The apical actin fringe contributes to localized cell wall deposition and polarized growth in the lily pollen tube.

In lily (Lilium formosanum) pollen tubes, pectin, a major component of the cell wall, is delivered through regulated exocytosis. The targeted transport and secretion of the pectin-containing vesicles may be controlled by the cortical actin fringe at the pollen tube apex. Here, we address the role of the actin fringe using three different inhibitors of growth: brefeldin A, latrunculin B, and potassium cyanide. Brefeldin A blocks membrane trafficking and inhibits exocytosis in pollen tubes; it also leads to the degradation of the actin fringe and the formation of an aggregate of filamentous actin at the base of the clear zone. Latrunculin B, which depolymerizes filamentous actin, markedly slows growth but allows focused pectin deposition to continue. Of note, the locus of deposition shifts frequently and correlates with changes in the direction of growth. Finally, potassium cyanide, an electron transport chain inhibitor, briefly stops growth while causing the actin fringe to completely disappear. Pectin deposition continues but lacks focus, instead being delivered in a wide arc across the pollen tube tip. These data support a model in which the actin fringe contributes to the focused secretion of pectin to the apical cell wall and, thus, to the polarized growth of the pollen tube.

The pollen tube clear zone: clues to the mechanism of polarized growth.

Pollen tubes usually exhibit a prominent region at their apex called the "clear zone" because it lacks light refracting amyloplasts. A robust, long clear zone often associates with fast growing pollen tubes, and thus serves as an indicator of pollen tube health. Nevertheless we do not understand how it arises or how it is maintained. Here we review the structure of the clear zone, and attempt to explain the factors that contribute to its formation. While amyloplasts and vacuolar elements are excluded from the clear zone, virtually all other organelles are present including secretory vesicles, mitochondria, Golgi dictyosomes, and the endoplasmic reticulum (ER). Secretory vesicles aggregate into an inverted cone appressed against the apical plasma membrane. ER elements move nearly to the extreme apex, whereas mitochondria and Golgi dictyosomes move less far forward. The cortical actin fringe assumes a central position in the control of clear zone formation and maintenance, given its role in generating cytoplasmic streaming. Other likely factors include the tip-focused calcium gradient, the apical pH gradient, the influx of water, and a host of signaling factors (small G-proteins). We think that the clear zone is an emergent property that depends on the interaction of several factors crucial for polarized growth.

Some retrospectives on early studies of plant microtubules.

We pay tribute to the seminal paper 'A microtubule in plant cell fine structure' by Myron C. Ledbetter and Keith R. Porter (1963) by summarizing the very limited knowledge of plant cell ultrastructure that we had prior to that publication, and, by way of our three retrospective accounts, show how this paper stimulated and influenced subsequent research on plant microtubules. Micrographs of historical interest are presented that are either previously unpublished or from primary publications.

Mother trees, altruistic fungi, and the perils of plant personification.

There are growing doubts about the true role of the common mycorrhizal networks (CMN or wood wide web) connecting the roots of trees in forests. We question the claims of a substantial carbon transfer from 'mother trees' to their offspring and nearby seedlings through the CMN. Recent reviews show that evidence for the 'mother tree concept' is inconclusive or absent. The origin of this concept seems to stem from a desire to humanize plant life but can lead to misunderstandings and false interpretations and may eventually harm rather than help the commendable cause of preserving forests. Two recent books serve as examples: The Hidden Life of Trees and Finding the Mother Tree.

Propidium iodide competes with Ca(2+) to label pectin in pollen tubes and Arabidopsis root hairs.

We have used propidium iodide (PI) to investigate the dynamic properties of the primary cell wall at the apex of Arabidopsis (Arabidopsis thaliana) root hairs and pollen tubes and in lily (Lilium formosanum) pollen tubes. Our results show that in root hairs, as in pollen tubes, oscillatory peaks in PI fluorescence precede growth rate oscillations. Pectin forms the primary component of the cell wall at the tip of both root hairs and pollen tubes. Given the electronic structure of PI, we investigated whether PI binds to pectins in a manner analogous to Ca(2+) binding. We first show that Ca(2+) is able to abrogate PI growth inhibition in a dose-dependent manner. PI fluorescence itself also relies directly on the amount of Ca(2+) in the growth solution. Exogenous pectin methyl esterase treatment of pollen tubes, which demethoxylates pectins, freeing more Ca(2+)-binding sites, leads to a dramatic increase in PI fluorescence. Treatment with pectinase leads to a corresponding decrease in fluorescence. These results are consistent with the hypothesis that PI binds to demethoxylated pectins. Unlike other pectin stains, PI at low yet useful concentration is vital and specifically does not alter the tip-focused Ca(2+) gradient or growth oscillations. These data suggest that pectin secretion at the apex of tip-growing plant cells plays a critical role in regulating growth, and PI represents an excellent tool for examining the role of pectin and of Ca(2+) in tip growth.

