Pollen Tube Cell Wall Research Articles

LRX Proteins Play a Crucial Role in Pollen Grain and Pollen Tube Cell Wall Development.

Leu-rich repeat extensins (LRXs) are chimeric proteins containing an N-terminal Leu-rich repeat (LRR) and a C-terminal extensin domain. LRXs are involved in cell wall formation in vegetative tissues and required for plant growth. However, the nature of their role in these cellular processes remains to be elucidated. Here, we used a combination of molecular techniques, light microscopy, and transmission electron microscopy to characterize mutants of pollen-expressed LRXs in Arabidopsis (Arabidopsisthaliana). Mutations in multiple pollen-expressed lrx genes cause severe defects in pollen germination and pollen tube growth, resulting in a reduced seed set. Physiological experiments demonstrate that manipulating Ca2+ availability partially suppresses the pollen tube growth defects, suggesting that LRX proteins influence Ca2+-related processes. Furthermore, we show that LRX protein localizes to the cell wall, and its LRR-domain (which likely mediates protein-protein interactions) is associated with the plasma membrane. Mechanical analyses by cellular force microscopy and finite element method-based modeling revealed significant changes in the material properties of the cell wall and the fine-tuning of cellular biophysical parameters in the mutants compared to the wild type. The results indicate that LRX proteins might play a role in cell wall-plasma membrane communication, influencing cell wall formation and cellular mechanics.

Efficient preparation of Arabidopsis pollen tubes for ultrastructural analysis using chemical and cryo-fixation

BackgroundThe pollen tube (PT) serves as a model system for investigating plant cell growth and morphogenesis. Ultrastructural studies are indispensable to complement data from physiological and genetic analyses, yet an effective method is lacking for PTs of the model plant Arabidopsis thaliana.MethodsHere, we present reliable approaches for ultrastructural studies of Arabidopsis PTs, as well as an efficient technique for immunogold detection of cell wall epitopes. Using different fixation and embedding strategies, we show the amount of PT ultrastructural details that can be obtained by the different methods. ResultsDozens of cross-sections can be obtained simultaneously by the approach, which facilitates and shortens the time for evaluation. In addition to in vitro-grown PTs, our study follows the route of PTs from germination, growth along the pistil, to the penetration of the dense stylar tissue, which requires considerable mechanical forces. To this end, PTs have different strategies from growing between cells but also between the protoplast and the cell wall and even within each other, where they share a partly common cell wall. The separation of PT cell walls in an outer and an inner layer reported for many plant species is less clear in Arabidopsis PTs, where these cell wall substructures are connected by a distinct transition zone.ConclusionsThe major advancement of this method is the effective production of a large number of longitudinal and cross-sections that permits obtaining a detailed and representative picture of pollen tube structures in an unprecedented way. This is particularly important when comparing PTs of wild type and mutants to identify even subtle alterations in cytoarchitecture. Arabidopsis is an excellent plant for genetic manipulation, yet the PTs, several-times smaller compared to tobacco or lily, represent a technical challenge. This study reveals a method to overcome this problem and make Arabidopsis PTs more amenable to a combination of genetic and ultrastructural analyses.

Physiological influence of silica on germinating pollen as shown by Raman spectroscopy.

The process of silicification in plants and the biochemical effects of silica in plant tissues are largely unknown. To study the molecular changes occurring in growing cells that are exposed to higher than normal concentration of silicic acid, Raman spectra of germinating pollen grains of three species (Pinus nigra, Picea omorika, and Camellia japonica) were analyzed in a multivariate classification approach that takes into account the variation of biochemical composition due to species, plant tissue structure, and germination condition. The results of principal component analyses of the Raman spectra indicate differences in the utilization of stored lipids, a changed mobilization of storage carbohydrates in the pollen grain bodies, and altered composition and/or structure of cellulose of the developing pollen tube cell walls. These biochemical changes vary in the different species.

