Flowers and mycorrhizal roots – closer than we think?

Roots and flowers are formed at the extreme ends of plants and they differ in almost every aspect of their development and function; even so, they exhibit surprising molecular commonalities. For example, the calcium and calmodulin-dependent protein kinase (CCaMK) plays a central role in root symbioses with fungi and bacteria, but is also highly expressed in developing anthers. Moreover, independent evidence from transcriptomics, phylogenomics, and genetics reveals common developmental elements in root symbioses and reproductive development. We discuss the significance of these overlaps, and we argue that an integrated comparative view of the two phenomena will stimulate research and provide new insight, not only into shared components, but also into the specific aspects of anther development and root symbioses

Structural evolution drives diversification of the large LRR-RLK gene family

Cells are continuously exposed to chemical signals that they must discriminate between and respond to appropriately. In embryophytes, the leucine‐rich repeat receptor‐like kinases (LRR‐RLKs) are signal receptors critical in development and defense. LRR‐RLKs have diversified to hundreds of genes in many plant genomes. Although intensively studied, a well‐resolved LRR‐RLK gene tree has remained elusive. To resolve the LRR‐RLK gene tree, we developed an improved gene discovery method based on iterative hidden Markov model searching and phylogenetic inference. We used this method to infer complete gene trees for each of the LRR‐RLK subclades and reconstructed the deepest nodes of the full gene family. We discovered that the LRR‐RLK gene family is even larger than previously thought, and that protein domain gains and losses are prevalent. These structural modifications, some of which likely predate embryophyte diversification, led to misclassification of some LRR‐RLK variants as members of other gene families. Our work corrects this misclassification. Our results reveal ongoing structural evolution generating novel LRR‐RLK genes. These new genes are raw material for the diversification of signaling in development and defense. Our methods also enable phylogenetic reconstruction in any large gene family

Plant phosphoinositide-dependent phospholipases C: Variations around a canonical theme

International audiencePhosphoinositide-specific phospholipase C (PI-PLC) cleaves, in a Ca 2þ-dependent manner, phosphati-dylinositol-4,5-bisphosphate (PI-4,5-P 2) into diacylglycerol (DAG) and inositol triphosphate (IP 3). PI-PLCs are multidomain proteins that are structurally related to the PI-PLCzs, the simplest animal PI-PLCs. Like these animal counterparts, they are only composed of EF-hand, X/Y and C2 domains. However, plant PI-PLCs do not have a conventional EF-hand domain since they are often truncated, while some PI-PLCs have no EF-hand domain at all. Despite this simple structure, plant PI-PLCs are involved in many essential plant processes, either associated with development or in response to environmental stresses. The action of PI-PLCs relies on the mediators they produce. In plants, IP 3 does not seem to be the sole active soluble molecule. Inositol pentakisphosphate (IP 5) and inositol hexakisphosphate (IP 6) also transmit signals, thus highlighting the importance of coupling PI-PLC action with inositol-phosphate kinases and phosphatases. PI-PLCs also produce a lipid molecule, but plant PI-PLC pathways show a peculiarity in that the active lipid does not appear to be DAG but its phosphorylated form, phosphatidic acid (PA). Besides, PI-PLCs can also act by altering their substrate levels. Taken together, plant PI-PLCs show functional differences when compared to their animal counterparts. However, they act on similar general signalling pathways including calcium homeostasis and cell phosphoproteome. Several important questions remain unanswered. The cross-talk between the soluble and lipid mediators generated by plant PI-PLCs is not understood and how the coupling between PI-PLCs and inositol-kinases or DAG-kinases is carried out remains to be established

The PTI1-like kinase ZmPti1a from maize (Zea mays L.) co-localizes with callose at the plasma membrane of pollen and facilitates a competitive advantage to the male gametophyte

BACKGROUND: The tomato kinase Pto confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato in a gene for gene manner. Upon recognition of specific avirulence factors the Pto kinase activates multiple signal transduction pathways culminating in induction of pathogen defense. The soluble cytoplasmic serine/threonine kinase Pti1 is one target of Pto phosphorylation and is involved in the hypersensitive response (HR) reaction. However, a clear role of Pti1 in plant pathogen resistance is uncertain. So far, no Pti1 homologues from monocotyledonous species have been studied. RESULTS: Here we report the identification and molecular analysis of four Pti1-like kinases from maize (ZmPti1a, -b, -c, -d). These kinase genes showed tissue-specific expression and their corresponding proteins were targeted to different cellular compartments. Sequence similarity, expression pattern and cellular localization of ZmPti1b suggested that this gene is a putative orthologue of Pti1 from tomato. In contrast, ZmPti1a was specifically expressed in pollen and sequestered to the plasma membrane, evidently owing to N-terminal modification by myristoylation and/or S-acylation. The ZmPti1a:GFP fusion protein was not evenly distributed at the pollen plasma membrane but accumulated as an annulus-like structure which co-localized with callose (1,3-β-glucan) deposition. In addition, co-localization of ZmPti1a and callose was observed during stages of pollen mitosis I and pollen tube germination. Maize plants in which ZmPti1a expression was silenced by RNA interference (RNAi) produced pollen with decreased competitive ability. Hence, our data provide evidence that ZmPti1a plays an important part in a signalling pathway that accelerates pollen performance and male fitness. CONCLUSION: ZmPti1a from maize is involved in pollen-specific processes during the progamic phase of reproduction, probably in crucial signalling processes associated with regions of callose deposition. Pollen-sporophyte interactions and pathogen induced HR show certain similarities. For example, HR has been shown to be associated with cell wall reinforcement through callose deposition. Hence, it is hypothesized that Pti1 kinases from maize act as general components in evolutionary conserved signalling processes associated with callose, however during different developmental programs and in different tissue types