Exocytosis precedes and predicts the increase in growth in oscillating pollen tubes.

We examined exocytosis during oscillatory growth in lily (Lilium formosanum and Lilium longiflorum) and tobacco (Nicotiana tabacum) pollen tubes using three markers: (1) changes in cell wall thickness by Nomarski differential interference contrast (DIC), (2) changes in apical cell wall fluorescence in cells stained with propidium iodide (PI), and (3) changes in apical wall fluorescence in cells expressing tobacco pectin methyl esterase fused to green fluorescent protein (PME-GFP). Using PI fluorescence, we quantified oscillatory changes in the amount of wall material from both lily and tobacco pollen tubes. Measurement of wall thickness by DIC was only possible with lily due to limitations of microscope resolution. PME-GFP, a direct marker for exocytosis, only provides information in tobacco because its expression in lily causes growth inhibition and cell death. We show that exocytosis in pollen tubes oscillates and leads the increase in growth rate; the mean phase difference between exocytosis and growth is -98 degrees +/- 3 degrees in lily and -124 degrees +/- 4 degrees in tobacco. Statistical analyses reveal that the anticipatory increase in wall material predicts, to a high degree, the rate and extent of the subsequent growth surge. Exocytosis emerges as a prime candidate for the initiation and regulation of oscillatory pollen tube growth.

Timing is everything: early degradation of abscission layer is associated with increased seed shattering in U.S. weedy rice.

BACKGROUND: Seed shattering, or shedding, is an important fitness trait for wild and weedy grasses. U.S. weedy rice (Oryza sativa) is a highly shattering weed, thought to have evolved from non-shattering cultivated ancestors. All U.S. weedy rice individuals examined to date contain a mutation in the sh4 locus associated with loss of shattering during rice domestication. Weedy individuals also share the shattering trait with wild rice, but not the ancestral shattering mutation at sh4; thus, how weedy rice reacquired the shattering phenotype is unknown. To establish the morphological basis of the parallel evolution of seed shattering in weedy rice and wild, we examined the abscission layer at the flower-pedicel junction in weedy individuals in comparison with wild and cultivated relatives. RESULTS: Consistent with previous work, shattering wild rice individuals possess clear, defined abscission layers at flowering, whereas non-shattering cultivated rice individuals do not. Shattering weedy rice from two separately evolved populations in the U.S. (SH and BHA) show patterns of abscission layer formation and degradation distinct from wild rice. Prior to flowering, the abscission layer has formed in all weedy individuals and by flowering it is already degrading. In contrast, wild O. rufipogon abscission layers have been shown not to degrade until after flowering has occurred. CONCLUSIONS: Seed shattering in weedy rice involves the formation and degradation of an abscission layer in the flower-pedicel junction, as in wild Oryza, but is a developmentally different process from shattering in wild rice. Weedy rice abscission layers appear to break down earlier than wild abscission layers. The timing of weedy abscission layer degradation suggests that unidentified regulatory genes may play a critical role in the reacquisition of shattering in weedy rice, and sheds light on the morphological basis of parallel evolution for shattering in weedy and wild rice.

Oscillatory growth in lily pollen tubes does not require aerobic energy metabolism.

Oscillatory tip growth in pollen tubes depends on prodigious amounts of energy. We have tested the hypothesis that oscillations in the electron transport chain lead to growth oscillations in lily (Lilium formosanum). Using three respiratory inhibitors, oligomycin, antimycin A, and cyanide, we find that pollen tube growth is much less sensitive to respiratory inhibition than respiration is. All three block respiration at concentrations severalfold lower than necessary to inhibit growth. Mitochondrial NAD(P)H and potentiometric JC-1 fluorescence, employed as markers for electron transport chain activity, rise rapidly in response to oligomycin, as expected. Pollen tube growth stops for several minutes before resuming. Subsequent growth has a lower mean rate, but continues to oscillate, albeit with a longer period. NAD(P)H fluorescence no longer exhibits coherent oscillations, and mitochondria no longer congregate directly behind the apex: they distribute evenly throughout the cell. Postinhibition growth relies on aerobic fermentation for energy production as revealed by an increase in ethanol in the media. These data suggest that oscillatory growth depends not on a single oscillatory pacemaker but rather is an emergent property arising from a number of stable limit cycles.

