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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. Supplementary Information The online version contains supplementary material available at 10.1007/s00709-021-01667-5.

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.

Tip growth is a specialized type of growth utilized by only a few cell types in the plant kingdom. Nevertheless, these cell types perform vital functions for plants. Pollen tubes are necessary for seed plant reproduction, root hairs are vital for nutrient acquisition, and protonemata comprise the

Dynein orients the spindle by pulling on astral microtubules from the cortex. In Saccharomyces cerevisiae, the microtubule-associated protein She1 specifically inhibits dynein in the mother compartment to promote spindle movements toward the bud. Zhu et al. demonstrate that She1 also stabilizes interpolar microtubules, ensuring spindle integrity during dynein-mediated 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.

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.

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.

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.

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.

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 Ca2+ binding. We first show that Ca2+ is able to abrogate PI growth inhibition in a dose-dependent manner. PI fluorescence itself also relies directly on the amount of Ca2+ in the growth solution. Exogenous pectin methyl esterase treatment of pollen tubes, which demethoxylates pectins, freeing more Ca2+-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 Ca2+ 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 Ca2+ in tip growth.

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° ± 3° in lily and –124° ± 4° 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.

The cytoskeleton is well recognized as an ever-present component of all eukaryotic cells; more recently, it has been identified in prokaryotic cells as well ([van den Ent et al., 2001][1]; [Wickstead and Gull, 2011][2]). The cytoskeleton gives order to a cell, and in large cells, where diffusion may

Although most people think of pollen merely as an allergen, its true biological function is to facilitate sexual reproduction in flowering plants. The angiosperm pollen grain, upon arriving at a receptive stigma, germinates, producing a tube that extends through the style to deliver its cargo to the ovule, thereby fertilizing the egg, and completing the life cycle of the plant. The pollen tube grows rapidly, exclusively at its tip, and produces a cell that is highly polarized both in its outward shape and its internal cytoplasmic organization. Recent studies reveal that the growth oscillates in rate. Many underlying physiological processes, including ionic fluxes and energy levels, also oscillate with the same periodicity as the growth rate, but usually not with the same phase. Current research focuses on these phase relationships in an attempt to decipher their hierarchical sequence and to provide a physiological explanation for the factors that govern pollen tube growth.

The location and changes in NAD(P)H have been monitored during oscillatory growth in pollen tubes of lily (Lilium formosanum) using the endogenous fluorescence of the reduced coenzyme (excitation, 360 nm; emission,400 nm). The strongest signal resides 20 to 40 microm behind the apex where mitochondria (stained with Mitotracker Green) accumulate. Measurements at 3-s intervals reveal that NAD(P)H-dependent fluorescence oscillates during oscillatory growth. Cross-correlation analysis indicates that the peaks follow growth maxima by 7 to 11 s or 77 degrees to 116 degrees, whereas the troughs anticipate growth maxima by 5 to 10 s or 54 degrees to 107 degrees. We have focused on the troughs because they anticipate growth and are as strongly correlated with growth as the peaks. Analysis of the signal in 10-microm increments along the length of the tube indicates that the troughs are most advanced in the extreme apex. However, this signal moves basipetally as a wave, being in phase with growth rate oscillations at 50 to 60 microm from the apex. We suggest that the changes in fluorescence are due to an oscillation between the reduced (peaks) and oxidized (troughs) states of the coenzyme and that an increase in the oxidized state [NAD(P)(+)] may be coupled to the synthesis of ATP. We also show that diphenyleneiodonium, an inhibitor of NAD(P)H dehydrogenases, causes an increase in fluorescence and a decrease in tube growth. Finally, staining with 5-(and-6)-chloromethyl-2',7'-dichlorohydrofluorescein acetate indicates that reactive oxygen species are most abundant in the region where mitochondria accumulate and where NAD(P)H fluorescence is maximal.

