Polarized NORTIA accumulation in response to pollen tube arrival at synergids promotes fertilization.

Signal-mediated regulation of protein trafficking is an elegant mechanism for controlling the delivery of molecules to a precise location for critical signaling events that occur over short time frames. During plant reproduction, the FERONIA receptor complex is critical for intercellular communication that leads to gamete delivery; however, the impact of the FERONIA signal transduction cascade on protein trafficking in synergid cells remains unknown. Live imaging of pollen tube reception has revealed that a key outcome of FERONIA signaling is polar accumulation of the MLO protein NORTIA at the filiform apparatus in response to signals from an arriving pollen tube. Artificial delivery of NORTIA to the filiform apparatus is sufficient to bypass the FERONIA signaling pathway and to promote interspecific pollen tube reception. We propose that polar accumulation of NORTIA leads to the production of a secondary booster signal to ensure that pollen tubes burst to deliver the sperm cells for double fertilization.

A transposon-mediated reciprocal translocation promotes environmental adaptation but compromises domesticability of wild soybeans.

Large structural variations frequently occur in higher plants; however, the impact of such variations on plant diversification, adaptation and domestication remains elusive. Here, we mapped and characterised a reciprocal chromosomal translocation in soybeans and assessed its effects on diversification and adaptation of wild (Glycine soja) and semiwild (Glycine gracilis) soybeans, and domestication of cultivated soybean (Glycine max), by tracing the distribution of the translocation in the USDA Soybean Germplasm Collection and population genetics analysis. We demonstrate that the translocation occurred through CACTA transposon-mediated chromosomal breakage in wild soybean c. 0.34 Ma and is responsible for semisterility in translocation heterozygotes and reduces their reproductive fitness. The translocation has differentiated Continental (i.e. China and Russia) populations from Maritime (i.e. Korea and Japan) populations of G. soja and predominately adapted to cold and dry climates. Further analysis revealed that the divergence of G. max from G. soja predates the translocation event and that G. gracilis is an evolutionary intermediate between G. soja and G. max. Our results highlight the effects of a chromosome rearrangement on the processes leading to plant divergence and adaptation, and provides evidence that suggests G. gracilis, rather than G. soja, as the ancestor of cultivated soybean.

Growing straight through walls.

The pollen tube in a flowering plant grows in a direction that is influenced by the mechanical properties of the stigma papillae and the organization of structures called cortical microtubules inside these cells.

Reactive Oxygen Species as Mediators of Gametophyte Development and Double Fertilization in Flowering Plants.

Reactive oxygen species (ROS) are toxic by-products of aerobic metabolism. In plants, they also function as important signaling molecules that regulate biotic and abiotic stress responses as well as plant growth and development. Recent studies have implicated ROS in various aspects of plant reproduction. In male gametophytes, ROS are associated with germline development as well as the developmentally associated programmed cell death of tapetal cells necessary for microspore development. ROS have a role in regulation of female gametophyte patterning and maintenance of embryo sac polarity. During pollination, ROS play roles in the generation of self-incompatibility response during pollen-pistil interaction, pollen tube growth, pollen tube burst for sperm release and fertilization. In this mini review, we provide an overview of ROS production and signaling in the context of plant reproductive development, from female and male gametophyte development to fertilization.

Specific Recruitment of Phosphoinositide Species to the Plant-Pathogen Interfacial Membrane Underlies Arabidopsis Susceptibility to Fungal Infection.

Different phosphoinositides enriched at the membranes of specific subcellular compartments within plant cells contribute to organelle identity, ensuring appropriate cellular trafficking and function. During the infection of plant cells, biotrophic pathogens such as powdery mildews enter plant cells and differentiate into haustoria. Each haustorium is enveloped by an extrahaustorial membrane (EHM) derived from the host plasma membrane. Little is known about the EHM biogenesis and identity. Here, we demonstrate that among the two plasma membrane phosphoinositides in Arabidopsis (Arabidopsis thaliana), PI(4,5)P2 is dynamically up-regulated at powdery mildew infection sites and recruited to the EHM, whereas PI4P is absent in the EHM. Lateral transport of PI(4,5)P2 into the EHM occurs through a brefeldin A-insensitive but actin-dependent trafficking pathway. Furthermore, the lower levels of PI(4,5)P2 in pip5k1 pip5k2 mutants inhibit fungal pathogen development and cause disease resistance, independent of cell death-associated defenses and involving impaired host susceptibility. Our results reveal that plant biotrophic and hemibiotrophic pathogens modulate the subcellular distribution of host phosphoinositides and recruit PI(4,5)P2 as a susceptibility factor for plant disease.

