Sexual reproduction in flowering plants involves intimate contact and continuous interactions between the growing pollen tube and the female reproductive structures. These interactions can trigger responses in distal regions of the flower well ahead of fertilization. While pollination-induced petal senescence has been studied extensively, less is known about how pollination is perceived at a distance in the ovary, and how specific this response is to various pollen genotypes. To address this question, we performed a global transcriptomic analysis in the ovary of a wild potato species, Solanum chacoense, at various time points following compatible, incompatible, and heterospecific pollinations. In all cases, pollen tube penetration in the stigma was initially perceived as a wounding aggression. Then, as the pollen tubes grew in the style, a growing number of genes became specific to each pollen genotype. Functional classification analyses revealed sharp differences in the response to compatible and heterospecific pollinations. For instance, the former induced reactive oxygen species (ROS)-related genes while the latter affected genes associated to ethylene signaling. In contrast, incompatible pollination remained more akin to a wound response. Our analysis reveals that every pollination type produces a specific molecular signature generating diversified and specific responses at a distance in the ovary in preparation for fertilization.
Fertilization-related kinase (FRK) is a group of the mitogen-activated protein kinase kinase kinase (MAP3K or MEKK) that has proliferated in Solanaceae species. Studies on the wild potato Solanum chacoense have shown that three ScFRKs are directly involved in female gametophyte development. Decreasing the expression of ScFRK1 and ScFRK3 by RNA interference lead to embryonic sac development arrest at the functional megaspore (FM) stage. As for ScFRK2, the first FRK studied, antisense and co-suppression lines showed no abnormality, while overexpression lines lead to a drastic decrease in seed numbers, presumably caused by a conversion of the ovule into a carpel-like structure. Here we show that in ScFRK2 overexpression lines, carpel-like structures from the ovule cannot explain the drastic decrease in seeds considering the low percentage of these carpel-like structures but occurs in early ovule development as observed in Scfrk1 and Scfrk3 knockdown mutants were most ovules are arrested at the FM stage. The highly similar phenotype from knockdown mutants (Scfrk1 and Scfrk3) and ScFRK2 overexpression lines suggests that these MAP kinases could operate antagonistically through a balance between ScFRK1 and 3 on one side and ScFRK2 on the other. This study strongly suggests the importance of the FRK family expression levels during early stages of ovule development in Solanum chacoense embryo sac.
When plants conquered land, they developed specialized organs, tissues, and cells in order to survive in this new and harsh terrestrial environment. New cell polymers such as the hydrophobic lipid-based polyesters cutin, suberin, and sporopollenin were also developed for protection against water loss, radiation, and other potentially harmful abiotic factors. Cutin and waxes are the main components of the cuticle, which is the waterproof layer covering the epidermis of many aerial organs of land plants. Although the in vivo functions of the group of lipid binding proteins known as lipid transfer proteins (LTPs) are still rather unclear, there is accumulating evidence suggesting a role for LTPs in the transfer and deposition of monomers required for cuticle assembly. In this review, we first present an overview of the data connecting LTPs with cuticle synthesis. Furthermore, we propose liverworts and mosses as attractive model systems for revealing the specific function and activity of LTPs in the biosynthesis and evolution of the plant cuticle.
Proteins defined by a key amino acid pattern are key players in the exchange of signals between bacteria, animals and plants, as well as important mediators for cell-cell communication within a single organism. Their description and characterization open the way to a better knowledge of molecular signalling in a broad range of organisms, and to possible application in medical and agricultural research. The contrasted pattern of evolution in these proteins makes it difficult to detect and cluster them with classical sequence-based search tools. Here, we introduce Key Aminoacid Pattern-based Protein Analyzer (KAPPA), a new multi-platform program to detect them in a given set of proteins, analyze their pattern and cluster them by comparison to reference patterns (ab initio search) or internal pairwise comparison (de novo search). In this study, we use the concrete example of cysteine-rich proteins (CRPs) to show that the similarity of two cysteine patterns can be precisely and efficiently assessed by a quantitative tool created for KAPPA: the κ-score. We also demonstrate the clear advantage of KAPPA over other classical sequence search tools for ab initio search of new CRPs. Eventually, we present de novo clustering and subclustering functionalities that allow to rapidly generate consistent groups of CRPs without a seed reference. KAPPA executables are available for Linux, Windows and Mac OS at http://kappa-sequence-search.sourceforge.net.