Lily (Lilium formosanum or Lilium longiflorum) pollen tubes, microinjected with a low concentration of the pH-sensitive dye bis-carboxyethyl carboxyfluorescein dextran, show oscillating pH changes in their apical domain relative to growth. An increase in pH in the apex precedes the fastest growth velocities, whereas a decline follows growth, suggesting a possible relationship between alkalinity and cell extension. A target for pH may be the actin cytoskeleton, because the apical cortical actin fringe resides in the same region as the alkaline band in lily pollen tubes and elongation requires actin polymerization. A pH-sensitive actin binding protein, actin-depolymerizing factor (ADF), together with actin-interacting protein (AIP) localize to the cortical actin fringe region. Modifying intracellular pH leads to reorganization of the actin cytoskeleton, especially in the apical domain. Acidification causes actin filament destabilization and inhibits growth by 80%. Upon complete growth inhibition, the actin fringe is the first actin cytoskeleton component to disappear. We propose that during normal growth, the pH increase in the alkaline band stimulates the fragmenting activity of ADF/AIP, which in turn generates more sites for actin polymerization. Increased actin polymerization supports faster growth rates and a proton influx, which inactivates ADF/AIP, decreases actin polymerization, and retards growth. As pH stabilizes and increases, the activity of ADF/AIP again increases, repeating the cycle of events.

Sperm delivery in flowering plants requires extensive pollen tube growth through the female sporophytic tissues of the pistil. The apical cell wall emerges as a central player in the control of pollen tube growth, since it provides strength to withstand the internal turgor pressure, while imparting sufficient plasticity to allow cell wall extension through the incorporation of new membrane and wall material. Within this scenario, pectin methylesterases (PMEs; EC 3.1.1.11) emerge as crucial regulators in determining the mechanical properties of pectins, the major component of the apical pollen tube wall. We previously identified NtPPME1, a pollen specific PME from Nicotiana tabacum. Here we show that silencing of NtPPME1 results in a mild but significant decrease of in vivo pollen tube growth while the overall PME activity in pollen is not significantly affected. Although the precise mechanisms responsible for the observed phenotype are not known, it seems likely that the cell must maintain a closely regulated level of PME activity in order to maintain the equilibrium between strength and plasticity in the apical cell wall. A relatively minor disturbance of this equilibrium, as caused by NtPPME1 silencing, compromises pollen tube growth.

Our present understanding implicates both calmodulin (CaM) and 3',5'-cyclicAMP (cAMP) in the regulation of pollen tube growth. However, downstream molecules of these signalling pathways and the cellular processes they modulate remain largely unknown. In order to elucidate the role of CaM, we mapped its activity in growing pollen tubes. 2-chloro-(epsilon-amino-Lys(75))-[6-4-(N,N'-diethylaminophenyl)-1,3,5-triazin-4-yl]-calmodulin (TA-CaM) and fluorescein-calmodulin (FL-CaM), fluorescent analogues of CaM, were loaded into pollen tubes and CaM activity was mapped by fluorescence ratio imaging. It was found that CaM activity exhibits a tip-focused gradient, similar to the distribution of cytosolic-free calcium ([Ca(2+)](c)). In long pollen tubes, apical CaM activity was also found to oscillate with a period similar to [Ca(2+)](c) (40-80 sec). This oscillatory behaviour was not observed in small pollen tubes or in tubes that had stopped growing. Changes in CaM activity within the dome of the pollen tube apex resulting from the photolysis of caged photolysis of RS-20 (a peptide antagonist of CaM) induced re-orientation of the growth axis, suggesting that CaM is also involved in the guidance mechanism. CaM activity was strongly modulated by intracellular changes in cAMP (induced by activators and antagonists of adenylyl cyclase). These results indicate that the action of this protein is dependent not solely on [Ca(2+)](c) but also on a cross-talk with other signalling pathways. A putative target of this cross-talk is the secretory machinery as observed in pollen tubes loaded with the FM (N-(3-triethylammoniumpropyl)-4-(4-dibutylamino)styryl)pyridinium dibromide 1-43 dye and exposed to different antagonists and activators of these molecules. Our data thus suggest that pollen tube growth and orientation depend on an intricate cross-talk between multiple signalling pathways in which CaM is a key element.

