Degradation of S-RNase in compatible pollen tubes of Solanum chacoense inferred by immunogold labeling.

The flowering plant Solanum chacoense uses an S-RNase-based self-incompatibility system in order to reject pollen that shares the same genes at the S-locus (S-haplotype) with the style (an incompatible reaction). Two different models have been advanced to explain how compatible pollen tubes are protected from the cytotoxic effects of the S-RNase, sequestration of the S-RNase in a vacuolar compartment or degradation of the S-RNase in the cytoplasm. Here, we examine the subcellular distribution of an S11-RNase 18 and 24 h post pollination (hpp) in compatible and incompatible crosses by immunogold labeling and transmission electron microscopy. We find that the S-RNase is present in the cytoplasm of both compatible and incompatible crosses by 18 hpp, but that almost all the cytoplasmic S-RNase is degraded by 24 hpp in compatible crosses. These results provide compelling evidence that S-RNases are degraded in compatible but not in incompatible pollen tubes.

Quantification of cellular penetrative forces using lab-on-a-chip technology and finite element modeling.

Tip-growing cells have the unique property of invading living tissues and abiotic growth matrices. To do so, they exert significant penetrative forces. In plant and fungal cells, these forces are generated by the hydrostatic turgor pressure. Using the TipChip, a microfluidic lab-on-a-chip device developed for tip-growing cells, we tested the ability to exert penetrative forces generated in pollen tubes, the fastest-growing plant cells. The tubes were guided to grow through microscopic gaps made of elastic polydimethylsiloxane material. Based on the deformation of the gaps, the force exerted by the elongating tubes to permit passage was determined using finite element methods. The data revealed that increasing mechanical impedance was met by the pollen tubes through modulation of the cell wall compliance and, thus, a change in the force acting on the obstacle. Tubes that successfully passed a narrow gap frequently burst, raising questions about the sperm discharge mechanism in the flowering plants.

Zm908p11, encoded by a short open reading frame (sORF) gene, functions in pollen tube growth as a profilin ligand in maize.

Double fertilization of flowering plants depends on the targeted transportation of sperm to the embryo sac by the pollen tube. Currently, little is known about the underlying molecular mechanisms that regulate pollen germination and pollen tube growth in maize (Zea mays). Here, a maize pollen-predominant gene Zm908, with several putative short open reading frames (sORFs), was isolated and characterized. The longest ORF of Zm908 encodes a small protein of 97 amino acids. This was designated as Zm908p11 and is distributed throughout the maize pollen tube. Western blot detected the small peptide in mature pollen. Quantitative reverse transcription-PCR and northern blot analysis revealed that Zm908p11 was expressed predominantly in mature pollen grains. Ectopic overexpression of full-length Zm908 and Zm908p11 in tobacco resulted in defective pollen, while transgenic tobacco plants with a site-specific mutation or a frameshift mutation of Zm908p11 showed normal pollen development. Overexpression of Zm908p11 in maize decreased pollen germination efficiency. Maize pollen cDNA library screening and protein-protein interaction assays demonstrated that Zm908p11 interacts with maize profilin 1 (ZmPRO1). A microarray analysis identified 273 up-regulated and 203 down-regulated genes in the overexpressing transgenic Zm908p11 pollen. Taken together, these results indicate that Zm908 functions as Zm908p11, and binds to profilins as a novel ligand, with a required role during pollen tube growth in maize. Accordingly, a model is proposed for the role of Zm908p11 during pollen tube growth in maize.

A signaling peptide locks pollen tube walls.

A protein-peptide complex generates and stabilizes a cell-wall carbohydrate lattice.

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.

Protein Analysis of Pollen Tubes after the Treatments of Membrane Trafficking Inhibitors Gains Insights on Molecular Mechanism Underlying Pollen Tube Polar Growth.

