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Flowering plants have evolved numerous intraspecific and interspecific prezygotic reproductive barriers to prevent production of unfavourable offspring1. Within a species, self-incompatibility (SI) is a widely utilized mechanism that rejects self-pollen2,3 to avoid inbreeding depression. Interspecific barriers restrain breeding between species and often follow the SI × self-compatible (SC) rule, that is, interspecific pollen is unilaterally incompatible (UI) on SI pistils but unilaterally compatible (UC) on SC pistils1,4–6. The molecular mechanisms underlying SI, UI, SC and UC and their interconnections in the Brassicaceae remain unclear. Here we demonstrate that the SI pollen determinant S-locus cysteine-rich protein/S-locus protein 11 (SCR/SP11)2,3 or a signal from UI pollen binds to the SI female determinant S-locus receptor kinase (SRK)2,3, recruits FERONIA (FER)7–9 and activates FER-mediated reactive oxygen species production in SI stigmas10,11 to reject incompatible pollen. For compatible responses, diverged pollen coat protein B-class12–14 from SC and UC pollen differentially trigger nitric oxide, nitrosate FER to suppress reactive oxygen species in SC stigmas to facilitate pollen growth in an intraspecies-preferential manner, maintaining species integrity. Our results show that SRK and FER integrate mechanisms underlying intraspecific and interspecific barriers and offer paths to achieve distant breeding in Brassicaceae crops.

Fertilization of an egg by multiple sperm (polyspermy) leads to lethal genome imbalance and chromosome segregation defects. In Arabidopsis thaliana , the block to polyspermy is facilitated by a mechanism that prevents polytubey (the arrival of multiple pollen tubes to one ovule). We show here that FERONIA, ANJEA, and HERCULES RECEPTOR KINASE 1 receptor-like kinases located at the septum interact with pollen tube–specific RALF6, 7, 16, 36, and 37 peptide ligands to establish this polytubey block. The same combination of RALF (rapid alkalinization factor) peptides and receptor complexes controls pollen tube reception and rupture inside the targeted ovule. Pollen tube rupture releases the polytubey block at the septum, which allows the emergence of secondary pollen tubes upon fertilization failure. Thus, orchestrated steps in the fertilization process in Arabidopsis are coordinated by the same signaling components to guarantee and optimize reproductive success.

Fertilization of an egg by multiple sperm (polyspermy) leads to lethal genome imbalance and chromosome segregation defects. In Arabidopsis thaliana, the block to polyspermy is facilitated by a mechanism that prevents polytubey (the arrival of multiple pollen tubes to one ovule). We show here that FERONIA, ANJEA, and HERCULES RECEPTOR KINASE 1 receptor-like kinases located at the septum interact with pollen tube–specific RALF6, 7, 16, 36, and 37 peptide ligands to establish this polytubey block. The same combination of RALF (rapid alkalinization factor) peptides and receptor complexes controls pollen tube reception and rupture inside the targeted ovule. Pollen tube rupture releases the polytubey block at the septum, which allows the emergence of secondary pollen tubes upon fertilization failure. Thus, orchestrated steps in the fertilization process in Arabidopsis are coordinated by the same signaling components to guarantee and optimize reproductive success.

Competing signal peptides hold the key When a pollen grain lands on a receptive flower's pistil, a complex dance leading to sexual reproduction begins. Liu et al. show some of the early steps that help to distinguish a compatible pollen grain from a random piece of dust. Normally, a stigmatic gatekeeper, the ANJEA–FERONIA receptor kinase complex, perceives signaling peptides produced by the stigma that drive the production of reactive oxygen species at the stigma papillae. Upon pollination, POLLEN COAT PROTEIN B-class peptides compete with those stigmatic peptides for binding to the stigmatic receptor kinase complex. The subsequent decline of stigmatic reactive oxygen species production allows hydration and opens the gates to pollen germination. Science , this issue p. 171

Explosive advances have been made in the molecular understanding of pollen-pistil interactions that underlie reproductive success in flowering plants in the past three decades. Among the most notable is the discovery of pollen tube attractants [1∗,2∗]. The roles these molecules play in facilitating conspecific precedence thus promoting interspecific genetic isolation are also emerging [3-5]. Male-female interactions during the prezygotic phase and contributions from the male and female gametophytes have been comprehensively reviewed recently. Here, we focus on key advances in understanding the mechanistic underpinnings of how these interactions overcome barriers at various pollen-pistil interfaces along the pollen tube growth pathway to facilitate fertilization by desirable mates.

Cell walls are at the front line of interactions between walled-organisms and their environment. They support cell expansion, ensure cell integrity and, for multicellular organisms such as plants, they provide cell adherence, support cell shape morphogenesis and mediate cell-cell communication. Wall-sensing, detecting perturbations in the wall and signaling the cell to respond accordingly, is crucial for growth and survival. In recent years, plant signaling research has suggested that a large family of receptor-like kinases (RLKs) could function as wall sensors partly because their extracellular domains show homology with malectin, a diglucose binding protein from the endoplasmic reticulum of animal cells. Studies of several malectin/malectin-like (M/ML) domain-containing RLKs (M/MLD-RLKs) from the model plant Arabidopsis thaliana have revealed an impressive array of biological roles, controlling growth, reproduction and stress responses, processes that in various ways rely on or affect the cell wall. Malectin homologous sequences are widespread across biological kingdoms, but plants have uniquely evolved a highly expanded family of proteins with ML domains embedded within various protein contexts. Here, we present an overview on proteins with malectin homologous sequences in different kingdoms, discuss the chromosomal organization of Arabidopsis M/MLD-RLKs and the phylogenetic relationship between these proteins from several model and crop species. We also discuss briefly the molecular networks that enable the diverse biological roles served by M/MLD-RLKs studied thus far.