In silico prediction of proteins related to xyloglucan fucosyltransferases in Solanaceae genomes

Two independent studies have shown that the cell wall of pollen tubes from tobacco and tomato species contained fucosylated xyloglucan (XyG). These findings are intriguing as many reports have shown that XyG of somatic cells of these species is not fucosylated but instead is arabinosylated. In order to produce fucosylated XyG, plants must express a functional galactoside α-2-fucosyltransferase. Here, using a bioinformatics approach, we show that several candidate genes coding for XyG fucosyltransferases are present in the genome of coffee and several Solanaceae species including tomato, tobacco, potato, eggplant and pepper. BLAST and protein alignments with the 2 well-characterized XyG fucosyltransferases from Arabidopsis thaliana and Pisum sativum revealed that at least 6 proteins from different Solanaceae species and from coffee displayed the 3 conserved motifs required for XyG fucosyltransferase activity.

Pollen tube cell walls of wild and domesticated tomatoes contain arabinosylated and fucosylated xyloglucan

In flowering plants, fertilization relies on the delivery of the sperm cells carried by the pollen tube to the ovule. During the tip growth of the pollen tube, proper assembly of the cell wall polymers is required to maintain the mechanical properties of the cell wall. Xyloglucan (XyG) is a cell wall polymer known for maintaining the wall integrity and thus allowing cell expansion. In most angiosperms, the XyG of somatic cells is fucosylated, except in the Asterid clade (including the Solanaceae), where the fucosyl residues are replaced by arabinose, presumably due to an adaptive and/or selective diversification. However, it has been shown recently that XyG of Nicotiana alata pollen tubes is mostly fucosylated. The objective of the present work was to determine whether such structural differences between somatic and gametophytic cells are a common feature of Nicotiana and Solanum (more precisely tomato) genera. XyGs of pollen tubes of domesticated (Solanum lycopersicum var. cerasiforme and var. Saint-Pierre) and wild (S. pimpinellifolium and S. peruvianum) tomatoes and tobacco (Nicotiana tabacum) were analysed by immunolabelling, oligosaccharide mass profiling and GC-MS analyses. Pollen tubes from all the species were labelled with the mAb CCRC-M1, a monoclonal antibody that recognizes epitopes associated with fucosylated XyG motifs. Analyses of the cell wall did not highlight major structural differences between previously studied N. alata and N. tabacum XyG. In contrast, XyG of tomato pollen tubes contained fucosylated and arabinosylated motifs. The highest levels of fucosylated XyG were found in pollen tubes from the wild species. The results clearly indicate that the male gametophyte (pollen tube) and the sporophyte have structurally different XyG. This suggests that fucosylated XyG may have an important role in the tip growth of pollen tubes, and that they must have a specific set of functional XyG fucosyltransferases, which are yet to be characterized.

Dynamic, high precision targeting of growth modulating agents is able to trigger pollen tube growth reorientation.

The pollen tube is the most rapidly growing cell in the plant kingdom and has the function to deliver the sperm cells for fertilization. The growing tip region of the cell behaves in a chemotropic manner to respond to the guidance cues emitted by the pistil and the female gametophyte, but how it perceives and responds to these directional triggers is virtually unknown. Quantitative assessment of chemotropic behavior can greatly be enhanced by the administration of pharmacological or other biologically active agents at subcellular precision, which is a technical challenge when the target area moves as it grows. We developed a laminar flow based microfluidic device that allows for continuous administration of two different solutions with a movable interface that permits the dynamic targeting of the growing pollen tube apex over prolonged periods of time. Asymmetric administration of calcium revealed that rather than following the highest calcium concentration as would be expected with simple chemotropic behavior, the pollen tube of Camellia targets an optimal concentration suggesting the presence of two superimposed mechanisms. Subcellular application of pectin methyl esterase (PME), an enzyme that modifies the growth behavior by rigidifying the pollen tube cell wall, caused the tube to turn away from the agent - providing important evidence for a previously proposed conceptual model of the growth mechanism.