Pollen tube elongation is characterized by a highly-polarized tip growth process dependent on an efficient vesicular transport system and largely mobilized by actin cytoskeleton. Pollen tubes are an ideal model system to study exocytosis, endocytosis, membrane recycling, and signaling network coordinating cellular processes, structural organization and vesicular trafficking activities required for tip growth. Proteomic analysis was applied to identify Nicotiana tabacum Differentially Abundant Proteins (DAPs) after in vitro pollen tube treatment with membrane trafficking inhibitors Brefeldin A, Ikarugamycin and Wortmannin. Among roughly 360 proteins separated in two-dimensional gel electrophoresis, a total of 40 spots visibly changing between treated and control samples were identified by MALDI-TOF MS and LC-ESI-MS/MS analysis. The identified proteins were classified according to biological processes, and most proteins were related to pollen tube energy metabolism, including ammino acid synthesis and lipid metabolism, structural features of pollen tube growth as well modification and actin cytoskeleton organization, stress response, and protein degradation. In-depth analysis of proteins corresponding to energy-related pathways revealed the male gametophyte to be a reliable model of energy reservoir and dynamics.

How to shape a cylinder: pollen tube as a model system for the generation of complex cellular geometry.

Expansive growth in plant cells is a formidable problem for biophysical studies, and the mechanical principles governing the generation of complex cellular geometries are still poorly understood. Pollen, the male gametophyte stage of the flowering plants, is an excellent model system for the investigation of the mechanics of complex growth processes. The initiation of pollen tube growth requires first of all, the spatially confined formation of a protuberance. This process must be controlled by the mechanical properties of the cell wall, since turgor is a non-vectorial force. In the elongating tube, cell wall expansion is confined to the apex of the cell, requiring the tubular region to be stabilized against turgor-induced tensile stress. Tip focused surface expansion must be coordinated with the supply of cell wall material to this region requiring the precise, logistical control of intracellular transport processes. The advantage of such a demanding mechanism is the high efficiency it confers on the pollen tube in leading an invasive way of life.

Overexpression of PwTUA1, a pollen-specific tubulin gene, increases pollen tube elongation by altering the distribution of alpha-tubulin and promoting vesicle transport.

Tubulin genes are intimately associated with cell division and cell elongation, which are central to plant secondary cell wall development. However, their roles in pollen tube polar growth remain elusive. Here, a TUA1 gene from Picea wilsonii, which is specifically expressed in pollen, was isolated. Semi-quantitative RT-PCR analysis showed that the amount of PwTUA1 transcript varied at each stage of growth of the pollen tube and was induced by calcium ions and boron. Transient expression analysis in P. wilsonii pollen indicated that PwTUA1 improved pollen germination and pollen tube growth. The pollen of transgenic Arabidopsis overexpressing PwTUA1 also showed a higher percentage of germination and faster growth than wild-type plants not only in optimal germination medium, but also in medium supplemented with elevated levels of exogenous calcium ions or boron. Immunofluorescence and electron microscopy showed alpha-tubulin to be enriched and more vesicles accumulated in the apex region in germinating transgenic Arabidopsis pollen compared with wild-type plants. These results demonstrate that PwTUA1 up-regulated by calcium ions and boron contributes to pollen tube elongation by altering the distribution of alpha-tubulin and regulating the deposition of pollen cell wall components during the process of tube growth. The possible role of PwTUA1 in microtubule dynamics and organization was discussed.

In situ assessment of mitochondrial calcium transport in tobacco pollen tubes.

Pollen tubes require functional mitochondria in order to achieve fast and sustained growth. In addition, cell wall expansion requires a calcium gradient in the tube apex formed by a dedicated array of calcium pumps and channels. Most studies have traditionally focused on the molecular aspects of calcium interactions and transport across the pollen tube plasmalemma. However, calcium transients across mitochondrial membranes from pollen tubes are beginning to be studied. Here, we report the presence of a ruthenium red-sensitive mitochondrial calcium uniporter-like activity in tobacco pollen tubes with functional oxidative phosphorylation. The present study provides a framework to measure in situ specifics of mitochondrial transport and respiration in pollen tubes from different plants. The relevance of a mitochondrial calcium uniporter for pollen tube growth is discussed.

IAA stimulates pollen tube growth and mediates the modification of its wall composition and structure in Torenia fournieri.