Self-incompatibility (SI) is a genetic mechanism flowering plants adopted to reject self-pollen and promote outcrossing. In the Brassicaceae family plants, the stigma tissue plays a key role in self-pollen recognition and rejection. We reported earlier in Chinese cabbage (Brassica rapa) that stigma tissue showed upregulated ethylene responses and programmed cell death (PCD) upon compatible pollination, but not in SI responses. Here, we show that SI is significantly compromised or completely lost in senescent flowers and young flowers of senescent plants. Senescence upregulates senescence-associated genes in B. rapa. Suppressing their expression in young stigmas by antisense oligodeoxyribonucleotide abolishes compatible pollination-triggered PCD and inhibits the growth of compatible pollen tubes. Furthermore, ethylene biosynthesis genes and response genes are upregulated in senescent stigmas, and increasing the level of ethylene or inhibiting its response increases or decreases the expression of senescence-associated genes, respectively. Our results show that senescence causes PCD in stigmatic papilla cells and is associated with the breakdown of SI in Chinese cabbage and in radish.

Timing a switch in tissue integrity In plants, sperm cells travel through the pollen tube as it grows toward the ovule. Successful fertilization depends on the pollen tube rupturing to release the sperm cells (see the Perspective by Stegmann and Zipfel). Ge et al. and Mecchia et al. elucidated the intercellular cross-talk that maintains pollen tube integrity during growth but destroys it at just the right moment. The signaling peptides RALF4 and RALF19, derived from the pollen tube, maintain its integrity as it grows. Once in reach of the ovule, a related signaling peptide, RALF34, which derives from female tissues, takes over and causes rupture of the pollen tube. Science , this issue p. 1596 , p. 1600 ; see also p. 1544

Summary Most plants in the Brassicaceae evolve self-incompatibility (SI) to avoid inbreeding and generate hybrid vigor. Self-pollen is recognized by the S-haplotype-specific interaction of the pollen ligand S-locus protein 11 (SP11) (also known as S-locus cysteine-rich protein [SCR]) and its stigma-specific S-locus receptor kinase (SRK). However, mechanistically much remains unknown about the signaling events that culminate in self-pollen rejection. Here, we show that self-pollen triggers high levels of reactive oxygen species (ROS) in stigma papilla cells to mediate SI in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis). We found that stigmatic ROS increased after self-pollination but decreased after compatible(CP)- pollination. Reducing stigmatic ROS by scavengers or suppressing the expression of respiratory burst oxidase homologs (Rbohs), which encode plant NADPH oxidases that produce ROS, both broke down SI. On the other hand, increasing the level of ROS inhibited the germination and penetration of compatible pollen on the stigma, mimicking an incompatible response. Furthermore, suppressing a B. rapa FERONIA (FER) receptor kinase homolog or Rac/Rop guanosine triphosphatase (GTPase) signaling effectively reduced stigmatic ROS and interfered with SI. Our results suggest that FER-Rac/Rop signaling-regulated, NADPH oxidase-produced ROS is an essential SI response leading to self-pollen rejection.

In angiosperms, two sperm cells are transported and delivered by the pollen tube to the ovule to achieve double fertilization. Extensive communication takes place between the pollen tube and the female tissues until the sperm cell cargo is ultimately released. During this process, a pollen tube surface-located receptor complex composed of ANXUR1/2 (ANX1/2) and Buddha's Paper Seal 1/2 (BUPS1/2) was reported to control the maintenance of pollen tube integrity by perceiving the autocrine peptide ligands rapid alkalinization factor 4 and 19 (RALF4/19). It was further hypothesized that pollen-tube rupture to release sperm is caused by the paracrine RALF34 peptide from the ovule interfering with this signaling pathway. In this study, we identified two Arabidopsis pollen-tube-expressed glycosylphosphatidylinositol-anchored proteins (GPI-APs), LORELEI-like-GPI-anchored protein 2 (LLG2) and LLG3, as co-receptors in the BUPS-ANX receptor complex. llg2 llg3 double mutants exhibit severe fertility defects. Mutant pollen tubes rupture early during the pollination process. Furthermore, LLG2 and LLG3 interact with ectodomains of both BUPSs and ANXURs, and this interaction is remarkably enhanced by the presence of RALF4/19 peptides. We further demonstrate that the N terminus (including a YISY motif) of the RALF4 peptide ligand interacts strongly with BUPS-ANX receptors but weakly with LLGs and is essential for its biological function, and its C-terminal region is sufficient for LLG binding. In conclusion, we propose that LLG2/3 serve as co-receptors during BUPS/ANX-RALF signaling and thereby further establish the importance of GPI-APs as key regulators in plant reproduction processes.