During plant sexual reproduction, continuous exchange of signals between the pollen and the pistil (stigma, style, and ovary) plays important roles in pollen recognition and selection, establishing breeding barriers and, ultimately, leading to optimal seed set. After navigating through the stigma and the style, pollen tubes (PTs) reach their final destination, the ovule. This ultimate step is also regulated by numerous signals emanating from the embryo sac (ES) of the ovule. These signals encompass a wide variety of molecules, but species-specificity of the pollen-ovule interaction relies mainly on secreted proteins and their receptors. Isolation of candidate genes involved in pollen-pistil interactions has mainly relied on transcriptomic approaches, overlooking potential post-transcriptional regulation. To address this issue, ovule exudates were collected from the wild potato species Solanum chacoense using a tissue-free gravity-extraction method (tf-GEM). Combined RNA-seq and mass spectrometry-based proteomics led to the identification of 305 secreted proteins, of which 58% were ovule-specific. Comparative analyses using mature ovules (attracting PTs) and immature ovules (not attracting PTs) revealed that the last maturation step of ES development affected almost half of the ovule secretome. Of 128 upregulated proteins in anthesis stage, 106 were not regulated at the mRNA level, emphasizing the importance of post-transcriptional regulation in reproductive development.
The fertilization-related kinase 1 (ScFRK1), a nuclear-localized mitogen-activated protein kinase kinase kinase (MAPKKK) from the wild potato species Solanum chacoense, belongs to a small group of pMEKKs that do not possess an extended Nor C-terminal regulatory domain. Initially selected based on its highly specific expression profile following fertilization, in situ expression analyses revealed that the ScFRK1 gene is also expressed early on during female gametophyte development in the integument and megaspore mother cell and, later, in the synergid and egg cells of the embryo sac. ScFRK1 mRNAs are also detected in pollen mother cells. Transgenic plants with lower or barely detectable levels of ScFRK1 mRNAs lead to the production of small fruits with severely reduced seed set, resulting from a concomitant decline in the number of normal embryo sacs produced. Megagametogenesis and microgametogenesis were affected, as megaspores did not progress beyond the functional megaspore (FG1) stage and the microspore collapsed around the first pollen mitosis. As for other mutants that affect embryo sac development , pollen tube guidance was severely affected in the ScFRK1 transgenic lines. Gametophyte to sporophyte communication was also affected, as observed from a marked change in the transcriptomic profiles of the sporophytic tissues of the ovule. The ScFRK1 MAPKKK is thus involved in a signalling cascade that regulates both male and female gamete development.
Small, secreted cysteine-rich proteins (CRPs) combine a highly stable cysteine spacing, ensuring conservation of their 3D structure and function, and hypervariable inter-cysteine blocks, allowing quick evolution of specific recognition domains. Interestingly, several CRPs were shown to control key pollen-pistil interactions in a species-specific way. The most emblematic example is perhaps the LURE defensin-like family, controlling directional guidance of pollen tubes (PTs) in Torenia and Arabidopsis. We chose wild potatoes (Solanum sect. Petota) as a case study to investigate the impact of rapid CRP divergence in plant speciation. Gathering ~200 close species with overlapping distribution areas, this taxon indeed exhibits strong reproductive isolation. Lab-on-a-chip microfluidic experiments carried out on 4 species show that species-preferential PT attraction is a key factor in this isolation. We suspect polymorphic CRPs to control this attraction. High-throughput sequencing technologies were applied to profile the ovule secretome as well as the reproductive transcriptomes of our 4 species of interest. To screen out candidate genes, we developped KAPPA, a sequence search algorithm specifically dedicated to CRPs, and obtained a set of 32 defensin-like groups expressed in ovules. Five promising chemoattractant candidates exhibiting (i) ovule-specific expression, (ii) down-regulation in guidance-defective ovules, and (iii) interspecific divergence were selected for further characterization. They are currently being investigated with on-gel assays and specific microfluidic devices tailored for Solanum PTs. This study will lead to a better understanding of CRP-mediated PT chemoattraction as one of the major species-specificity checkpoints that must be “unlocked” by pollen tubes in the pistil.