ROS regulation of polar growth in plant cells

Root hair cells and pollen tubes, like fungal hyphae, possess a typical tip or polar cell expansion with growth limited to the apical dome. Cell expansion needs to be carefully regulated to produce a correct shape and size. Polar cell growth is sustained by oscillatory feedback loops comprising three main components that together play an important role regulating this process. One of the main components are reactive oxygen species (ROS) that, together with calcium ions (Ca(2+)) and pH, sustain polar growth over time. Apoplastic ROS homeostasis controlled by NADPH oxidases as well as by secreted type III peroxidases has a great impact on cell wall properties during cell expansion. Polar growth needs to balance a focused secretion of new materials in an extending but still rigid cell wall in order to contain turgor pressure. In this review, we discuss the gaps in our understanding of how ROS impact on the oscillatory Ca(2+) and pH signatures that, coordinately, allow root hair cells and pollen tubes to expand in a controlled manner to several hundred times their original size toward specific signalsFil: Mangano, Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Denita Juárez, Silvina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Estevez, Jose Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Cytological characterization and allelism testing of anther developmental mutants identified in a screen of maize male sterile lines.

Proper regulation of anther differentiation is crucial for producing functional pollen, and defects in or absence of any anther cell type result in male sterility. To deepen understanding of processes required to establish premeiotic cell fate and differentiation of somatic support cell layers a cytological screen of maize male-sterile mutants has been conducted which yielded 42 new mutants including 22 mutants with premeiotic cytological defects (increasing this class fivefold), 7 mutants with postmeiotic defects, and 13 mutants with irregular meiosis. Allelism tests with known and new mutants confirmed new alleles of four premeiotic developmental mutants, including two novel alleles of msca1 and single new alleles of ms32, ms8, and ocl4, and two alleles of the postmeiotic ms45. An allelic pair of newly described mutants was found. Premeiotic mutants are now classified into four categories: anther identity defects, abnormal anther structure, locular wall defects and premature degradation of cell layers, and/or microsporocyte collapse. The range of mutant phenotypic classes is discussed in comparison with developmental genetic investigation of anther development in rice and Arabidopsis to highlight similarities and differences between grasses and eudicots and within the grasses

Defensin-Like ZmES4 Mediates Pollen Tube Burst in Maize via Opening of the Potassium Channel KZM1

Species-preferential osmotic pollen tube burst and sperm discharge in maize involve induced opening of the pollen tube-expressed potassium channel KZM1 by the egg apparatus-derived defensin-like protein ZmES4