Regulation of pollen tube growth by reversible protein phosphorylation

The tip-growth of pollen tube is regulated by diverse signaling and metabolic processes, including Rop GTPase signaling pathway, phosphatidylinositol signaling pathway, Ca(2+) signaling, actin dynamics, vesicular trafficking, and cell wall re-modulation. These processes are regulated by reversible protein phosphorylation events: (1) The activities of Rop regulatory proteins (GEF, GDI, and GAP) that regulate Rop GTPase are variable under different protein phosphorylation states. In addition, various protein kinases function as the downstream effectors of Rop GTPase, and participate in the downstream pathways of Rop signaling. (2) Reversible protein phosphorylation can activate/inactivate the plasma membrane (PM) Ca(2+) channels and/or Ca(2+) pump, as well as control the release of intracellular Ca(2+), thereby regulating the formation of Ca(2+) gradient in pollen tube tip. In addition, protein kinases function as Ca(2+) sensors and phosphorylate the target proteins involved in the downstream regulatory pathways of Ca(2+) signaling. (3) The dynamics of actin polymerization and depolymerization are regulated by reversible phosphorylation of actin binding proteins (e.g., ADF and profilin). (4) Reversible protein phosphorylation regulates the activities of endo/exocytosis-related proteins and PM phospholipid metabolism that are involved in the membrane trafficking. (5) Reversible protein phosphorylation participates in the pollen tube cell wall re-modulation through regulating the function and subcellular distribution of cytoplasmic serine/threonine protein kinase and sucrose synthase. (6) The activities of transcription regulatory protein and eukaryotic translation initiation factor are regulated by protein phosphorylation, modulating the RNA transcription and protein synthesis. In this review, we present an overview of the functions of protein reversible phosphorylation in the aforementioned processes during the pollen tube tip-growth.

Sucrose concentration in the growth medium affects the cell wall composition of tobacco pollen tubes.

The cell wall of pollen tubes is organized in both spatial and temporal order to allow the pollen tube to grow according to external conditions. The deposition of methyl-esterified and acid pectins in addition to callose/cellulose occurs according to a series of temporally succeeding events. In this work, we attempted to determine how the composition of the external growth medium (in terms of osmolarity) could affect the deposition of cell wall components. Pollen tubes of tobacco were grown in a hypotonic medium and then analyzed for the distribution of pectins and callose/cellulose [as well as for the distribution of the enzyme callose synthase (CALS)]. The data indicate that pollen tubes grown in a hypotonic medium show changes of the initial growth rate followed by modification of the deposition of acid pectins and, to a lesser extent, of CALS. These observations indicate that, under the osmolarity determined by the growth medium, pollen tubes adapt their cell wall to the changing conditions of growth.

The cell wall pectic polymer rhamnogalacturonan-II is required for proper pollen tube elongation: implications of a putative sialyltransferase-like protein.

Rhamnogalacturonan-II (RG-II) is one of the pectin motifs found in the cell wall of all land plants. It contains sugars such as 2-keto-3-deoxy-d-lyxo-heptulosaric acid (Dha) and 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo), and within the wall RG-II is mostly found as a dimer via a borate diester cross-link. To date, little is known regarding the biosynthesis of this motif. Here, after a brief review of our current knowledge on RG-II structure, biosynthesis and function in plants, this study explores the implications of the presence of a Golgi-localized sialyltransferase-like 2 (SIA2) protein that is possibly involved in the transfer of Dha or Kdo in the RG-II of Arabidopsis thaliana pollen tubes, a fast-growing cell type used as a model for the study of cell elongation. Two heterozygous mutant lines of arabidopsis (sia2-1+/- and qrt1 × sia2-2+/-) were investigated. sia2-2+/- was in a quartet1 background and the inserted T-DNA contained the reporter gene β-glucuronidase (GUS) under the pollen-specific promoter LAT52. Pollen germination and pollen tube phenotype and growth were analysed both in vitro and in vivo by microscopy. Self-pollination of heterozygous lines produced no homozygous plants in the progeny, which may suggest that the mutation could be lethal. Heterozygous mutants displayed a much lower germination rate overall and exhibited a substantial delay in germination (20 h of delay to reach 30 % of pollen grain germination compared with the wild type). In both lines, mutant pollen grains that were able to produce a tube had tubes that were either bursting, abnormal (swollen or dichotomous branching tip) or much shorter compared with wild-type pollen tubes. In vivo, mutant pollen tubes were restricted to the style, whereas the wild-type pollen tubes were detected at the base of the ovary. This study highlights that the mutation in arabidopsis SIA2 encoding a sialyltransferase-like protein that may transfer Dha or Kdo on the RG-II motif has a dramatic effect on the stability of the pollen tube cell wall.