Species that propagate by sexual reproduction actively guard against the fertilization of an egg by multiple sperm (polyspermy). Flowering plants rely on pollen tubes to transport their immotile sperm to fertilize the female gametophytes inside ovules. In Arabidopsis, pollen tubes are guided by cysteine-rich chemoattractants to target the female gametophyte1,2. The FERONIA receptor kinase has a dual role in ensuring sperm delivery and blocking polyspermy3. It has previously been reported that FERONIA generates a female gametophyte environment that is required for sperm release4. Here we show that FERONIA controls several functionally linked conditions to prevent the penetration of female gametophytes by multiple pollen tubes in Arabidopsis. We demonstrate that FERONIA is crucial for maintaining de-esterified pectin at the filiform apparatus, a region of the cell wall at the entrance to the female gametophyte. Pollen tube arrival at the ovule triggers the accumulation of nitric oxide at the filiform apparatus in a process that is dependent on FERONIA and mediated by de-esterified pectin. Nitric oxide nitrosates both precursor and mature forms of the chemoattractant LURE11, respectively blocking its secretion and interaction with its receptor, to suppress pollen tube attraction. Our results elucidate a mechanism controlled by FERONIA in which the arrival of the first pollen tube alters ovular conditions to disengage pollen tube attraction and prevent the approach and penetration of the female gametophyte by late-arriving pollen tubes, thus averting polyspermy.

SUMMARY Cells maintain integrity despite changes in their mechanical properties elicited during growth and environmental stress. How cells sense their physical state and compensate for cell-wall damage is poorly understood, particularly in plants. Here we report that FERONIA (FER), a plasma-membrane-localized receptor kinase from Arabidopsis, is necessary for the recovery of root growth after exposure to high salinity, a widespread soil stress. The extracellular domain of FER displays tandem regions of homology with malectin, an animal protein known to bind diglucose in vitro and important for protein quality control in the endoplasmic reticulum. The presence of malectin-like domains in FER and related receptor kinases has led to widespread speculation that they interact with cell-wall polysaccharides and can potentially serve a wall-sensing function. Results reported here show that salinity causes softening of the cell wall and that FER is necessary to sense these defects. When this function is disrupted in the fer mutant, root cells explode dramatically during growth recovery. Similar defects are observed in the mur1 mutant, which disrupts pectin cross-linking. Furthermore, fer cell-wall integrity defects can be rescued by treatment with calcium and borate, which also facilitate pectin cross-linking. Sensing of these salinity-induced wall defects might therefore be a direct consequence of physical interaction between the extracellular domain of FER and pectin. FER-dependent signaling elicits cell-specific calcium transients that maintain cell-wall integrity during salt stress. These results reveal a novel extracellular toxicity of salinity, and identify FER as a sensor of damage to the pectin-associated wall., In Brief For plant cells, growth requires maintenance of cell-wall integrity. Feng et al. show that salinity weakens the cell wall, which triggers FER-mediated calcium signaling to prevent root cells from bursting during growth under salt stress. The extracellular domain of FER physically interacts with pectin, indicating a potential sensing mechanism.

In flowering plants, sperm-containing pollen tubes are guided towards ovules by attractants from the female reproductive organ. Receptors for the attractant molecule AtLURE1 have now been found. See Letters p.241 & p.245 In flowering plants, the female gametophyte secretes chemoattractant peptides to guide pollen tube growth so that it delivers the immobile sperm to the ovule-enclosed female gametophyte. Two papers published in this issue of Nature report the identification of male pollen tube cell-surface receptors for one of these female attractants, LURE1, in the model plant Arabidopsis thaliana. Wei-Cai Yang and colleagues show that LURE1 is perceived by a receptor-like kinase complex, the heteromer MDIS1–MIK. Tetsuya Higashiyama and Hidenori Takeuchi report that pollen-specific receptor-like kinase 6 (PRK6) is required for sensing LURE1, and PRK6 acts together with other PRK family receptors. Both groups demonstrate that by engineering pollen tubes of the sister species Capsella rubella to express a component of the A. thaliana receptors — either MDIS1 or PRK6 — the reproductive isolation barrier between the two species is partially broken down.

Polar auxin transport, facilitated by the combined activities of auxin influx and efflux carriers to maintain asymmetric auxin distribution, is essential for plant growth and development. Here, we show that Arabidopsis (Arabidopsis thaliana) RopGEF1, a guanine nucleotide exchange factor and activator of Rho GTPases of plants (ROPs), is critically involved in polar distribution of auxin influx carrier AUX1 and differential accumulation of efflux carriers PIN7 and PIN2 and is important for embryo and early seedling development when RopGEF1 is prevalently expressed. Knockdown or knockout of RopGEF1 induces embryo defects, cotyledon vein breaks, and delayed root gravity responses. Altered expression from the auxin response reporter DR5rev:GFP in the root pole of RopGEF1-deficient embryos and loss of asymmetric distribution of DR5rev:GFP in their gravistimulated root tips suggest that auxin distribution is affected in ropgef1 mutants. This is reflected by the polarity of AUX1 being altered in ropgef1 embryos and roots, shifting from the normal apical membrane location to a basal location in embryo central vascular and root protophloem cells and also reduced PIN7 accumulation at embryos and altered PIN2 distribution in gravistimulated roots of mutant seedlings. In establishing that RopGEF1 is critical for AUX1 localization and PIN differential accumulation, our results reveal a role for RopGEF1 in cell polarity and polar auxin transport whereby it imapcts auxin-mediated plant growth and development.