The effects of several hormones on pollen tube growth were compared in Torenia fournieri and it was found that IAA was the most effective, stimulating pollen tube growth and causing the shank part of pollen tubes to be slender and straighter. The role of IAA was investigated by studying the changes in ultrastructure and PM H(+)-ATPase distribution in the pollen tubes and the modification of the tube wall. Using the fluorescent marker FM4-64, together with transmission electron microscopy, it was shown that secretory vesicles and mitochondria increased in IAA-treated tubes. Immunolocalization and fluorescence labelling, together with Fourier-transform infrared analysis, detected that IAA enhanced the level of PM H(+)-ATPase and the synthesis of pectins, and reduced the cellulose density in pollen tubes. Importantly, to observe the orientation of cellulose microfibrils in pollen tubes in situ, atomic force microscopy was used to examine the 'intact' tube wall. Atomic force microscopy images showed that cellulose microfibrils were parallel to each other in the subapical region of IAA-treated tubes, but disorganized in control tubes. All results provided new insights into the functions of cellulose microfibrils in pollen tube growth and direction, and revealed that the IAA-induced changes of pollen tubes were attributed to the increase in secretory vesicles, mitochondria, and PM H(+)-ATPase, and the modification of pectin and cellulose microfibrils in the tube wall.

Olive pollen profilin (Ole e 2 allergen) co-localizes with highly active areas of the actin cytoskeleton and is released to the culture medium during in vitro pollen germination.

Pollen allergens offer a dual perspective of study: some of them are considered key proteins for pollen physiology, but they are also able to trigger allergy symptoms in susceptible humans after coming in contact with their tissues. Profilin (Ole e 2 allergen) has been characterized, to some extent, as one of the major allergens from Olea europaea L. pollen, a highly allergenic species in the Mediterranean countries. In order to obtain clues regarding the biological role of this protein, we have analyzed both its cellular localization and the organization of actin throughout pollen hydration and early pollen tube germination. The localization of the cited proteins was visualized by confocal laser scanning microscopy immunofluorescence using different antibodies. Upon pollen hydration and pollen germination, a massive presence of profilin was detected close to the site of pollen tube emergence, forming a ring-like structure around the 'effective' apertural region. Profilin was also detected in the pollen exine of the germinating pollen grains and in the germination medium. After using a permeabilization-enhanced protocol for immunolocalization, profilin was also localized in the cytoplasm of the pollen tube, particularly at both the proximal and apical ends. Noticeable accumulations of actin were observed in the cytoplasm of the pollen tube; particularly, in both the apical region and the area immediately close to the aperture. Actin filaments were not observed, probably due to the need of further enhanced fixation procedures. The ultrastructural localization of profilin showed the presence of the protein in the cytoplasm of both the mature pollen grain and the pollen tube. The results shown here could be interpreted as signs of a massive dissociation of the actin-profilin complexes, mobilization of actin monomers, and therefore, an intense activity of the actin cytoskeleton. The extensive release of allergenic proteins from the pollen grain into the surrounding aqueous media, as described here for profilin, may help us to understand the mechanisms by which these allergens might come in contact with the human mucosa, therefore triggering the symptoms of allergy.

Single measurement detection of individual cell ionic oscillations using an n-type semiconductor - electrolyte interface.

Pollen tubes are used as models in studies on the type of tip-growth in plants. They are an example of polarised and rapid growth because pollen tubes are able to quickly invade the flower pistil in order to accomplish fertilisation. How different ionic fluxes are perceived, processed or generated in the pollen tube is still not satisfactorily understood. In order to measure the H+, K+, Ca2+ and Cl- fluxes of a single pollen tube, we developed an Electrical Lab on a Photovoltaic-Chip (ELoPvC) on which the evolving cell was immersed in an electrolyte of a germination medium. Pollen from Hyacinthus orientalis L. was investigated ex vivo. We observed that the growing cell changed the (redox) potential in the medium in a periodic manner. This subtle measurement was feasible due to the effects that were taking place at the semiconductor-liquid interface. The experiment confirmed the existence of the ionic oscillations that accompany the periodic extension of pollen tubes, thereby providing - in a single run - the complete discrete frequency spectrum and phase relationships of the ion gradients and fluxes, while all of the metabolic and enzymatic functions of the cell life cycle were preserved. Furthermore, the global 1/fα characteristic of the power spectral density, which corresponds to the membrane channel noise, was found.