In many cases, actin filaments are arranged into bundles and serve as tracks for cytoplasmic streaming in plant cells. We have isolated an actin-filament bundling protein, which is composed of 115-kDa polypeptide (P-115-ABP), from the germinating pollen of lily, Lilium longiflorum [Nakayasu et al. (1998) BIOCHEM: Biophys. Res. Commun. 249: 61]. P-115-ABP shared similar antigenicity with a plant 135-kDa actin-filament bundling protein (P-135-ABP), a plant homologue of villin. A full-length cDNA clone (ABP115; accession no. AB097407) was isolated from an expression cDNA library of lily pollen by immuno-screening using antisera against P-115-ABP and P-135-ABP. The amino acid sequence of P-115-ABP deduced from this clone showed high homology with those of P-135-ABP and four villin isoforms of Arabidopsis thaliana (AtVLN1, AtVLN2, AtVLN3 and AtVLN4), especially AtVLN4, indicating that P-115-ABP can also be classified as a plant villin. The P-115-ABP isolated biochemically from the germinating lily pollen was able to arrange F-actin filaments with uniform polarity into bundles and this bundling activity was suppressed by Ca2+-calmodulin (CaM), similar to the actin-filament bundling properties of P-135-ABP. The P-115-ABP type of plant villin was widely distributed in plant cells, from algae to land plants. In root hair cells of Hydrocharis dubia, this type of plant villin was co-localized with actin-filament bundles in the transvacuolar strands and the sub-cortical regions. Microinjection of the antiserum against P-115-ABP into living root hair cells caused the disappearance of transvaculor strands and alteration of the route of cytoplasmic streaming. In internodal cells of Chara corallina in which the P-135-ABP type of plant villin is lacking, the P-115-ABP type showed co-localization with actin-filament cables anchored on the intracellular surface of chloroplasts. These results indicated that plant villins are widely distributed and involved in the organization of actin filaments into bundles throughout the plant kingdom.

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.

Calcium ions (Ca(2+)), protons (H(+)), and borate (B(OH)(4)(-)) are essential ions in the control of tip growth of pollen tubes. All three ions may interact with pectins, a major component of the expanding pollen tube cell wall. Ca(2+ )is thought to bind acidic residues, and cross-link adjacent pectin chains, thereby strengthening the cell wall. Protons are loosening agents; in pollen tube walls they may act through the enzyme pectin methylesterase (PME), and either reduce demethylation or stimulate hydrolysis of pectin. Finally, borate cross-links monomers of rhamnogalacturonan II (RG-II), and thus stiffens the cell wall. It is demonstrated here that changing the extracellular concentrations of Ca(2+), H(+) and borate affect not only the average growth rate of lily pollen tubes, but also influence the period of growth rate oscillations. The most dramatic effects are observed with increasing concentrations of Ca(2+) and borate, both of which markedly reduce the rate of growth of oscillating pollen tubes. Protons are less active, except at pH 7.0 where growth is inhibited. It is noteworthy, especially with borate, that the faster growing tubes exhibit the shorter periods of oscillation. The results are consistent with the idea that binding of Ca(2+) and borate to the cell wall may act at a similar level to alter the mechanical properties of the apical cell wall, with optimal concentrations being high enough to impart sufficient rigidity to the wall so as to prevent bursting in the face of cell turgor, but low enough to allow the wall to stretch quickly during periods of accelerating growth.

Pollen tube elongation is a polarized cell growth process that transports the male gametes from the stigma to the ovary for fertilization inside the ovules. Actomyosin-driven intracellular trafficking and active actin remodeling in the apical and subapical regions of pollen tubes are both important aspects of this rapid tip growth process. Actin-depolymerizing factor (ADF) and cofilin are actin binding proteins that enhance the depolymerization of microfilaments at their minus, or slow-growing, ends. A pollen-specific ADF from tobacco, NtADF1, was used to dissect the role of ADF in pollen tube growth. Overexpression of NtADF1 resulted in the reduction of fine, axially oriented actin cables in transformed pollen tubes and in the inhibition of pollen tube growth in a dose-dependent manner. Thus, the proper regulation of actin turnover by NtADF1 is critical for pollen tube growth. When expressed at a moderate level in pollen tubes elongating in in vitro cultures, green fluorescent protein (GFP)–tagged NtADF1 (GFP-NtADF1) associated predominantly with a subapical actin mesh composed of short actin filaments and with long actin cables in the shank. Similar labeling patterns were observed for GFP-NtADF1–expressing pollen tubes elongating within the pistil. A Ser-6-to-Asp conversion abolished the interaction between NtADF1 and F-actin in elongating pollen tubes and reduced its inhibitory effect on pollen tube growth significantly, suggesting that phosphorylation at Ser-6 may be a prominent regulatory mechanism for this pollen ADF. As with some ADF/cofilin, the in vitro actin-depolymerizing activity of recombinant NtADF1 was enhanced by slightly alkaline conditions. Because a pH gradient is known to exist in the apical region of elongating pollen tubes, it seems plausible that the in vivo actin-depolymerizing activity of NtADF1, and thus its contribution to actin dynamics, may be regulated spatially by differential H+ concentrations in the apical region of elongating pollen tubes.