Small, secreted cysteine-rich proteins (CRPs) combine a highly stable cysteine spacing, ensuring conservation of their 3D structure and function, and hypervariable inter-cysteine blocks, allowing quick evolution of specific recognition domains. Interestingly, several CRPs were shown to control key pollen-pistil interactions, including self-incompatibility (SCR/SP11, PrsS), pollen-style interactions (SCA/LTP5, LeSTIG1), sperm discharge (ZmES4), and gamete fusion (EC1). The most emblematic example of species-specific CRP function in reproduction remains however the LURE family, controlling directional guidance of pollen tubes (PTs) in Torenia and Arabidopsis. We chose wild potatoes (Solanum sect. Petota) as a case study to investigate the impact of rapid CRP divergence in plant speciation. Gathering ~200 close species with overlapping distribution areas, this taxon indeed exhibits strong reproductive isolation. Lab-on-a-chip microfluidic experiments carried out on 4 species show that species-preferential PT attraction is a key factor in this isolation. We suspect polymorphic CRPs to control this attraction. Next-generation RNA sequencing led us to obtain a set of 33,852 CDS-containing transcripts expressed in ovules. Moreover, an original secretomic approach allowed to directly identify 305 proteins in ovule exudates. To screen out candidate genes, we developped KAPPA, a sequence search algorithm specifically dedicated to CRPs, and obtained a set of 27 CRP families, including defensin-, LTP-, ECA1- and thionin- like proteins, but also groups with new cysteine patterns. Candidate chemoattractant CRPs are currently being investigated with on-gel and microfluidic pollen tube attraction assays, based on a combination of three major features of interest: (i) ovule-specific expression; (ii) down-regulation in guidance-defective (immature and embryo sac-less) ovules); (iii) interspecific divergence, as verified by finding their orthologs in close species and detecting signatures of positive selection such as accumulation of non-synonymous mutations (dN/dS > 1). The current study will lead to a better understanding of CRP-mediated PT chemoattraction as one of the major species-specificity checkpoints that must be “unlocked” by pollen tubes in the pistil.
Gathering 196 species, wild potatoes (Solanum sect. Petota) are a striking example of plant diversification. Although they coexist sympatrically throughout all Latin America, and have the physiological ability to hybridize with close species, they display strong reproductive isolation barriers. Experiments carried out in our laboratory show that pollen-pistil interactions are the cornerstone of this isolation: in a heterospecific pistil, pollen tubes can be slowed down and/or their receptivity to female chemoattractive cues can be impaired. How is this so finely regulated? Most of plant sexual interactions described to date involve binding of pollinic receptors to female protein ligands expressed in various locations all along the pollen tube path in the pistil or vice versa. We hypothesize that rapid divergence of these reproductive proteins, driven by directional selective pressures, is the engine of interspecific isolation. This could especially take the form of species-specific aminoacid changes accumulating within binding sites, thus impairing mutual recognition in case of an interspecific cross. To identify candidate proteins, we used next-generation sequencing technologies to obtain transcriptomes from ovules, pistils and pollen tubes of four closely related Solanum species species, and screened for coding sequences displaying a non-synonymous substitution rate (dN) greater than the synonymous one (dS), which is a commonly used, robust signature of directional selection. This approach, that had scarcely been applied to plants so far, led to us to identify a wide range of candidates including proteins similar to well-known key players of pollen-pistil interactions —such as the ovule-expressed FERONIA (FER) receptor kinase and the glycosylphosphatidylinositol-anchored protein LORELEI (LRE), stylar adhesion proteins or pollen tube calcium channels— but also totally uncharacterized proteins including several families of small secreted cysteine-rich proteins (CRPs). CRPs are of outstanding interest since they display a dual evolutive pattern, involving a highly conserved cysteine backbone delimitating blocks of extensively divergent aminoacids. Several CRPs were shown to control various aspects of pollen-pistil interactions in other species, especially stigmatic and ovular chemoattraction cues. Using KAPPA, a sequence search program we recently developed to detect these peculiar proteins, we identified new groups of Solanaceous-specific CRPs undergoing rapid divergence between species. These are promising candidates as ligands of pollinic or style receptors. Our study points out that reproductive isolation relies on a coordinated set of pistil species-specific checkpoints that need to be successively “unlocked” by pollen tubes to achieve fertilization. Further functional characterization of these proteins paves the way to a better understanding of the speciation processes in action among wild potatoes.