Natural fumigation as a mechanism for volatile transport between flower organs.

Plants synthesize volatile organic compounds (VOCs) to attract pollinators and beneficial microorganisms, to defend themselves against herbivores and pathogens, and for plant-plant communication. In general, VOCs accumulate in and are emitted from the tissue of their biosynthesis. However, using biochemical and reverse genetic approaches, we demonstrate a new physiological phenomenon: inter-organ aerial transport of VOCs via natural fumigation. Before petunia flowers open, a tube-specific terpene synthase produces sesquiterpenes, which are released inside the buds and then accumulate in the stigma, potentially defending the developing stigma from pathogens. These VOCs also affect reproductive organ development and seed yield, which are previously unknown functions of terpenoid compounds.

A genome-wide association study reveals a novel regulator of ovule number and fertility in Arabidopsis thaliana.

Ovules contain the female gametophytes which are fertilized during pollination to initiate seed development. Thus, the number of ovules that are produced during flower development is an important determinant of seed crop yield and plant fitness. Mutants with pleiotropic effects on development often alter the number of ovules, but specific regulators of ovule number have been difficult to identify in traditional mutant screens. We used natural variation in Arabidopsis accessions to identify new genes involved in the regulation of ovule number. The ovule numbers per flower of 189 Arabidopsis accessions were determined and found to have broad phenotypic variation that ranged from 39 ovules to 84 ovules per pistil. Genome-Wide Association tests revealed several genomic regions that are associated with ovule number. T-DNA insertion lines in candidate genes from the most significantly associated loci were screened for ovule number phenotypes. The NEW ENHANCER of ROOT DWARFISM (NERD1) gene was found to have pleiotropic effects on plant fertility that include regulation of ovule number and both male and female gametophyte development. Overexpression of NERD1 increased ovule number per fruit in a background-dependent manner and more than doubled the total number of flowers produced in all backgrounds tested, indicating that manipulation of NERD1 levels can be used to increase plant productivity.

Contribution of isopentenyl phosphate to plant terpenoid metabolism.

Plant genomes encode isopentenyl phosphate kinases (IPKs) that reactivate isopentenyl phosphate (IP) via ATP-dependent phosphorylation, forming the primary metabolite isopentenyl diphosphate (IPP) used generally for isoprenoid/terpenoid biosynthesis. Therefore, the existence of IPKs in plants raises unanswered questions concerning the origin and regulatory roles of IP in plant terpenoid metabolism. Here, we provide genetic and biochemical evidence showing that IP forms during specific dephosphorylation of IPP catalysed by a subset of Nudix superfamily hydrolases. Increasing metabolically available IP by overexpression of a bacterial phosphomevalonate decarboxylase (MPD) in Nicotiana tabacum resulted in significant enhancement in both monoterpene and sesquiterpene production. These results indicate that perturbing IP metabolism results in measurable changes in terpene products derived from both the methylerythritol phosphate (MEP) and mevalonate (MVA) pathways. Moreover, the unpredicted peroxisomal localization of bacterial MPD led us to discover that the step catalysed by phosphomevalonate kinase (PMK) imposes a hidden constraint on flux through the classical MVA pathway. These complementary findings fundamentally alter conventional views of metabolic regulation of terpenoid metabolism in plants and provide new metabolic engineering targets for the production of high-value terpenes in plants.

Cellular distribution of secretory pathway markers in the haploid synergid cells of Arabidopsis thaliana.

In flowering plants, cell-cell communication plays a key role in reproductive success, as both pollination and fertilization require pathways that regulate interactions between many different cell types. Some of the most critical of these interactions are those between the pollen tube (PT) and the embryo sac, which ensure the delivery of sperm cells required for double fertilization. Synergid cells function to attract the PT through secretion of small peptides and in PT reception via membrane-bound proteins associated with the endomembrane system and the cell surface. While many synergid-expressed components regulating PT attraction and reception have been identified, few tools exist to study the localization of membrane-bound proteins and the components of the endomembrane system in this cell type. In this study, we describe the localization and distribution of seven fluorescent markers that labelled components of the secretory pathway in synergid cells of Arabidopsis thaliana. These markers were used in co-localization experiments to investigate the subcellular distribution of the two PT reception components LORELEI, a GPI-anchored surface protein, and NORTIA, a MILDEW RESISTANCE LOCUS O protein, both found within the endomembrane system of the synergid cell. These secretory markers are useful tools for both reproductive and cell biologists, enabling the analysis of membrane-associated trafficking within a haploid cell actively involved in polar transport.

Arabidopsis thaliana MLO genes are expressed in discrete domains during reproductive development.