Pollen tube elongation depends on the secretion of large amounts of membrane and cell wall materials at the pollen tube tip to sustain rapid growth. A large family of RAS-related small GTPases, Rabs or Ypts, is known to regulate both anterograde and retrograde trafficking of transport vesicles between different endomembrane compartments and the plasma membrane in mammalian and yeast cells. Studies on the functional roles of analogous plant proteins are emerging. We report here that a tobacco pollen-predominant Rab2, NtRab2, functions in the secretory pathway between the endoplasmic reticulum and the Golgi in elongating pollen tubes. Green fluorescent protein–NtRab2 fusion protein localized to the Golgi bodies in elongating pollen tubes. Dominant-negative mutations in NtRab2 proteins inhibited their Golgi localization, blocked the delivery of Golgi-resident as well as plasmalemma and secreted proteins to their normal locations, and inhibited pollen tube growth. On the other hand, when green fluorescent protein–NtRab2 was over-expressed in transiently transformed leaf protoplasts and epidermal cells, in which NtRab2 mRNA have not been observed to accumulate to detectable levels, these proteins did not target efficiently to Golgi bodies. Together, these observations indicate that NtRab2 is important for trafficking between the endoplasmic reticulum and the Golgi bodies in pollen tubes and may be specialized to optimally support the high secretory demands in these tip growth cells.

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.

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.

Inositol 1,4,5 trisphosphate [Ins(1,4,5)P3] is produced from the hydrolysis of phosphatidylinositol 4,5 bisphosphate, and as part of a second-messenger signal transduction mechanism, induces release of Ca2+ from internal stores in both plant and animal systems. It is less well established how the active Ins(1,4,5)P3 is inactivated. Studies in animal cells have demonstrated two separate metabolic pathways. Ins(1,4,5)P3 can be hydrolyzed by a 5-phosphatase or phosphorylated by a 3-kinase, resulting in the formation of Ins(1,4)P2 and Ins(1,3,4,5)P4, respectively, neither of which is able to mobilize intracellular Ca2+. Plant cell extracts have been reported to have hydrolytic and kinase activities that produce Ins(1,4)P2, and Ins(4,5)P2 and Ins(1,4,5,6)P4 from Ins(1,4,5)P3. These results offer little insight into the enzyme activities in the intact plant cell since the observed activities might be confined to intracellular compartments that have little if any impact on the signaling events within the cytosol that require Ins(1,4,5)P3. To resolve the mechanism of Ins(1,4,5)P3 inactivation, we microinjected stamen hair cells of Tradescantia virginiana L. with nonhydrolysable analogs of Ins(1,4,5)P3 that have been previously shown to cause Ca2+ release from intracellular stores. Our results indicate a sustained cytosolic [Ca2+] increase when cells were injected with the 5-phosphatase-insensitive 5-monophosphorothioate derivative of Ins(1,4,5)P3, in contrast to a brief transient when injected with the 3-kinase-insensitive 3-fluoro-3-deoxy Ins(1,4,5)P3 analog. We conclude that the 5-phosphatase pathway is the preferred pathway for Ins(1,4,5)P3 inactivation in the stamen hair cells of Tradescantia.