In Solanaceae, the S-specific interaction between the pistil S-RNase and the pollen S-Locus F-box protein controls self-incompatibility (SI). Although this interaction defines the specificity of the pollen rejection response, the identification of three pistil essential modifier genes unlinked to the S-locus (HT-B, 120K, and NaStEP) unveils a higher degree of complexity in the pollen rejection pathway. We showed previously that NaStEP, a stigma protein with homology with Kunitz-type protease inhibitors, is essential to SI in Nicotiana spp. During pollination, NaStEP is taken up by pollen tubes, where potential interactions with pollen tube proteins might underlie its function. Here, we identified NaSIPP, a mitochondrial protein with phosphate transporter activity, as a novel NaStEP-interacting protein. Coexpression of NaStEP and NaSIPP in pollen tubes showed interaction in the mitochondria, although when expressed alone, NaStEP remains mostly cytosolic, implicating NaSIPP-mediated translocation of NaStEP into the organelle. The NaSIPP transcript is detected specifically in mature pollen of Nicotiana spp.; however, in self-compatible plants, this gene has accumulated mutations, so its coding region is unlikely to produce a functional protein. RNA interference suppression of NaSIPP in Nicotiana spp. pollen grains disrupts the SI by preventing pollen tube inhibition. Taken together, our results are consistent with a model whereby the NaStEP and NaSIPP interaction, in incompatible pollen tubes, might destabilize the mitochondria and contribute to arrest pollen tube growth.

Current research into the FERONIA family of receptor kinases highlights both questions and opportunities for understanding signaling strategies in plant growth and survival.

Auxin functions as a key morphogen in regulating plant growth and development. Studies on auxin-regulated gene expression and on the mechanism of polar auxin transport and its asymmetric distribution within tissues have provided the basis for realizing the molecular mechanisms underlying auxin function. In eukaryotes, members of the Ras and Rho subfamilies of the Ras superfamily of small GTPases function as molecular switches in many signaling cascades that regulate growth and development. Plants do not have Ras proteins, but they contain Rho-like small G proteins called RACs or ROPs that, like fungal and metazoan Rhos, are regulators of cell polarity and may also undertake some Ras functions. Here, we discuss the advances made over the last decade that implicate RAC/ROPs as mediators for auxin-regulated gene expression, rapid cell surface-located auxin signaling, and directional auxin transport. We also describe experimental data indicating that auxin–RAC/ROP crosstalk may form regulatory feedback loops and theoretical modeling that attempts to connect local auxin gradients with RAC/ROP regulation of cell polarity. We hope that by discussing these experimental and modeling studies, this perspective will stimulate efforts to further refine our understanding of auxin signaling via the RAC/ROP molecular switch.

Plant RHO GTPases (RAC/ROPs) mediate multiple extracellular signals ranging from hormone to stress and regulate diverse cellular processes important for polarized cell growth, differentiation, development, reproduction, and responses to the environment. They shuttle between the GDP-bound inactive state and the GTP-bound activated state and their activation is predominantly mediated by a family of guanine nucleotide exchange factors (GEFs) referred to as ROPGEFs. Using the Arabidopsis ROPGEF1 as bait, we identified members of a receptor-like kinase (RLK) family as potential upstream regulators for RAC/ROP signaling. NADPH oxidase-derived reactive oxygen species (ROS) are emerging as important regulators for growth and development and play a crucial role in mediating RAC/ROP-regulated root hair development, a polarized cell growth process. We therefore screened T-DNA insertion mutants in these RLKs for root hair defects and found that mutations in one of them, At3g51550 encoding the FERONIA (FER) receptor-like kinase, induced severe root hair defects. We show that the fer phenotypes correlated with reduced levels of active RAC/ROPs and NADPH oxidase-dependent, auxin-regulated ROS accumulation in roots and root hairs and that up-regulating RAC/ROP signaling in fer countered the mutant phenotypes. Taken together, these observations strongly support FER as an upstream regulator for the RAC/ROP-signaled pathway that controls ROS-mediated root hair development. Moreover, FER was pulled down by ROP2 GTPase in a guanine nucleotide-regulated manner implying a dynamic signaling complex involving FER, a ROPGEF, and a RAC/ROP.

Pollen tubes are highly polarized plant cells specialized in delivering sperm for fertilization. Pollen tube growth is rapid, occurs exclusively at the tip, and can reach distances thousands of times the diameter of the pollen grain without cell division, thus representing an excellent model system for studying asymmetric cell growth. In flowering plants, pollen tube growth is dependent on the actin cytoskeleton, which supports an efficient vesicle trafficking system to deliver membrane and cell-wall materials to the tube tip. A highly dynamic subapical actin structure and an apical vesicular zone are known to be critical for the tip-growth process. How this apical organization is maintained, how the subapical actin structure is assembled, and direct evidence for its functional coupling with tip growth remain to be established. Here, we show that a tip-located, cell membrane-anchored actin-nucleating protein, the Arabidopsis formin homology5 (FH5), stimulates actin assembly from the subapical membrane, provides actin filaments for vesicular trafficking to the apical dome, and mediates assembly of the subapical actin structure. Moreover, FH5-expressing pollen tubes provided a unique opportunity to demonstrate that assembly of the subapical actin structure is concomitant with the acquisition of rapid tip growth, providing further support for their functional coupling. Together, our results show that FH5 plays a pivotal role in establishing the subapical actin and apical vesicular organization critical for tip-focused growth in pollen tubes.