KEY MESSAGE: MLOs in Plant Reproduction. The MILDEW RESISTANCE LOCUS-O (MLO) protein family, comprised of 15 members, plays roles in diverse cell-cell communication processes such as powdery mildew susceptibility, root thigmomorphogenesis, and pollen tube reception. The NORTIA (NTA, AtMLO7) gene is expressed in the synergid cells of the female gametophyte where it functions in intercellular communication with the pollen tube. Discrepancies between previously published promoter::GUS and promoter::gene-GUS constructs expression patterns led us to explore the regulation of NTA expression. Here we found via NTApro::gNTA-GUS truncations that sequences within the NTA gene negatively regulate its expression in the stomata and carpel walls. This led to the hypothesis that other MLO family members may also have additional regulatory sequences within the gene. MLOpro::gMLO-GUS constructs were examined for each family member focusing specifically on flowers in order to determine whether other MLOs could play a role in reproductive cell-cell communication. Notably, several MLOs were expressed in the pollen, in the stigma, in the pollinated style, and in the synergids and central cell. These findings indicate that other MLOs in addition to NTA could play a role in reproduction. Previous studies on the MLO family showed that phylogenetically related MLOs had redundant functions in powdery mildew infection and root thigmomorphogenesis; however, MLO expression in reproductive tissues did not strictly follow phylogenetic relationships, indicating that MLOs from different evolutionary origins may have been recruited for function in sexual reproduction.

Cell type-dependent localization of MLO proteins.

Mildew resistance locus O (MLO) proteins are transmembrane proteins that mediate cell-cell communication in plants. We recently demonstrated the importance of subcellular localization to MLO function during pollen tube reception. NORTIA (NTA), the MLO protein involved in this process, localizes to the Golgi of the synergid cell before interaction with the pollen tube. MLO proteins that can substitute for NTA's function in this pathway all partially localize with the same Golgi marker in the synergid cell. In this study, we report that MLO subcellular localization is cell type-dependent, with different distributions of some MLOs observed when ectopically expressed in the epidermal cells of tobacco and Arabidopsis compared to synergids. This dependency may be due to co-factors that influence MLO function within a given cell type, providing an intriguing new target for understanding MLO distribution and subsequent function in their respective processes.

MILDEW RESISTANCE LOCUS O Function in Pollen Tube Reception Is Linked to Its Oligomerization and Subcellular Distribution.

Sexual reproduction in flowering plants requires communication between synergid cells and a tip-elongating pollen tube (PT) for the successful delivery of sperm cells to the embryo sac. The reception of the PT relies on signaling within the synergid cell that ultimately leads to the degeneration of the receptive synergid and PT rupture, releasing the sperm cells for double fertilization. In Arabidopsis (Arabidopsis thaliana), NORTIA, a member of the MILDEW RESISTANCE LOCUS O (MLO) family of proteins, plays a critical role in the communication processes regulating PT reception. In this study, we determined that MLO function in PT reception is dependent on MLO protein localization into a Golgi-associated compartment before PT arrival, indicating that PT-triggered regulation of the synergid secretory system is important for synergid function during pollination. Additionally, a structure-function analysis revealed that MLO homooligomerization, mediated by the amino-terminal region of the protein, and carboxyl-terminal tail identity both contribute to MLO activity during PT reception.

TURAN and EVAN mediate pollen tube reception in Arabidopsis Synergids through protein glycosylation.

Pollen tube (PT) reception in flowering plants describes the crosstalk between the male and female gametophytes upon PT arrival at the synergid cells of the ovule. It leads to PT growth arrest, rupture, and sperm cell release, and is thus essential to ensure double fertilization. Here, we describe TURAN (TUN) and EVAN (EVN), two novel members of the PT reception pathway that is mediated by the FERONIA (FER) receptor-like kinase (RLK). Like fer, mutations in these two genes lead to PT overgrowth inside the female gametophyte (FG) without PT rupture. Mapping by next-generation sequencing, cytological analysis of reporter genes, and biochemical assays of glycoproteins in RNAi knockdown mutants revealed both genes to be involved in protein N-glycosylation in the endoplasmic reticulum (ER). TUN encodes a uridine diphosphate (UDP)-glycosyltransferase superfamily protein and EVN a dolichol kinase. In addition to their common role during PT reception in the synergids, both genes have distinct functions in the pollen: whereas EVN is essential for pollen development, TUN is required for PT growth and integrity by affecting the stability of the pollen-specific FER homologs ANXUR1 (ANX1) and ANX2. ANX1- and ANX2-YFP reporters are not expressed in tun pollen grains, but ANX1-YFP is degraded via the ER-associated degradation (ERAD) pathway, likely underlying the anx1/2-like premature PT rupture phenotype of tun mutants. Thus, as in animal sperm-egg interactions, protein glycosylation is essential for the interaction between the female and male gametophytes during PT reception to ensure fertilization and successful reproduction.