L’isolement reproductif des 196 espèces de pommes de terre sauvages d’Amérique latine (Solanum sect. Petota) est une énigme. En effet, malgré la faiblesse apparente des barrières pré- et post-zygotiques à l'hybridation, les espèces « pures » sont largement dominantes en nature, les hybrides restant rares. Comment résoudre ce paradoxe ? La clé du problème pourrait résider entre le moment de la pollinisation et la fécondation. Nous supposons que la communication entre le pistil (femelle) et le tube pollinique (mâle) serait plus efficace lorsque les deux partenaires sont de même espèce. Cette communication repose sur l’interaction de couples de protéines mâle-femelle, dont la divergence interspécifique serait un moteur important de l’isolement reproductif : plus les partenaires sexuels appartiennent à des espèces proches, meilleure sera l’interaction de leurs protéines reproductives. Notre intérêt porte principalement sur les protéines riches en cystéines (CRP), connues pour contrôler à différents niveaux les communications pollen-pistil. Nous avons développé un outil informatique, KAPPA, qui nous permet de détecter les CRP divergentes dans le transcriptome des tissus reproducteurs de Solanum chacoense et de trois espèces proches. Une fois les protéines candidates identifiées, nous aurons recours à la biologie moléculaire pour en caractériser la structure, la fonction et les interactions.
Wild potatoes (Solanum sect. Petota) gather dozens of species distributed throughout all Latin America. They share three interesting features: (i) their populations coexist in vast sympatric areas; (ii) their flowering period overlap; and (iii) most of them are physiologically able to hybridize with closely related species. Logically, hybrids should be abundant in nature. Yet, floristic inventories done in Argentina and mixed pollination assays carried out in our greenhouses show that hybridization rates are lower than expected. Why? We hypothesize that species-specificity mechanisms act at the molecular level to favour fertilization of ovules by conspecific pollen to the detriment of heterospecific one. These mechanisms would rely on male-female protein pairs, whose correct interaction – depending on the phylogenetic proximity between both sexual partners – determines reproductive success. One such example is the control of pollen tube guidance and attraction. If this phenomenon is confirmed, we would not only better understand the molecular control of sexual reproduction, but also decipher the genetic events underlying speciation of wild potatoes. Using next-generation sequencing technologies we sequenced the ovule transcriptome from three closely related species (Solanum chacoense, S. tarijense and S. gandarillasii), and are now isolating candidate genes using a broad range of bioinformatic tools. The same analysis will also be applied to the pollen transcriptome from the same species. Once candidate genes are isolated, functional analyses through transgenesis will be performed to determine their role in plant reproduction. Here we will analyse the cysteine-rich proteins family (CRPs) that has already been shown to control multiple aspects of pollen-pistil interactions.
Sexual reproduction in flowering plants involves intimate contact and continuous interactions between the growing pollen tube and the female reproductive structures. These interactions can affect gynoecia gene expression well ahead of fertilization. Genes modulated from a distance in the ovary represent one class of such genes. But how precisely can the ovary interprets pollination from a distance in preparation for fertilization? To address this question, transcriptomic analyses were performed on Solanum chacoense ovaries following compatible, incompatible, and heterospecific pollinations, as well as from wounding and touch treatments. In all pollination types, early detection of pollen tubes was perceived as a wounding aggression. As the pollen tubes grew along the style, more and more genes became specific for each pollination type, producing a specific transcriptomic signature. Interestingly, conspecific pollination was marked with regulation of genes related to GA-, ROS-, calcium-, and secondary metabolite-mediated signaling, as well as cell cycle and gene expression, while incompatible pollination remained more akin to a wound response. Our analysis reveals that these pollination types are specifically recognized in the style, and relay this information from a distance to the ovary ahead of fertilization. Similarly to plant–pathogen recognition, it is proposed that every pollen–pistil interaction produces pollen-associated molecular signatures generating diversified and specific responses that convey the current plant pollination status to the ovule in preparation for fertilization.