The LI-rings in pollen tubes

Monoclonal antibodies, which recognize the specific epitopes for pectins and arabinogalactan proteins, in connection with confocal laser scanning microscopy demonstrated the presence of a ring-like structure in the cell wall of pollen tubes of flowering plants.

Apical F‐actin‐regulated exocytic targeting of NtPPME1 is essential for construction and rigidity of the pollen tube cell wall

In tip-confined growing pollen tubes, delivery of newly synthesized cell wall materials to the rapidly expanding apical surface requires spatial organization and temporal regulation of the apical F-actin filament and exocytosis. In this study, we demonstrate that apical F-actin is essential for the rigidity and construction of the pollen tube cell wall by regulating exocytosis of Nicotiana tabacum pectin methylesterase (NtPPME1). Wortmannin disrupts the spatial organization of apical F-actin in the pollen tube tip and inhibits polar targeting of NtPPME1, which subsequently alters the rigidity and pectic composition of the pollen tube cell wall, finally causing growth arrest of the pollen tube. In addition to mechanistically linking cell wall construction and apical F-actin, wortmannin can be used as a useful tool for studying endomembrane trafficking and cytoskeletal organization in pollen tubes.

An unconventional road for the secretion of transglutaminase in pollen tubes?

The transglutaminase (TGase) is present in the pollen tube where it most likely participates in the regulation of different activities including the organization of cytoskeletal elements (microtubules and actin filaments). In addition to a cytosolic form of TGase, new data suggest the existence of TGase forms associated with the internal membranes and with the cell wall of pollen tubes. This different localization extends the functional range of pollen TGase but also raises the question how TGase can be precisely (and in harmony with the pollen tube growth) redistributed in different cellular compartments. The discovery that TGase exists as different isoforms may suggest a pathway to achieve this result.

Electrophoretic profiling and immunocytochemical detection of pectins and arabinogalactan proteins in olive pollen during germination and pollen tube growth

Cell wall pectins and arabinogalactan proteins (AGPs) are important for pollen tube growth. The aim of this work was to study the temporal and spatial dynamics of these compounds in olive pollen during germination. Immunoblot profiling analyses combined with confocal and transmission electron microscopy immunocytochemical detection techniques were carried out using four anti-pectin (JIM7, JIM5, LM5 and LM6) and two anti-AGP (JIM13 and JIM14) monoclonal antibodies. Pectin and AGP levels increased during olive pollen in vitro germination. (1 → 4)-β-d-Galactans localized in the cytoplasm of the vegetative cell, the pollen wall and the apertural intine. After the pollen tube emerged, galactans localized in the pollen tube wall, particularly at the tip, and formed a collar-like structure around the germinative aperture. (1 → 5)-α-l-Arabinans were mainly present in the pollen tube cell wall, forming characteristic ring-shaped deposits at regular intervals in the sub-apical zone. As expected, the pollen tube wall was rich in highly esterified pectic compounds at the apex, while the cell wall mainly contained de-esterified pectins in the shank. The wall of the generative cell was specifically labelled with arabinans, highly methyl-esterified homogalacturonans and JIM13 epitopes. In addition, the extracellular material that coated the outer exine layer was rich in arabinans, de-esterified pectins and JIM13 epitopes. Pectins and AGPs are newly synthesized in the pollen tube during pollen germination. The synthesis and secretion of these compounds are temporally and spatially regulated. Galactans might provide mechanical stability to the pollen tube, reinforcing those regions that are particularly sensitive to tension stress (the pollen tube-pollen grain joint site) and mechanical damage (the tip). Arabinans and AGPs might be important in recognition and adhesion phenomena of the pollen tube and the stylar transmitting cells, as well as the egg and sperm cells.

Cell Wall Composition, Biosynthesis and Remodeling during Pollen Tube Growth.