Apical pollen tube wall curvature correlates with growth and indicates localized changes in the yielding of the cell wall.

The shape of the apical region of lily pollen tube changes rhythmically as the growth rate of the tube oscillates becoming alternately more prolate then back to oblate. We quantified shape change by calculating the curvature of the cross-sectional edge of the pollen tube tip and cross-correlating curvature changes with growth rate. The apical region takes the form of a partial elliptical spheroid, with variation in the length and location of the minor axis. During oscillation curvature profiles show a sharp increase in curvature at the "shoulders" of the apex when oblate, 4-7 µm from the flatter central zone. As the tip becomes more prolate, the "shoulders" decrease rapidly in curvature and move towards the growth axis as curvature at the tip increases. We understand curvature changes to represent differential changes in local wall expansion rates, driven by uniform turgor pressure and mediated by changes in wall polysaccharides. To become more oblate, the tip region must become less extensible than the "shoulder" region. And, as the tip becomes more prolate, the increased curvature must be due to increased local expansion. We found that changes in the growth velocity of the "shoulders" of the cell measured as the progress of the cell edge along the growth axis are cyclically out of phase with growth velocity at the tip such that the shoulder regions lag for part of the oscillation cycle, then "catch up" as the growth rate at the tip reaches a maximum and begins to decline. In this way the cell becomes oblate. Cell shape and growth rate oscillate in concert and are functionally related. Spatial change in edge growth rate points to important cellular locations for further investigation of vesicle movement and exocytosis, calcium gradients, and actin dynamics in lily pollen tubes.

Gland cell responses to feeding in Drosera capensis, a carnivorous plant.

Glands of Drosera absorb and transport nutrients from captured prey, but the mechanism and dynamics remain unclear. In this study, we offered animal proteins in the form of fluorescent albumin (FITC-BSA) and observed the reactions of the glands by live cell imaging and fluorescence microscopy. The ultrastructure of these highly dynamic processes was also assessed in high-pressure frozen and freeze substituted (HPF-FS) cells. HPF-FS yielded excellent preservation of the cytoplasm of all cell types, although the cytosol looked different in gland cells as compared to endodermoid and stalk cells. Especially prominent were the ER and its contacts with the plasma membrane, plasmodesmata, and other organelles as well as continuities between organelles. Also distinct were actin microfilaments in association with ER and organelles. Application of FITC-BSA to glands caused the formation of fluorescent endosomes that pinched off the plasma membrane. Endosomes fused to larger aggregates, and accumulated in the bulk cytoplasm around the nucleus. They did not fuse with the cell sap vacuole but remained for at least three days; in addition, fluorescent vesicles also proceeded through endodermoid and transfer cells to the epidermal and parenchymal cells of the tentacle stalk.

Calcium at the cell wall-cytoplast interface.

Attention is given to the role of Ca(2+) at the interface between the cell wall and the cytoplast, especially as seen in pollen tubes. While the cytoplasm directs the synthesis and deposition of the wall, it is less well appreciated that the wall exerts considerable self control and influences activities of the cytoplasm. Ca(2+) participates as a crucial factor in this two way communication. In the cytoplasm, a [Ca(2+)] above 0.1 microM, regulates myriad processes, including secretion of cell wall components. In the cell wall Ca(2+), at 10 microM to 10 mM, binds negative charges on pectins and imparts structural rigidity to the wall. The plasma membrane occupies a pivotal position between these two compartments, where selective channels regulate influx of Ca(2+), and specific carriers pump the ion back into the wall. In addition we draw attention to different factors, which either respond to the wall or are present in the wall, and usually generate elevated [Ca(2+)] in the cytoplasm. These factors include: (i) stretch activated channels; (ii) calmodulin; (iii) annexins; (iv) wall associated kinases; (v) oligogalacturonides; and (vi) extracellular adenosine 5'-triphosphate. Together they provide evidence for a rich and multifaceted system of communication between the cytoplast and cell wall, with Ca(2+) as a carrier of information.