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.

Plants use cell surface-resident receptor-like kinases (RLKs) to sense diverse extrinsic and intrinsic cues and elicit distinct biological responses. In Arabidopsis, the ERECTA family RLKs recognize EPIDERMAL PATTERNING FACTORS (EPFs) to specify stomatal patterning. However, little is known about the molecular link between ERECTA activation and intracellular signaling. We report here that the SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) family RLKs regulate stomatal patterning downstream of EPF ligands and upstream of a MAP kinase cascade. EPF ligands induce the heteromerization of ERECTA and SERK family RLKs. SERKs and ERECTA family RLKs transphosphorylate each other. In addition, SERKs associate with the receptor-like protein (RLP) TMM, a signal modulator of stomata development, in a ligand-independent manner, suggesting that ERECTA, SERKs and TMM form a multi-protein receptorsome consisting of different RLKs and RLP perceiving peptide ligands in regulating stomatal patterning. In contrast to the differential requirement of individual SERK members in plant immunity, cell death control and BR signaling, all four functional SERKs are essential but with unequal genetic contributions to stomatal patterning with descending order of importance from SERK3/BAK1, SERK2, SERK1 to SERK4. Although BR signaling connects stomatal development via multiple components, the function of SERKs in stomatal patterning is uncoupled from their involvement in BR signaling. Our results reveal that the SERK family is a shared key module in diverse Arabidopsis signaling receptorsomes and different combinatorial codes of individual SERK members regulate distinct functions.

Emerging evidence indicates that some reactive oxygen species (ROS), such as the superoxide anion radical and hydrogen peroxide (H2O2), are central regulators of plant responses to biotic and abiotic stresses. Thus, the cellular levels of ROS are thought to be tightly regulated by an efficient and elaborate pro- and antioxidant system that modulates the production and scavenging of ROS. Until recently, studies of ROS in plant cells have been limited to biochemical assays and the use of fluorescent probes, however, the irreversible oxidation of these fluorescent probes makes it impossible to visualize dynamic changes in ROS levels. In this work, we describe the use of Hyper, a recently developed live cell probe for H2O2 measurements in living cells, to monitor oxidative stress in Arabidopsis roots subjected to aluminum treatment. Hyper consists of a circularly permuted YFP (cpYFP) inserted into the regulatory domain of the Escherichia coli hydrogen peroxide-binding protein (OxyR), and is a H2O2-specific ratiometric, and therefore quantitative, probe that can be expressed in plant and animal cells. Now we demonstrate that H2O2 levels drop sharply in the elongation zone of roots treated with aluminum. This response could contribute to root growth arrest and provides evidence that H2O2 is involved in early Al sensing.

Pollen tube growth is a polarized growth process whereby the tip-growing tubes elongate within the female reproductive tissues to deliver sperm cells to the ovules for fertilization. Efficient and regulated membrane trafficking activity incorporates membrane and deposits cell wall molecules at the tube apex and is believed to underlie rapid and focused growth at the pollen tube tip. Rab GTPases, key regulators of membrane trafficking, are candidates for important roles in regulating pollen tube growth. We show that a green fluorescent protein–tagged Nicotiana tabacum pollen-expressed Rab11b is localized predominantly to an inverted cone-shaped region in the pollen tube tip that is almost exclusively occupied by transport vesicles. Altering Rab11 activity by expressing either a constitutive active or a dominant negative variant of Rab11b in pollen resulted in reduced tube growth rate, meandering pollen tubes, and reduced male fertility. These mutant GTPases also inhibited targeting of exocytic and recycled vesicles to the pollen tube inverted cone region and compromised the delivery of secretory and cell wall proteins to the extracellular matrix. Properly regulated Rab11 GTPase activity is therefore essential for tip-focused membrane trafficking and growth at the pollen tube apex and is pivotal to reproductive success.

Plant Rac-like GTPases have been classified phylogenetically into two major groups-class I and class II. Several pollen-expressed class I Rac-like GTPases have been shown to be important regulators of polar pollen tube growth. The functional participation by some of the class I and all of the class II Arabidopsis Rac-like GTPases in pollen tube growth remains to be explored. It is shown that at least four members of the Arabidopsis Rac GTPase family are expressed in pollen, including a class II Rac, AtRac7. However, when over-expressed as fusion proteins with GFP, both pollen- and non-pollen-expressed AtRacs interfered with the normal pollen tube tip growth process. These observations suggest that these AtRacs share similar biochemical activities and may integrate into the pollen cellular machinery that regulates the polar tube growth process. Therefore, the functional contribution by individual Rac GTPase to the pollen tube growth process probably depends to a considerable extent on their expression characteristics in pollen. Among the Arabidopsis Racs, GFP-AtRac7 showed association with the cell membrane and Golgi bodies, a pattern distinct from all previously reported localization for other plant Racs. Over-expressing GFP-AtRac7 also induced the broadest spectrum of pollen tube growth defects, including pollen tubes that are bifurcated, with diverted growth trajectory or a ballooned tip. Transgenic plants with multiple copies of the chimeric Lat52-GFP-AtRac7 showed severely reduced seed set, probably many of these defective pollen tubes were arrested, or reduced in their growth rates that they did not arrive at the ovules while they were still receptive for fertilization. These observations substantiate the importance of Rac-like GTPases to sexual reproduction.