The pollen tube is a fast tip-growing cell carrying the two sperm cells to the ovule allowing the double fertilization process and seed setting. To succeed in this process, the spatial and temporal controls of pollen tube growth within the female organ are critical. It requires a massive cell wall deposition to promote fast pollen tube elongation and a tight control of the cell wall remodeling to modify the mechanical properties. In addition, during its journey, the pollen tube interacts with the pistil, which plays key roles in pollen tube nutrition, guidance and in the rejection of the self-incompatible pollen. This review focuses on our current knowledge in the biochemistry and localization of the main cell wall polymers including pectin, hemicellulose, cellulose and callose from several pollen tube species. Moreover, based on transcriptomic data and functional genomic studies, the possible enzymes involved in the cell wall remodeling during pollen tube growth and their impact on the cell wall mechanics are also described. Finally, mutant analyses have permitted to gain insight in the function of several genes involved in the pollen tube cell wall biosynthesis and their roles in pollen tube growth are further discussed.

Regulation of Pollen Tube Growth by Transglutaminase.

In pollen tubes, cytoskeleton proteins are involved in many aspects of pollen germination and growth, from the transport of sperm cells to the asymmetrical distribution of organelles to the deposition of cell wall material. These activities are based on the dynamics of the cytoskeleton. Changes to both actin filaments and microtubules are triggered by specific proteins, resulting in different organization levels suitable for the different functions of the cytoskeleton. Transglutaminases are enzymes ubiquitous in all plant organs and cell compartments. They catalyze the post-translational conjugation of polyamines to different protein targets, such as the cytoskeleton. Transglutaminases are suggested to have a general role in the interaction between pollen tubes and the extracellular matrix during fertilization and a specific role during the self-incompatibility response. In such processes, the activity of transglutaminases is enhanced, leading to the formation of cross-linked products (including aggregates of tubulin and actin). Consequently, transglutaminases are suggested to act as regulators of cytoskeleton dynamics. The distribution of transglutaminases in pollen tubes is affected by both membrane dynamics and the cytoskeleton. Transglutaminases are also secreted in the extracellular matrix, where they may take part in the assembly and/or strengthening of the pollen tube cell wall.

Quantification of the Young's modulus of the primary plant cell wall using Bending-Lab-On-Chip (BLOC)

Biomechanical and mathematical modeling of plant developmental processes requires quantitative information about the structural and mechanical properties of living cells, tissues and cellular components. A crucial mechanical property of plant cells is the mechanical stiffness or Young's modulus of its cell wall. Measuring this property in situ at single cell wall level is technically challenging. Here, a bending test is implemented in a chip, called Bending-Lab-On-a-Chip (BLOC), to quantify this biomechanical property for a widely investigated cellular model system, the pollen tube. Pollen along with culture medium is introduced into a microfluidic chip and the growing pollen tube is exposed to a bending force created through fluid loading. The flexural rigidity of the pollen tube and the Young's modulus of the cell wall are estimated through finite element modeling of the observed fluid-structure interaction. An average value of 350 MPa was experimentally estimated for the Young's modulus in longitudinal direction of the cell wall of Camellia pollen tubes. This value is in agreement with the result of an independent method based on cellular shrinkage after plasmolysis and with the mechanical properties of in vitro reconstituted cellulose-callose material.

The cell wall of the Arabidopsis pollen tube--spatial distribution, recycling, and network formation of polysaccharides.

The pollen tube is a cellular protuberance formed by the pollen grain, or male gametophyte, in flowering plants. Its principal metabolic activity is the synthesis and assembly of cell wall material, which must be precisely coordinated to sustain the characteristic rapid growth rate and to ensure geometrically correct and efficient cellular morphogenesis. Unlike other model species, the cell wall of the Arabidopsis (Arabidopsis thaliana) pollen tube has not been described in detail. We used immunohistochemistry and quantitative image analysis to provide a detailed profile of the spatial distribution of the major cell wall polymers composing the Arabidopsis pollen tube cell wall. Comparison with predictions made by a mechanical model for pollen tube growth revealed the importance of pectin deesterification in determining the cell diameter. Scanning electron microscopy demonstrated that cellulose microfibrils are oriented in near longitudinal orientation in the Arabidopsis pollen tube cell wall, consistent with a linear arrangement of cellulose synthase CESA6 in the plasma membrane. The cellulose label was also found inside cytoplasmic vesicles and might originate from an early activation of cellulose synthases prior to their insertion into the plasma membrane or from recycling of short cellulose polymers by endocytosis. A series of strategic enzymatic treatments also suggests that pectins, cellulose, and callose are highly cross linked to each other.