From the stigma to the ovary, small secreted proteins play pivotal roles in pollen-pistil interactions. They ensure proper pollen tube (PT) growth (germination, elongation, and guidance) and contribute to the establishment of breeding barriers through species-specific interactions. Among them, ovule-secreted proteins (OSPs) were shown to be of particular interest since they control the very last and most critical steps of pollen-pistil interactions such as PT chemoattraction and reception. So far, selection of candidate genes for pollen-ovule interactions has mostly relied on transcriptomics, especially by comparing RNA expression levels between sexually functional and non-functional ovules, or by single cell type analyses. Yet, these approaches do not take into account potential post-transcriptional regulation steps, such as the control of translation initiation, protein storage and secretion. To address this issue, we first developed a new tissue-free gravity-extraction method (tf-GEM) to collect ovule proteinaceous exudates from the wild potato species Solanum chacoense. This, combined with RNA-seq and mass spectrometry-based proteomics led to the identification of 305 OSPs, of which more than half were classified as ovule-specific. Functional annotation showed that these OSPs are predicted to control a broad range of reproductive functions, in particular PT growth and guidance —with several cysteine-rich peptides, a GABA-transaminase, and a PELPIII-like protein— as well as late-acting self-incompatibility S-RNase. Moreover, label-free protein quantification revealed that, from the transition of slightly immature ovules 2 days before anthesis to mature ovules, during which the capacity to attract PTs is acquired, 106 OSPs exhibited a highly significant increase in secretion levels (+14-fold on average) without being regulated at the mRNA level, emphasizing the importance of post-transcriptional regulation of reproductive proteins.
Small, secreted cysteine-rich proteins (CRPs) are known to control key aspects of sexual plant reproduction such as sporophytic self-incompatibility (SCR/SP11), pollen-style interactions (SCA/LTP5, LeSTIG1), as well as pollen tube guidance (TfLUREs, AtLUREs) and reception (EC1, ZmES4) by the female gametophyte. CRPs have a dual sequence structure, involving (i) a highly conserved cysteine spacing pattern –even between distant species– to maintain the disulfide bonds configuration, and (ii) inter-cysteine blocks that can diverge to a large extent. CRPs can thus exhibit a fast evolutive speed while conserving their 3D structure to maintain their function, which explain why several sexual CRPs such as LUREs exhibit a strong species-specificity. Due to this rapid evolution, detection of orthologs and paralogs of already known CRPs with common bioinformatics software has been laborious up to now. In addition, the cysteine backbone itself can evolve and give rise to totally new families of CRPs, but no tool was available so far to detect and cluster CRPs de novo, without relying on a set of reference proteins. KAPPA fills this gap, providing a simple algorithm specifically dedicated to CRPs and other proteins defined by a ‘key aminoacid’. KAPPA precisely and efficiently assesses the similarity of two CRPs using a new quantitative index called κ-score, and utilizes this value as a criterion to detect all CRPs similar to a seed reference, or to build de novo groups of similar CRPs thanks to an ad hoc recursive clustering function. Application of KAPPA to detection and clustering of well-known CRPs such as defensins or lipid-transfer proteins clearly demonstrated its superiority over classical sequence search methods. Its use on proteomic data available from model organisms will then benefit not only sexual plant reproduction research, but also various areas involving CRPs such as medicine (innate immunity), agronomy (resistance to pathogens), and toxicology (snake venoms).