Pollen tube elongation is a rapid tip growth process that is driven by a dynamic actin cytoskeleton. A ubiquitous family of actin binding proteins, actin-depolymerizing factors (ADFs)/cofilins, bind to actin filaments, induce severing, enhance depolymerization from their slow-growing end, and are important for maintaining actin dynamics in vivo. ADFs/cofilins are regulated by multiple mechanisms, among which Rho small GTPase–activated phosphorylation at a terminal region Ser residue plays an important role in regulating their actin binding and depolymerizing activity, affecting actin reorganization. We have shown previously that a tobacco pollen-specific ADF, NtADF1, is important for maintaining normal pollen tube actin cytoskeleton organization and growth. Here, we show that tobacco pollen grains accumulate phosphorylated and nonphosphorylated forms of ADFs, suggesting that phosphorylation could be a regulatory mechanism for their activity. In plants, Rho-related Rac/Rop GTPases have been shown to be important regulators for pollen tube growth. Overexpression of Rac/Rop GTPases converts polar growth into isotropic growth, resulting in pollen tubes with ballooned tips and a disrupted actin cytoskeleton. Using the Rac/Rop GTPase–induced defective pollen tube phenotype as a functional assay, we show that overexpression of NtADF1 suppresses the ability of NtRac1, a tobacco Rac/Rop GTPase, to convert pollen tube tip growth to isotropic growth. This finding suggests that NtADF1 acts in a common pathway with NtRac1 to regulate pollen tube growth. A mutant form of NtADF1 with a nonphosphorylatable Ala substitution at its Ser-6 position [NtADF1(S6A)] shows increased activity, whereas the mutant NtADF1(S6D), which has a phospho-mimicking Asp substitution at the same position, shows reduced ability to counteract the effect of NtRac1. These observations suggest that phosphorylation at Ser-6 of NtADF1 could be important for its integration into the NtRac1 signaling pathway. Moreover, overexpression of NtRac1 diminishes the actin binding activity of green fluorescent protein (GFP)–NtADF1 but has little effect on the association of GFP-NtADF1(S6A) with actin cables in pollen tubes. Together, these observations suggest that NtRac1-activated activity regulates the actin binding and depolymerizing activity of NtADF1, probably via phosphorylation at Ser-6. This notion is further supported by the observation that overexpressing a constitutively active NtRac1 in transformed pollen grains significantly increases the ratio of phosphorylated to nonphosphorylated ADFs. Together, the observations reported here strongly support the idea that NtRac1 modulates NtADF1 activity through phosphorylation at Ser-6 to regulate actin dynamics.

The auxin indole-3-acetic acid is a key plant hormone essential for a broad range of growth and developmental processes. Here, we show that auxin activates Rac-like GTPases (referred to as Rac/Rop GTPases), and they in turn stimulate auxin-responsive gene expression. In particular, we show that overexpressing a wild-type tobacco Rac/Rop GTPase, NtRac1, and its constitutively active mutant form activates auxin-responsive gene expression. On the other hand, overexpressing dominant-negative NtRac1 and Rac-negative regulators, or reducing the endogenous NtRac1 level, suppresses auxin-induced gene expression. Furthermore, overexpression of NtRac1 activity or suppression of its expression in transgenic seedlings induces phenotypes that are similar to auxin-related defects. Together, our results show that a subset of plant Rac/Rop GTPases functions in mediating the auxin signal to downstream responsive genes.

Pollen tube elongation is a polarized cell growth process that transports the male gametes from the stigma to the ovary for fertilization inside the ovules. Actomyosin-driven intracellular trafficking and active actin remodeling in the apical and subapical regions of pollen tubes are both important aspects of this rapid tip growth process. Actin-depolymerizing factor (ADF) and cofilin are actin binding proteins that enhance the depolymerization of microfilaments at their minus, or slow-growing, ends. A pollen-specific ADF from tobacco, NtADF1, was used to dissect the role of ADF in pollen tube growth. Overexpression of NtADF1 resulted in the reduction of fine, axially oriented actin cables in transformed pollen tubes and in the inhibition of pollen tube growth in a dose-dependent manner. Thus, the proper regulation of actin turnover by NtADF1 is critical for pollen tube growth. When expressed at a moderate level in pollen tubes elongating in in vitro cultures, green fluorescent protein (GFP)–tagged NtADF1 (GFP-NtADF1) associated predominantly with a subapical actin mesh composed of short actin filaments and with long actin cables in the shank. Similar labeling patterns were observed for GFP-NtADF1–expressing pollen tubes elongating within the pistil. A Ser-6-to-Asp conversion abolished the interaction between NtADF1 and F-actin in elongating pollen tubes and reduced its inhibitory effect on pollen tube growth significantly, suggesting that phosphorylation at Ser-6 may be a prominent regulatory mechanism for this pollen ADF. As with some ADF/cofilin, the in vitro actin-depolymerizing activity of recombinant NtADF1 was enhanced by slightly alkaline conditions. Because a pH gradient is known to exist in the apical region of elongating pollen tubes, it seems plausible that the in vivo actin-depolymerizing activity of NtADF1, and thus its contribution to actin dynamics, may be regulated spatially by differential H+ concentrations in the apical region of elongating pollen tubes.

