the cellular and molecular mechanisms. Most likely, the majority of the flowering species will never find their place as models species since they cannot compete as regards easiness to grow, maintain and analyse. Yet, an historical perspective shows beyond doubt that some of these species may bear variations to the basic models which may open new avenues and question established paradigms. Such was the case of Arabidopsis, for instance, often neglected and rejected for funding because of its condition of weed (Goldberg 1996).

In the first stages of root nodule morphogenesis, there is evidence that the actin cytoskeleton participates in the formation of preinfection threads and induction of cortical cell divisions (Bakhuizen, 1988). It has been reported that actin becomes fragmented in curled root hairs fromViciae hirsuta when infected with Rhizobium (Ridge et al. 1992). Although Allen et al., (1994) have reported that in curled root hairs actin foci were observed close to the tip, when exposed to Nod factors, the question of how actin cytoskeleton responded to Nod factors, during the first minutes of exposure, remains unanswered. We are interested in analyzing this response using as a model, the symbiosis P. vulgaris-R. etli. On the other hand, it is a known fact that Ca2+ is indispensable for the infection process as suggested by the use of aureomycin. The increased fluorescence normally found at the root hair tip due to the presence of Ca2+ has suggested its possible role in the formation and growth of the infection thread (Sethi and Reporter 1981). Recent studies have explored ion movement as an indicator of root hair activity related to signal perception. It has been found that alfalfa root hairs displayed a depolarization of the cytoplasmic membrane in response to R. meliloti Nod factors (Ehrhardt et al., 1992; Felle et al., 1995; Kurkdjian, 1995). Albeit the ionic basis for this behaviour is unknown, there is some evidence that variable currents of H+ and Ca++ ions, are present outside root hairs from alfalfa (legume forming indeterminate nodules) which had been exposed to Nod factors (Allen et al., 1994). In alfalfa root hair cells, a calcium spiking response around the nucleus, that initiated 10 min after Nod factor exposure and remained up to 60 min has been reported (Ehrhardt et al., (1996). To elucidate the calcium response in root hair cells from a legume forming determinate nodules, P. vulgaris, we isolated and purified Nod factors from R. etli (Cardenas et al., 1995), and studied their effect on intracellular and extracellular Ca2+ current influxes in this legume. We used two approaches, dextran-linked calcium selective dyes injected directly into P. vulgaris root hairs, and an ion selective vibrating probe.

We present data on the geographic distribution, morphology, and biology of the Brazilian funnel-eared bat, Natalus (Gervais, 1856), with new records for the Brazilian state of Sergipe, filling a gap of approximately 800 km in the distribution of the species in Brazilian Northeast.

This study provides the first record of Uroderma magnirostrum Davis, 1968 from the state of Sergipe in the Brazilian northeast, based on the capture of two specimens, one male and one female. The morphometric data and morphological characters were consistent with those recorded for the species at other Brazilian sites. This record extends the distributional range of the species within South America approximately 220 km eastwards.

Two specimens of bats deposited in the mammal collection of Universidade Federal da Paraíba have been found: one young female of Artibeus planirostris and one adult male of Trachops cirrhosus. These reports represent the first record of these species for the state of Sergipe, northeastern Brazil.

SUMMARYThe ultrastructural study of microsporogenesis in Ophrys lutea revealed that the formation of cytomictic channels in the callose wall was followed by the transfer of organelles and chromatin (cytomixis) through them. The transfer of chromatin was first observed on zygo-pachytene, and throughout the other stages of prophase I. Even though restricted to some nuclear areas, cytomixis was observed at different stages of chromatin condensation. Some abnormalities were detected as a consequence of cytomixis. The significance of cytomixis as a possible meiotic event is discussed.

International audience; BACKGROUND: Current views on the control of cell development are anchored on the notion that phenotypes are defined by networks of transcriptional activity. The large amounts of information brought about by transcriptomics should allow the definition of these networks through the analysis of cell-specific transcriptional signatures. Here we test this principle by applying an analogue to comparative anatomy at the cellular level, searching for conserved transcriptional signatures, or conserved small gene-regulatory networks (GRNs) on root hairs (RH) and pollen tubes (PT), two filamentous apical growing cells that are a striking example of conservation of structure and function in plants. RESULTS: We developed a new method for isolation of growing and mature root hair cells, analysed their transcriptome by microarray analysis, and further compared it with pollen and other single cell transcriptomics data. Principal component analysis shows a statistical relation between the datasets of RHs and PTs which is suggestive of a common transcriptional profile pattern for the apical growing cells in a plant, with overlapping profiles and clear similarities at the level of small GTPases, vesicle-mediated transport and various specific metabolic responses. Furthermore, cis-regulatory element analysis of co-regulated genes between RHs and PTs revealed conserved binding sequences that are likely required for the expression of genes comprising the apical signature. This included a significant occurrence of motifs associated to a defined transcriptional response upon anaerobiosis. CONCLUSIONS: Our results suggest that maintaining apical growth mechanisms synchronized with energy yielding might require a combinatorial network of transcriptional regulation. We propose that this study should constitute the foundation for further genetic and physiological dissection of the mechanisms underlying apical growth of plant cells.