Microtubule cross-linking activity of She1 ensures spindle stability for spindle positioning.

Dynein mediates spindle positioning in budding yeast by pulling on astral microtubules (MTs) from the cell cortex. The MT-associated protein She1 regulates dynein activity along astral MTs and directs spindle movements toward the bud cell. In addition to localizing to astral MTs, She1 also targets to the spindle, but its role on the spindle remains unknown. Using function-separating alleles, live-cell spindle assays, and in vitro biochemical analyses, we show that She1 is required for the maintenance of metaphase spindle stability. She1 binds and cross-links MTs via a C-terminal MT-binding site. She1 can also self-assemble into ring-shaped oligomers. In cells, She1 stabilizes interpolar MTs, preventing spindle deformations during movement, and we show that this activity is regulated by Ipl1/Aurora B phosphorylation during cell cycle progression. Our data reveal how She1 ensures spindle integrity during spindle movement across the bud neck and suggest a potential link between regulation of spindle integrity and dynein pathway activity.

Perturbation Analysis of Calcium, Alkalinity and Secretion during Growth of Lily Pollen Tubes.

Pollen tubes grow by spatially and temporally regulated expansion of new material secreted into the cell wall at the tip of the tube. A complex web of interactions among cellular components, ions and small molecule provides dynamic control of localized expansion and secretion. Cross-correlation studies on oscillating lily (Lilium formosanum Wallace) pollen tubes showed that an increase in intracellular calcium follows an increase in growth, whereas the increase in the alkaline band and in secretion both anticipate the increase in growth rate. Calcium, as a follower, is unlikely to be a stimulator of growth, whereas the alkaline band, as a leader, may be an activator. To gain further insight herein we reversibly inhibited growth with potassium cyanide (KCN) and followed the re-establishment of calcium, pH and secretion patterns as growth resumed. While KCN markedly slows growth and causes the associated gradients of calcium and pH to sharply decline, its removal allows growth and vital processes to fully recover. The calcium gradient reappears before growth restarts; however, it is preceded by both the alkaline band and secretion, in which the alkaline band is slightly advanced over secretion. Thus the pH gradient, rather than the tip-focused calcium gradient, may regulate pollen tube growth.

The role of nod factor substituents in actin cytoskeleton rearrangements in Phaseolus vulgaris.

In order to define the symbiotic role of some of the chemical substituents in the Rhizobium etli Nod factors (NFs), we purified Nod metabolites secreted by the SM25 strain, which carries most of the nodulation genes, and SM17 with an insertion in nodS. These NFs were analyzed for their capabilities to induce root hair curling and cytoskeletal rearrangements. The NFs secreted by strain SM17 lack the carbamoyl and methyl substituents on the nonreducing terminal residue and an acetyl moiety on the fucosyl residue on the reducing-terminal residue as determined by mass spectrometry. We have reported previously that the root hair cell actin cytoskeleton from bean responds with a rapid fragmentation of the actin bundles within 5 min of NF exposure, and also is accompanied by increases in the apical influxes and intracellular calcium levels. In this article, we report that methyl-bearing NFs are more active in inducing root hair curling and actin cytoskeleton rearrangements than nonmethylated NFs. However, the carbamoyl residue on the nonreducing terminal residue and the acetyl group at the fucosyl residue on the reducing terminal residue do not seem to have any effect on root hair curling induction or in actin cytoskeleton rearrangement.

Control of pollen tube growth: role of ion gradients and fluxes.

Pollen tube growth attracts our attention as a model system for studying cell elongation in plants. The process is fast, it is confined to the tip of the tube, and it is crucial for sexual reproduction in plants. In the enclosed review we focus on the control of pollen tube growth, giving special attention to the role of ions, especially calcium and protons. During the last decade technical advances have made it possible to detect localized intracellular gradients, and extracellular fluxes of calcium and protons in the apical domain. Other ions, notably potassium and chloride, are also receiving attention. An important development has been the realization that pollen tube growth oscillates in rate; in addition, the ion gradients and fluxes oscillate in magnitude. Although all the ionic oscillations show the same period as that of the growth rate, with the exception of extracellular chloride efflux, they are not in phase with growth. Considerable effort is devoted to the elucidation of these different phase relationships, with the view that a hierarchical order may provide clues about those events that are primary vs. secondary in growth control. Attention is also given to the targets for the ions, for example, the secretory system, the cytoskeleton, the cell wall, in an attempt to provide a global understanding of pollen tube growth. Contents Summary 539 I. Introduction 540 II. Ion gradients and flux patterns 541 III. Oscillations 544 IV. The need for a Ca2+ store 547 V. Intracellular targets for Ion activity 549 VI. Extracellular targets for ions: the cell wall 552 VII. Ions in navigation 554 VIII. Role of ions in self-incompatibility 555 IX. The plasma membrane; site of global coordination and control 556 X. A model for pollen tube growth 557 IX. Conclusions 558 Acknowledgements 559 References 559.