Pollen tube elongation depends on the secretion of large amounts of membrane and cell wall materials at the pollen tube tip to sustain rapid growth. A large family of RAS-related small GTPases, Rabs or Ypts, is known to regulate both anterograde and retrograde trafficking of transport vesicles between different endomembrane compartments and the plasma membrane in mammalian and yeast cells. Studies on the functional roles of analogous plant proteins are emerging. We report here that a tobacco pollen-predominant Rab2, NtRab2, functions in the secretory pathway between the endoplasmic reticulum and the Golgi in elongating pollen tubes. Green fluorescent protein–NtRab2 fusion protein localized to the Golgi bodies in elongating pollen tubes. Dominant-negative mutations in NtRab2 proteins inhibited their Golgi localization, blocked the delivery of Golgi-resident as well as plasmalemma and secreted proteins to their normal locations, and inhibited pollen tube growth. On the other hand, when green fluorescent protein–NtRab2 was over-expressed in transiently transformed leaf protoplasts and epidermal cells, in which NtRab2 mRNA have not been observed to accumulate to detectable levels, these proteins did not target efficiently to Golgi bodies. Together, these observations indicate that NtRab2 is important for trafficking between the endoplasmic reticulum and the Golgi bodies in pollen tubes and may be specialized to optimally support the high secretory demands in these tip growth cells.

In flowering plants, sperm are transported inside pollen tubes to the female gametophyte for fertilization. The female gametophyte induces rupture of the penetrating pollen tube, resulting in sperm release and rendering them available for fertilization. Here we utilize the Arabidopsis FERONIA (FER) receptor kinase mutants, whose female gametophytes fail to induce pollen tube rupture, to decipher the molecular mechanism of this critical male-female interactive step. We show that FER controls the production of high levels of reactive oxygen species at the entrance to the female gametophyte to induce pollen tube rupture and sperm release. Pollen tube growth assays in vitro and in the pistil demonstrate that hydroxyl free radicals are likely the most reactive oxygen molecules, and they induce pollen tube rupture in a Ca(2+)-dependent process involving Ca(2+) channel activation. Our results provide evidence for a RHO GTPase-based signalling mechanism to mediate sperm release for fertilization in plants.

Rac-like GTPases or Rho-related GTPases from plants (RAC/ROPs) are important components of hormone signalling pathways in plants. Based on phylogeny, several groups can be distinguished, and the underlying premise is that members of different groups perform distinct functions in the plant. AtRAC7/ROP9 is phylogenetically unique among 11 Arabidopsis RAC/ROPs, and here it was shown that it functions as a modulator of auxin and abscisic acid (ABA) signalling, a dual role not previously assigned to these small GTPases. Plants with reduced levels of AtRAC7/ROP9 had increased sensitivity to auxin and were less sensitive to ABA. On the other hand, overexpressing AtRAC7/ROP9 activated ABA-induced gene expression but repressed auxin-induced gene expression. In addition, both hormones regulated the activity of the AtRAC7/ROP9 promoter, suggesting a feedback mechanism to modulate the signalling output from the AtRAC7/ROP9-controlled molecular switch. High levels of AtRAC7/ROP9 were detected specifically in embryos and lateral roots, underscoring the important role of this protein during embryo development and lateral root formation. These results place AtRAC7/ROP9 as an important signal transducer in recently described pathways that integrate auxin and ABA signalling in the plant.

Pollen germination and tube growth underlie reproductive success in flowering plants. Pollen germination, whether occurring on the stigmatic surface of the pistil or under defined medium conditions in vitro, involves producing a polarized outgrowth that extends at the tip, producing a pollen tube. RAC/ROPs, the RHO GTPases of plants, are molecular switches that pivotally control the polarized pollen tube growth process. However, how RAC/ROP activities respond to conditions during germination and tube growth to signal the needed cellular events has not been examined.

Reactive oxygen species (ROS) are highly reactive reduced oxygen molecules that play a myriad of roles in animal and plant cells. In plant cells, the production of ROS occurs as a result of aerobic metabolism during respiration and photosynthesis. Therefore mitochondria, chloroplasts, and peroxisomes constitute an important source of ROS. However, they can be produced in response to many physiological stimuli such as pathogen attack, hormone signaling, abiotic stresses, or during cell wall organization and plant morphogenesis. Monitoring ROS in plant cells has been limited to biochemical assays and use of fluorescent probes, however, the irreversible oxidation of the fluorescent dyes make it impossible to visualize dynamic changes of ROS. Hyper is a recently developed live cell probe for H2O2 and consists of a circularly permutated YFP (cpYFP) inserted into the regulatory domain of the Escherichia coli hydrogen peroxide (H2O2) sensor OxyR rendering it a H2O2 specific ratiometric, and therefore quantitative probe. Herein, we describe a protocol for using Hyper as a dynamic probe for H2O2 in Arabidopsis with virtually unlimited potential to detect H2O2 throughout the plant and under a broad range of developmental and environmental conditions.