Membrane proteins involved in flowering plant gamete interactions

Flowering plants have evolved a unique mode of sexual reproduction termed double fertilization, where two female gametes, the egg cell and the central cell, get fertilized by one sperm cell each. The fertilized egg cell gives rise to the zygote, whereas fertilization of the central cell results in formation of the nutrient-storing and embryo-nourishing endosperm. Successful seed formation requires both fertilization events, but molecular mechanisms regulating gamete recognition, attachment and fusion are poorly understood. Prior to this work, only three proteins residing or acting on plant gamete surfaces were known: The sperm membrane protein GAMETE EXPRESSED2 (GEX2) is involved in pre-fusion attachment whereas HAPLESS2 (HAP2) functions as membrane fusogen. Cysteine-rich EGG CELL1 (EC1) peptides are secreted by the egg cell and act as sperm activating molecules by inducing HAP2 re-localization from endomembrane compartments to the sperm surface. In order to identify novel sperm cell surface proteins with a role in gamete interactions, a protocol for bulk sperm cell isolation from maize was established in this work. Sperm cells were used for RNA-seq transcriptomic profiling and proteomics approaches. The protein composition of membrane-enriched sperm microsomal fractions was analyzed by high-throughput LC-MS/MS. Combined with publicly available Arabidopsis sperm transcriptome data, the maize sperm cell proteome and transcriptome data were used to select candidate genes, or gene families, for functional studies in Arabidopsis. Translational reporter activity in sperm cells was detected for 5 candidates. Assuming functional redundancies, CRISPR/Cas9 was established and genome edited mutants were screened for fertilization defects. Loss-of-function mutants of two sperm-expressed DOMAIN OF UNKNOWN FUNCTION 679 Membrane Proteins (DMP8 and DMP9) displayed severe reproductive defects. dmp8,9 double mutants showed impaired male fertility and up to four unfused dmp8,9 sperm cells were frequently observed close to the female gametes. Beside fertilization failure, also aborted seeds were observed in dmp8,9 siliques, caused by single fertilization events. Quantification revealed that dmp8,9 sperm cells preferentially fertilize the central cell. To address whether gamete adhesion or fusion is impaired in sperm cells lacking functional DMP8 and DMP9, in situ cell adhesion assays were performed and showed that dmp8,9 sperm cells frequently manage to attach to the egg cell, suggesting DMP8 and DMP9 act after gamete attachment but before or during fusion. DMPs are short four-span transmembrane proteins conserved in Viridiplantae (green plants). To gain insight about functionally important DMP9 regions and evolutionary conserved protein function, complementation of the dmp8,9 mutant was performed. 8 Deletion constructs expressing truncated DMP9 versions failed to rescue the mutant phenotype. Complementation with most ancient DMP from Chlamydomonas reinhardtii (CrDMP) was not successful, however a putative DMP9 ortholog from Amborella trichopoda (AmTrDMP) managed to partially rescue the fusion-defective dmp8,9 phenotype, suggesting functional conservation at least in flowering plants. The biochemical function of DMPs is not yet known, but Arabidopsis thaliana DMP1 was suggested to be involved in membrane remodeling. Transient overexpression of DMP9-GFP in tobacco leaf epidermis cells induced DMP1-like membrane remodeling events, which are likely artificial. Mutant dmp8,9 sperm cells were furthermore used as a tool to investigate sperm-induced events in the egg cell. Upon sperm cell arrival, the Arabidopsis egg cell secretes EC1 proteins to render the sperm cells competent for fusion but the precise moment of triggered EC1 secretion is not yet known. Quantification of EC1-GFP signals in unfertilized egg cells and in those with adjacent, unfused dmp8,9 sperm cells gave evidence that the secretion of EC1-GFP by the egg cell does not depend on DMP8/9 and is upstream of sperm adhesion. Furthermore, in mammals two plasma membrane-localized TETRASPANINS (TETs) are crucial for gamete interactions and accumulate at the egg-sperm contact site to form fusion competent membrane patches. To investigate whether a similar scenario holds true during flowering plant gamete interactions, the subcellular distribution of egg- and central cell-expressed Arabidopsis thaliana TET9-GFP was monitored during interaction with dmp8,9 sperm cells. A role for TET9 in the formation of fusion-competent sites seems unlikely, as TET9-GFP remained uniformly distributed at the egg- and central cell plasma membrane with attached but unfused dmp8,9 sperm cells. This suggests that TETs expressed in angiosperm gametes might fulfil different, yet unknown functions. In summary, two sperm-expressed membrane proteins were discovered that function after gamete attachment to facilitate gamete fusion. It will be an important task for the future to unravel the biochemical-mechanistic function of DMP8 and DMP9, which may involve membrane remodeling, supporting the activity of the fusogen HAP2, or the activation or delivery of HAP2 to the sperm cell surface. The maize sperm transcriptomic and proteomic datasets that have been generated in this work will serve as valuable resources for the identification of more membrane-localized proteins with a role during gamete interaction and fusion

A computational-based update on microRNAs and their targets in barley (Hordeum vulgare L.)

<p>Abstract</p> <p>Background</p> <p>Many plant species have been investigated in the last years for the identification and characterization of the corresponding miRNAs, nevertheless extensive studies are not yet available on barley (at the time of this writing). To extend and to update information on miRNAs and their targets in barley and to identify candidate polymorphisms at miRNA target sites, the features of previously known plant miRNAs have been used to systematically search for barley miRNA homologues and targets in the publicly available ESTs database. Matching sequences have then been related to Unigene clusters on which most of this study was based.</p> <p>Results</p> <p>One hundred-fifty-six microRNA mature sequences belonging to 50 miRNA families have been found to significantly match at least one EST sequence in barley. As expected on the basis of phylogenetic relations, miRNAs putatively orthologous to those of <it>Triticum </it>are significantly over-represented inside the set of identified barley microRNA mature sequences. Many previously known and several putatively new miRNA/target pairs have been identified. When the predicted microRNA targets were grouped into functional categories, biological processes previously known to be regulated by miRNAs, such as development and response to biotic and abiotic stress, have been highlighted and most of the target molecular functions were related to transcription regulation. Candidate microRNA coding genes have been reported and genetic variation (SNPs/indels) both in functional regions of putative miRNAs (mature sequence) and at miRNA target sites has been found.</p> <p>Conclusions</p> <p>This study has provided an update of the information on barley miRNAs and their targets representing a foundation for future studies. Many of previously known plant microRNAs have homologues in barley with expected important roles during development, nutrient deprivation, biotic and abiotic stress response and other important physiological processes. Putative polymorphisms at miRNA target sites have been identified and they can represent an interesting source for the identification of functional genetic variability.</p