Next-generation sequencing technologies challenge researchers with increasing amounts of data. ‘Big data’ itself becomes a biological issue! For instance, transcriptomics and proteomics now make it easy to describe the compendium of proteins expressed in a given cell or tissue. Bioinformatic tools such as the well-known programs BLAST and HMMER have become indispensable to select sequences relevant to a give n research project. However, peculiar classes of proteins still remain very difficult to grasp with these ‘classical’ algorithms. Cysteine-rich proteins (CRPs) are part of them. They are involved in highly specific recognition functions in humans, animals and plants, and can thus exhibit a fast evolutive speed while conserving their 3D structure to maintain their function. This is why CRPs have a dual sequence structure: the cysteine spacing pattern is highly conserved – even between distant species – to ma intain the disulfide bonds configuration, while other aminoacids can diverge to a large extent. Consequently, sequence identity can be as low as 15% within a given family of CRPs. Detection of orthologs and paralogs of already known CRPs with common bioinf ormatics software has thus been laborious up to now. In addition, the cysteine backbone itself can evolve and give rise to totally new families of CRPs, but no tool was available so far to detect and cluster CRPs de novo, without relying on a set of refere nce proteins. KAPPA fills this gap, providing a simple algorithm specifically dedicated to CRPs and other proteins defined by a ‘key aminoacid’. KAPPA precisely and efficiently assesses the similarity of two CRPs using a new quantitative index called κ-score. This value is computed taking into account various aspects of cysteine spacing consistency (identity and coverage of inter-cysteine blocks, conservation of cysteines themselves) adjusted by means of user-defined stringency parameters. KAPPA then utili zes the κ-score as a criterion to detect all CRPs similar to a seed reference, or to build de novo groups of similar CRPs thanks to an ad hoc recursive clustering function. Application of KAPPA to detection and clustering of well-known CRPs such as defens ins or lipid-transfer proteins clearly demonstrated its superiority over classical sequence search methods. Its use on proteomic data available from model organisms will then benefit various research areas involving CRPs: e.g. medicine (innate immunity), a gronomy (resistance to pathogens), toxicology (snake venoms), or molecular biology (signaling molecules).
Wild potatoes (Solanum sect. Petota) gather dozens of species distributed throughout all Latin America. They share three interesting features: (i) their populations coexist in vast sympatric areas; (ii) their flowering period overlap; and (iii) most of them are physiologically able to hybridize with closely related species. Logically, hybrids should be abundant in nature. Yet, floristic inventories done in Argentina and mixed pollination assays carried out in our greenhouses show that hybridization rates are lower than expected. Why? We hypothesize that species-specificity mechanisms act at the molecular level to favor fertilization of ovules by conspecific pollen to the detriment of heterospecific one. These mechanisms would rely on male-female protein pairs, whose correct interaction – depending on the phylogenetic proximity between both sexual partners – determines reproductive success. Such mechanisms would include numerous checkpoints along the pollen tube growth path. For example, our data show that pollen tubes grow more slowly in heterospecific styles. Further down the path, pollen tubes are also less attracted by heterospecific ovules. Thus pollen tube growth optimization and guidance are crucial for reproductive success. If such phenomenon were confirmed, we would not only better understand the molecular control of sexual reproduction, but also decipher the genetic events underlying speciation of wild potatoes. Using next-generation sequencing technologies we sequenced the ovule transcriptome from three closely related species (Solanum chacoense, Solanum tarijense and Solanum gandarillasii), and are now isolating candidate genes using a broad range of bioinformatic tools. In a first approach, we have chosen to analyze small, secreted peptides, especially the cysteine-rich proteins family (CRPs) that has already been shown to control multiple aspects of pollen-pistil interactions. Using Hidden Markov Models-based sequence search and an original de novo CRP-finding algorithm, we have already detected several clusters of interspecifically divergent proteins with conserved cysteine backbones. By means of differential gene expression analyses between wild type and embryo sac-less mutants, we aim at isolating proteins specifically expressed in the female gametophyte. The same analysis is also being applied to the pollen tube transcriptome from the same species. Once candidate genes are isolated, functional analyses through transgenesis will be performed to determine their role in plant reproduction.
Wild potatoes (Solanum sect. Petota) gather about 200 species that coexist in vast sympatric areas in Latin America. Although they can often hybridize with close relatives, “pure” species are still largely dominant in nature. Why? We hypothesize that species-specificity mechanisms act at the molecular level to favor fertilization of ovules by conspecific pollen to the detriment of heterospecific one. These mechanisms would rely on male-female protein pairs, whose correct interaction – depending on the phylogenetic proximity between both sexual partners – determines reproductive success. Such mechanisms would include numerous checkpoints along the pollen tube growth path. For example, our data show that pollen tubes grow more slowly and are less attracted by ovules in heterospecific female tissues. Here, we aim at discovering the genes responsible for this intraspecific preference. To isolate them, we are exploring the ovule, pollen and style transcriptomes from Solanum chacoense and its two closest relatives. We have chosen to focus first on small, secreted peptides, especially cysteine-rich proteins (CRPs) that are known to control multiple aspects of pollen-pistil interactions. Using HMM-based sequence search and an original de novo CRP-finding algorithm, we have detected several clusters of interspecifically divergent proteins that are now to be assessed functionally.