Summary Pollination induces many physiological responses in the flower, including deterioration and death in specific pistil cell types. It is shown here that within the style of tobacco, pollination-induced cell deterioration was restricted to the transmitting tissue while the surrounding cortical tissue was not affected. It was distinct from general senescence since exogenously applying the senescence-inducing hormone ethylene, or its precursor aminocyclopropane-1-carboxylic acid (ACC), to the flower or the pistil induced overall deterioration in the entire flower. Furthermore, both pollen tube growth and ethylene action were needed for the entire spectrum of cellular changes associated with this pollination-induced transmitting tissue deterioration process. It is also shown that pollination-induced mRNA poly(A) tail-shortening for at least three major classes of transmitting tissue-specific mRNAs. As is commonly observed for poly(A) tail-shortened mRNAs, the levels of two of these three mRNA classes decline after pollination. On the other hand, the third class of mRNAs, transmitting tissue-specific (TTs) mRNAs, was maintained at a very high level subsequent to pollination, even after substantial poly(A)-tail shortening. TTS mRNAs encode a pollen tube growth-promoting and -attracting protein needed for optimal in vivo pollen tube growth. The specific preservation of TTS mRNAs in the deteriorating transmitting tissue cells suggests that these cells can distinguish molecules needed in the pollinated styles from those that are dispensable, and protect them from degradation. It is suggested that the pollination-induced mRNA poly(A) tail-shortening and cell death are programmed processes suited to the post-pollination transmitting tissue environment. Results showing that ACC is a candidate signal molecule for the pollination-induced mRNA-shortening which is accentuated by ethylene and mediated via a protein phosphorylation-dependent signal transduction pathway are also presented.

Transmitting tissue-specific (TTS) protein is a pollen tube growth-promoting and attracting glycoprotein located in the stylar transmitting tissue extracellular matrix of the pistil of tobacco. The TTS protein backbones have a deduced molecular mass of about 28 kDa, whereas the glycosylated stylar TTS proteins have apparent molecular masses ranging between 50 and 100 kDa. TTS mRNAs and proteins are ectopically produced in transgenic tobacco plants that express either a cauliflower mosaic virus (CaMV) 35S promoter-TTS2 transgene or a CaMV 35S-promoter-NAG1 (NAG1 = Nicotiana tabacum Agamous gene) transgene. However, the patterns of TTS mRNA and protein accumulation and the quality of the TTS proteins produced are different in these two types of transgenic plants. In 35S-TTS transgenic plants, TTS mRNAs and proteins accumulate constitutively in vegetative and floral tissues. However, the ectopically expressed TTS proteins in these transgenic plants accumulate as underglycosylated protein species with apparent molecular masses between 30 and 50 kDa. This indicates that the capacity to produce highly glycosylated TTS proteins is restricted to the stylar transmitting tissue. In 35S-NAG transgenic plants, NAG1 mRNAs accumulate constitutively in vegetative and floral tissues, and TTS mRNAs are induced in the sepals of these plants. Moreover, highly glycosylated TTS proteins in the 50- to 100-kDa molecular mass range accumulate in the sepals of these transgenic, 35S-NAG plants. These results show that the tobacco NAGI gene, together with other yet unidentified regulatory factors, control the expression of TTS genes and the cellular capacity to glycosylate TTS proteins, which are normally expressed very late in the pistil developmental pathway and function in the final stage of floral development. The sepals in the transgenic 35S-NAG plants also support efficient pollen germination and tube growth, similar to what normally occurs in the pistil, and this ability correlates with the accumulation of the highest levels of the 50- to 100-kDa glycosylated TTS proteins.

Pollen tubes elongate directionally in the extracellular matrix of pistil tissues to transport the male gametes from the apically located stigma to the basally located ovary for fertilization. The molecular mechanisms underlying directional pollen tube growth in the pistil are poorly understood. We have purified a glycoprotein, TTS, from tobacco stylar transmitting tissue, which supports pollen tube growth between the stigma and the ovary. TTS proteins belong to the arabinogalactan protein family, and they polymerize readily in vitro in a head-to-tail fashion into oligomeric forms. TTS proteins stimulate pollen tube growth in vitro and attract pollen tubes grown in a semi-in vivo culture system. In vivo, the pollen tube growth rate is reduced in transgenic plants that have significantly reduced levels of TTS proteins as a result of either antisense suppression or sense cosuppression. These results identify TTS protein as a pistil component that positively contributes to pollen tube growth.

The plant cell wall provides form and integrity to the cell as well as a dynamic interface between a cell and its environment. Therefore mechanisms capable of policing changes in the cell wall, signaling cellular responses including those that would feedback regulate cell wall properties are expected to play important roles in facilitating growth and ensuring survival. Discoveries in the last few years that the Arabidopsis THESEUS 1 receptor-like kinase (RLK) may function as a sensor for cell wall defects to regulate growth and that its relatives FERONIA and ANXURs regulate pollen tube integrity imply strongly that they play key roles in cell wall-related processes. Furthermore, FERONIA acts as a cell surface regulator for RAC/ROP GTPases and activates production of reactive oxygen species which are, respectively, important molecular switches and mediators for diverse processes. These findings position the THESEUS 1/FERONIA family RLKs as surface regulators and potential cell wall sensors capable of broadly and profoundly impacting cellular pathways in response to diverse signals.