Effect of heat shock on the chilling sensitivity of trichomes and petioles of African violet (Saintpaulia ionantha).

Chilling at 6 degrees C caused an immediate cessation of protoplasmic streaming in trichomes from African violets (Saintpaulia ionantha), and a slower aggregation of chloroplasts in the cells. Streaming slowly recovered upon warming to 20 degrees C, reaching fairly stable rates after 4, 15, 25 and 35 min for tissue chilled for 2 min and for 2, 14 and 24 h, respectively. The rate of ion leakage from excised petioles into an isotonic 0.2 M mannitol solution increased after 12 h of chilling and reached a maximum after 3 days of chilling. A heat shock at 45 degrees C for 6 min reduced chilling-induced rates of ion leakage from excised 1-cm petiole segments by over 50%, namely to levels near that from non-chilled control tissue. Heat-shock treatments themselves had no effect on the rate of ion leakage from non-chilled petiole segments. Protoplasmic streaming was stopped by 1 min of heat shock at 45 degrees C, but slowly recovered to normal levels after about 30 min Chloroplasts aggregation was prevented by a 1 or 2 min 45 degrees C heat-shock treatment administered 1.5 h before chilling, but heat-shock treatments up to 6 min only slightly delayed the reduction in protoplasmic streaming caused by chilling. Tradescantia virginiana did not exhibit symptoms associated with chilling injury in sensitive species (i.e. cessation of protoplasmic streaming in stamen hairs and increased ion leakage from leaf tissue).

Fertilization in Torenia fournieri: Actin organization and nuclear behavior in the central cell and primary endosperm.

Studies of the living embryo sacs of Torenia fournieri reveal that the actin cytoskeleton undergoes dramatic changes that correlate with nuclear migration within the central cell and the primary endosperm. Before pollination, actin filaments appear as short bundles randomly distributed in the cortex of the central cell. Two days after anthesis, they become organized into a distinct actin network. At this stage the secondary nucleus, which is located in the central region of the central cell, possesses an associated array of short actin filaments. Soon after pollination, the actin filaments become fragmented in the micropylar end and the secondary nucleus is located next to the egg apparatus. After fertilization, the primary endosperm nucleus moves away from the egg cell and actin filaments reorganize into a prominent network in the cytoplasm of the primary endosperm. Disruption of the actin cytoskeleton with latrunculin A and cytochalasin B indicates that actin is involved in the migration of the nucleus in the central cell. Our data also suggest that the dynamics of actin cytoskeleton may be responsible for the reorganization of the central cell and primary endosperm cytoplasm during fertilization.

Control of cell wall extensibility during pollen tube growth.

In this review, we address the question of how the tip-growing pollen tube achieves its rapid rate of elongation while maintaining an intact cell wall. Although turgor is essential for growth to occur, the local expansion rate is controlled by local changes in the viscosity of the apical wall. We focus on several different structures and underlying processes that are thought to be major participants including exocytosis, the organization and activity of the actin cytoskeleton, calcium and proton physiology, and cellular energetics. We think that the actin cytoskeleton, in particular the apical cortical actin fringe, directs the flow of vesicles to the apical domain, where they fuse with the plasma membrane and contribute their contents to the expanding cell wall. While pH gradients, as generated by a proton-ATPase located on the plasma membrane along the side of the clear zone, may regulate rapid actin turnover and new polymerization in the fringe, the tip-focused calcium gradient biases secretion towards the polar axis. The recent data showing that exocytosis of new wall material precedes and predicts the process of cell elongation provide support for the idea that the intussusception of newly secreted pectin contributes to decreases in apical wall viscosity and to cell expansion. Other prime factors will be the localization and activity of the enzyme pectin methyl-esterase, and the chelation of calcium by pectic acids. Finally, we acknowledge a role for reactive oxygen species in the control of wall viscosity.