Functional analysis of related CrRLK1L receptor-like kinases in pollen tube reception.

The Catharanthus roseus Receptor-Like Kinase 1-like (CrRLK1L) family of 17 receptor-like kinases (RLKs) has been implicated in a variety of signaling pathways in Arabidopsis, ranging from pollen tube (PT) reception and tip growth to hormonal responses. The extracellular domains of these RLKs have malectin-like domains predicted to bind carbohydrate moieties. Domain swap analysis showed that the extracellular domains of the three members analyzed (FER, ANX1, HERK1) are not interchangeable, suggesting distinct upstream components, such as ligands and/or co-factors. In contrast, their intercellular domains are functionally equivalent for PT reception, indicating that they have common downstream targets in their signaling pathways. The kinase domain is necessary for FER function, but kinase activity itself is not, indicating that other kinases may be involved in signal transduction during PT reception.

She's the boss: signaling in pollen tube reception.

In angiosperms, the sperm cells are carried within the pollen tubes (male gametophytes) to the female gametophyte so that double fertilization can occur. The female gametophyte exerts control over the male, with specialized cells known as synergids guiding the pollen tubes and controlling their behavior when they enter the female gametophyte so that the sperm cells can be delivered to the egg and central cell. Upon pollen tube arrival at the ovule, signal transduction cascades mediated by receptor-like kinases are initiated in both the synergid and the tip of the pollen tube, leading to synergid cell death and pollen tube rupture. In this review, we discuss the role of these receptors and of newly discovered members of the pollen tube reception pathway.

The walls have ears: the role of plant CrRLK1Ls in sensing and transducing extracellular signals.

In plants, organ formation and cell elongation require the constant adjustment of the dynamic and adaptable cell wall in response to environmental cues as well as internal regulators, such as light, mechanical stresses, pathogen attacks, phytohormones, and other signaling molecules. The molecular mechanisms that perceive these cues and translate them into cellular responses to maintain integrity and remodelling of the carbohydrate-rich cell wall for the coordination of cell growth are still poorly understood. In the last 3 years, the function of six membrane-localized receptor-like kinases (RLKs) belonging to the CrRLK1L family has been linked to the control of cell elongation in vegetative and reproductive development. Moreover, the presence of putative carbohydrate-binding domains in the extracellular domains of these CrRLK1Ls makes this receptor family an excellent candidate for coordinating cell growth, cell-cell communication, and constant cell wall remodelling during the plant life cycle.

Conserved molecular components for pollen tube reception and fungal invasion.

During sexual reproduction in flowering plants such as Arabidopsis, a tip-growing pollen tube (PT) is guided to the synergid cells of the female gametophyte, where it bursts and releases the two sperm. Here we show that PT reception and powdery mildew (PM) infection, which involves communication between a tip-growing hypha and a plant epidermal cell, share molecular components. NORTIA (NTA), a member of the MLO family originally discovered in the context of PM resistance, and FERONIA (FER), a receptor-like kinase, both control PT reception in synergids. Homozygous fer mutants also display PM resistance, revealing a new function for FER and suggesting that conserved components, such as FER and distinct MLO proteins, are involved in both PT reception and PM infection.

PAMP (pathogen-associated molecular pattern)-induced changes in plasma membrane compartmentalization reveal novel components of plant immunity.

Plasma membrane compartmentalization spatiotemporally regulates cell-autonomous immune signaling in animal cells. To elucidate immediate early protein dynamics at the plant plasma membrane in response to the bacterial pathogen-associated molecular pattern (PAMP) flagellin (flg22) we employed quantitative mass spectrometric analysis on detergent-resistant membranes (DRMs) of Arabidopsis thaliana suspension cells. This approach revealed rapid and profound changes in DRM protein composition following PAMP treatment, prominently affecting proton ATPases and receptor-like kinases, including the flagellin receptor FLS2. We employed reverse genetics to address a potential contribution of a subset of these proteins in flg22-triggered cellular responses. Mutants of three candidates (DET3, AHA1, FER) exhibited a conspicuous defect in the PAMP-triggered accumulation of reactive oxygen species. In addition, these mutants showed altered mitogen-activated protein kinase (MAPK) activation, a defect in PAMP-triggered stomatal closure as well as altered bacterial infection phenotypes, which revealed three novel players in elicitor-dependent oxidative burst control and innate immunity. Our data provide evidence for dynamic elicitor-induced changes in the membrane compartmentalization of PAMP signaling components.