A signaling peptide locks pollen tube walls.

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

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.

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.

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.

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.

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.

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.

SB401, a pollen-specific protein from Solanum berthaultii, binds to and bundles microtubules and F-actin.

We characterize a novel, pollen-specific, microtubule-associated protein, SB401, found in Solanum berthaultii. This protein binds to and bundles taxol-stabilized microtubules and enhances tubulin polymerization in a concentration-dependent manner, particularly at lower temperatures. Electron microscopy revealed that the protein decorates the entire length of microtubules. Cross-linking and electrophoresis studies showed that SB401 protein forms dimers, and suggest that dimerization could account for bundling. Double immunofluorescent staining of pollen tubes of S. berthaultii showed that SB401 protein co-localized with cortical microtubule bundles. SB401 protein also binds to and bundles actin filaments, and could connect actin filaments to microtubules. SB401 protein had a much higher affinity for microtubules than for actin filaments. In the presence of both cytoskeletal elements, the protein preferentially bound microtubules to form bundles. These results demonstrate that SB401 protein may have important roles in organizing the cytoskeleton in pollen tubes.

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.