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces.

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 µm) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 µm gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

Characterization of size-dependent mechanical properties of tip-growing cells using a lab-on-chip device.

Quantification of mechanical properties of tissues, living cells, and cellular components is crucial for the modeling of plant developmental processes such as mechanotransduction. Pollen tubes are tip-growing cells that provide an ideal system to study the mechanical properties at the single cell level. In this article, a lab-on-a-chip (LOC) device is developed to quantitatively measure the biomechanical properties of lily (Lilium longiflorum) pollen tubes. A single pollen tube is fixed inside the microfluidic chip at a specific orientation and subjected to compression by a soft membrane. By comparing the deformation of the pollen tube at a given external load (compressibility) and the effect of turgor pressure on the tube diameter (stretch ratio) with finite element modeling, its mechanical properties are determined. The turgor pressure and wall stiffness of the pollen tubes are found to decrease considerably with increasing initial diameter of the pollen tubes. This observation supports the hypothesis that tip-growth is regulated by a delicate balance between turgor pressure and wall stiffness. The LOC device is modular and adaptable to a variety of cells that exhibit tip-growth, allowing for the straightforward measurement of mechanical properties.

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.

In vivo cross-linking combined with mass spectrometry analysis reveals receptor-like kinases and Ca(2+) signalling proteins as putative interaction partners of pollen plasma membrane H(+) ATPases.

During fertilisation in plants, pollen grains germinate and generate a pollen tube which grows through the style tissue to the egg apparatus delivering the two sperm cells for fertilisation. For this process, adaption to specific environmental conditions and communication between male and female organs are essential, requiring the sensing of internal and external signals which are translated into tube growth. The plasma membrane (PM) H(+) ATPase energises the pollen plasma membrane for nutrient, ion and water uptake, but additionally, its activity directly affects the germination frequency and drives the elongation of pollen tubes. A combination of in vivo cross-linking with para-formaldehyde, immunoaffinity purification of cross-linked PM H(+) ATPase complexes and subsequent mass spectrometry analysis revealed putative interaction partners of the PM H(+) ATPase of lily pollen, which are possibly involved in the perception and transduction of intra- and extracellular signals. Major interactions partners included (i) membrane-localised receptor-like kinases (RLKs) with the leucine-rich repeat RLKs (LRR-RLKs) forming the largest group, (ii) interacting protein kinases, phosphatases, WD-40 domain proteins and 14-3-3 proteins that may transduce intracellular, phosphorylation-dependent signals and (iii) specific cytosolic Ca(2+) signatures may be decoded by interacting Ca(2+) sensor proteins, calmodulin and calmodulin-like proteins, and Ca(2+)-dependent protein kinases, which were all identified as interaction partners of the PM H(+) ATPase in lily pollen. These identified interaction partners suggest new putative regulation mechanisms of the PM H(+) ATPase in general and new insights in regulating pollen tube growth rates in particular. Furthermore, the optimised experimental strategy can be applied to other non-model organisms to identify membrane protein interactions. BIOLOGICAL SIGNIFICANCE: Membrane proteomics is still very challenging due to the low abundance and poor solubility of membrane proteins. Furthermore, membrane protein interaction studies in a non-model organism like Lilium longiflorum require an unbiased preparation and detection approach. The presented strategy to identify putative interaction partners of the PM H(+) ATPase by using a combination of different biochemical techniques, i.e. in vivo crosslinking, immunoaffinity purification and mass spectrometry without the need of genetic engineering, transformation or other molecular biology techniques can be easily transferred to other protein interaction studies. The well characterised interaction of the PM H(+) ATPase with regulating 14-3-3 proteins served as an intrinsic control to proof the suitability and reliability of the presented strategy, whilst newly identified interaction partners may indicate novel regulation mechanisms of the PM H(+) ATPase.

Calcium entry into pollen tubes.