Chemically Mediated Mechanical Expansion of the Pollen Tube Cell Wall

Morphogenesis of plant cells is tantamount to the shaping of the stiff cell wall that surrounds them. To this end, these cells integrate two concomitant processes: 1), deposition of new material into the existing wall, and 2), mechanical deformation of this material by the turgor pressure. However, due to uncertainty regarding the mechanisms that coordinate these processes, existing models typically adopt a limiting case in which either one or the other dictates morphogenesis. In this report, we formulate a simple mechanism in pollen tubes by which deposition causes turnover of cell wall cross-links, thereby facilitating mechanical deformation. Accordingly, deposition and mechanics are coupled and are both integral aspects of the morphogenetic process. Among the key experimental qualifications of this model are: its ability to precisely reproduce the morphologies of pollen tubes; its prediction of the growth oscillations exhibited by rapidly growing pollen tubes; and its prediction of the observed phase relationships between variables such as wall thickness, cell morphology, and growth rate within oscillatory cells. In short, the model captures the rich phenomenology of pollen tube morphogenesis and has implications for other plant cell types.

Developmental evolution of flowering plant pollen tube cell walls: callose synthase (CalS) gene expression patterns

BackgroundA number of innovations underlie the origin of rapid reproductive cycles in angiosperms. A critical early step involved the modification of an ancestrally short and slow-growing pollen tube for faster and longer distance transport of sperm to egg. Associated with this shift are the predominantly callose (1,3-β-glucan) walls and septae (callose plugs) of angiosperm pollen tubes. Callose synthesis is mediated by callose synthase (CalS). Of 12 CalS gene family members in Arabidopsis, only one (CalS5) has been directly linked to pollen tube callose. CalS5 orthologues are present in several monocot and eudicot genomes, but little is known about the evolutionary origin of CalS5 or what its ancestral function may have been.ResultsWe investigated expression of CalS in pollen and pollen tubes of selected non-flowering seed plants (gymnosperms) and angiosperms within lineages that diverged below the monocot/eudicot node. First, we determined the nearly full length coding sequence of a CalS5 orthologue from Cabomba caroliniana (CcCalS5) (Nymphaeales). Semi-quantitative RT-PCR demonstrated low CcCalS5 expression within several vegetative tissues, but strong expression in mature pollen. CalS transcripts were detected in pollen tubes of several species within Nymphaeales and Austrobaileyales, and comparative analyses with a phylogenetically diverse group of sequenced genomes indicated homology to CalS5. We also report in silico evidence of a putative CalS5 orthologue from Amborella. Among gymnosperms, CalS5 transcripts were recovered from germinating pollen of Gnetum and Ginkgo, but a novel CalS paralog was instead amplified from germinating pollen of Pinus taeda.ConclusionThe finding that CalS5 is the predominant callose synthase in pollen tubes of both early-diverging and model system angiosperms is an indicator of the homology of their novel callosic pollen tube walls and callose plugs. The data suggest that CalS5 had transient expression and pollen-specific functions in early seed plants and was then recruited to novel expression patterns and functions within pollen tube walls in an ancestor of extant angiosperms.

How do microtubules affect deposition of cell wall polysaccharides in the pollen tube?

Callose is the primary polysaccharide present in the so-called secondary layer of the pollen tube cell wall while the content of cellulose in such layer is usually lower. Despite its lower quantity, cellulose might be potentially able to establish the growth direction of pollen tubes. Microtubules have been shown to regulate the deposition of callose synthase in the distal regions of pollen tubes related to the synthesis of callose plugs. However, the interplay between microtubules and cellulose synthase in the pollen tube is unclear. Here, the hypothetical role of microtubules and microtubule-based motor proteins in controlling the insertion of cellulose synthase in relation with growth and directionality of pollen tubes is discussed.