Growing pollen tubes require calcium to maintain a tip-focused cytosolic gradient and as a constituent of the constantly expanding cell wall. Advances in cell and molecular biology as well as electrophysiology implicate several candidate channels and receptors in the flow of calcium into the cell. In this review we discuss the channels that have been identified and consider the role of the growing tip cell wall acting as a sink for calcium thus accounting for differences in oscillatory phase between influx measured on the outside of the cell and changes in tip concentration inside the cell. We also briefly draw attention to uptake mechanisms that restrict and shape the calcium signature in the growing pollen tube.

Primary effect of bromoxynil to induce plant cell death may be cytosol acidification.

Bromoxynil, 3,5-dibromo-4-hydroxybenzonitrile, is a commonly used herbicide and is also used as a tool to trigger rapid cell death in basic botany. However, the primary effect inducing cell death is not known. Bromoxynil inhibited the cytoplasmic streaming and killed cells in Chara corallina when it was applied in the acidic external medium. At higher pH, bromoxynil was inert even at high concentrations. It was speculated that bromoxynil in the protonated form enters the cell and acidifies the cytosol by releasing H(+). Experiments using analogues of bromoxynil supported this possibility. Acidification of the cytosol by bromoxynil was confirmed by experiments using pollen tubes. Based on the acidity of the apoplast, the herbicide action of bromoxynil in higher plants was discussed.

Integrative proteomic and cytological analysis of the effects of extracellular Ca(2+) influx on Pinus bungeana pollen tube development.

Ca (2+) is an essential ion in the control of pollen germination and tube growth. However, the control of pollen tube development by Ca (2+) signaling and its interactions with cytoskeletal components, energy-providing pathways, and cell-expansion machinery remain elusive. Here, we used nifedipine (Nif) to study Ca (2+) functions in differential protein expression and other cellular processes in Pinus bungeana pollen tube growth. Proteomics analysis indicated that 50 proteins showed differential expression with varying doses of Nif. Thirty-four of these were homologous to previously reported proteins and were classified into different functional categories closely related to tip-growth machinery. Blocking the L-type Ca (2+) channel with Nif in the pollen tube membrane induced several early alterations within a short time, including a reduction of extracellular Ca (2+) influx and a subsequently dramatic decrease in cytosolic free Ca (2+) concentration ([Ca (2+)] c), concomitant with ultrastructural abnormalities and changes in the abundance of proteins involved in energy production and signaling. Secondary alterations included actin filament depolymerization, disrupted patterns of endocytosis/exocytosis, and cell wall remodeling, along with changes in the proteins involved in these processes. These results suggested that extracellular Ca (2+) influx was necessary for the maintenance of the typical tip-focused [Ca (2+)] c gradient in the P. bungeana pollen tube, and that reduced adenosine triphosphate production (ATP), depolymerization of the cytoskeleton, and abnormal endocytosis/exocytosis, together with enhanced rigidity of cell walls, were responsible for the growth arrest observed in pollen tubes treated with Nif.

Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifolia.

The pollen tube has been widely used to study the mechanisms underlying polarized tip growth in plants. A steep tip-to-base gradient of free cytosolic calcium ([Ca(2+)](cyt)) is essential for pollen-tube growth. Local Ca(2+) influx mediated by Ca(2+)-permeable channels plays a key role in maintaining this [Ca(2+)](cyt) gradient. Here, we developed a protocol for successful isolation of spheroplasts from pollen tubes of Pyrus pyrifolia and identified a hyperpolarization-activated cation channel using the patch-clamp technique. We showed that the cation channel conductance displayed a strong selectivity for divalent cations, with a relative permeability sequence of barium (Ba(2+)) approximately Ca(2+) > magnesium (Mg(2+)) > strontium (Sr(2+)) > manganese (Mn(2+)). This channel conductance was selective for Ca(2+) over chlorine (Cl(-)) (relative permeability P(Ca)/P(Cl) = 14 in 10 mm extracellular Ca(2+)). We also showed that the channel was inhibited by the Ca(2+) channel blockers lanthanum (La(3+)) and gadolinium (Gd(3+)). Furthermore, channel activity depended on extracellular pH and pollen viability. We propose that the Ca(2+)-permeable channel is likely to play a role in mediating Ca(2+) influx into the growing pollen tubes to maintain the [Ca(2+)](cyt) gradient.