Actin microfilaments, which are prominent in pollen tubes, have been implicated in the growth process; however, their mechanism of action is not well understood. In the present work we have used profilin and DNAse I injections, as well as latrunculin B and cytochalasin D treatments, under quantitatively controlled conditions, to perturb actin microfilament structure and assembly in an attempt to answer this question. We found that a approximately 50% increase in the total profilin pool was necessary to half-maximally inhibit pollen tube growth, whereas a approximately 100% increase was necessary for half-maximal inhibition of cytoplasmic streaming. DNAse I showed a similar inhibitory activity but with a threefold more pronounced effect on growth than streaming. Latrunculin B, at only 1--4 nM in the growth medium, has a similar proportion of inhibition of growth over streaming to that of profilin. The fact that tip growth is more sensitive than streaming to the inhibitory substances and that there is no correlation between streaming and growth rates suggests that tip growth requires actin assembly in a process independent of cytoplasmic streaming.

In many types of plant cell, bundles of actin filaments (AFs) are generally involved in cytoplasmic streaming and the organization of transvacuolar strands. Actin cross-linking proteins are believed to arrange AFs into the bundles. In root hair cells of Hydrocharis dubia (Blume) Baker, a 135-kDa polypeptide cross-reacted with an antiserum against a 135-kDa actin-bundling protein (135-ABP), a villin homologue, isolated from lily pollen tubes. Immunofluorescence microscopy revealed that the 135-kDa polypeptide co-localized with AF bundles in the transvacuolar strand and in the sub-cortical region of the cells. Microinjection of antiserum against 135-ABP into living root hair cells induced the disappearance of the transvacuolar strand. Concomitantly, thick AF bundles in the transvacuolar strand dispersed into thin bundles. In the root hair cells, AFs showed uniform polarity in the bundles, which is consistent with the in-vitro activity of 135-ABP. These results suggest that villin is a factor responsible for bundling AFs in root hair cells as well as in pollen tubes, and that it plays a key role in determining the direction of cytoplasmic streaming in these cells.

The concentration of cytoplasmic free calcium ([Ca(2+)](cyt)) required to close higher plant plasmodesmata was investigated using corn (Zea mays L. cv. Black Mexican Sweet) suspension-culture cells. Physiological elevations of [Ca(2+)](cyt) were applied by cold treatment, and ion injection was also used to increase [Ca(2+)](cyt), by diffusion (for small increases) or by iontophoresis (for larger increases). The impact of such treatments on [Ca(2+)](cyt) was measured by ratiometric ion imaging. Intercellular communication during treatments was monitored using our recently developed electrophysiological technique that allows the electrical resistance of plasmodesmata and the plasma membranes of a sister-cell pair to be measured. A 4-fold increase in the calculated resistance of single plasmodesmata was observed in response to cold treatment that caused a 2-fold increase in average [Ca(2+)](cyt) (from 107 to 210 nM). In response to iontophoresis of Ca(2+), plasmodesmata were observed to go from "open" (low resistance) to "shut" (high resistance) and then back "open" within 10 s. Our results thus indicate that higher plant plasmodesmata respond quickly to physiological changes in [Ca(2+)](cyt).

Application of Nod factors to growing, responsive root hairs of the bean Phaseolus vulgaris induces marked changes in both the intracellular cytosolic free calcium (Ca2+) and in the influx of extracellular [Ca2+]. The intracellular [Ca2+], which has been measured by ratiometric imaging in cells microinjected with fura-2-dextran (70 kDa), elevates within 5 min from approximately 400 nM to 1500 nM in localised zones in the root hair apex. Of particular note is the observation that the elevated regions of [Ca2+] appear to shift position during short time intervals. Increases in and fluctuations of the intracellular [Ca2+] are also observed in the perinuclear region after 10-15 min treatment with Nod factors. The extracellular Ca2+ flux, detected with the non-invasive, calcium specific vibrating electrode, is inwardly directed and also increases quickly in response to Nod factors from 13 pmol cm-2 s-1 to 28 pmol cm-2 s-1. Chitin-oligomers, which are structurally similar but biologically inactive when compared to the active Nod factors, fail to elicit changes in either intracellular or extracellular Ca2+. The similar timing and location of the intracellular elevations and the increased extracellular influx provide support for the idea that Ca2+ participates in secretion and cell wall remodelling, which occur in anticipation of root hair deformation and curling.

Cytoplasmic calcium concentration ([Ca2+]i) and extracellular calcium (Ca2+o) influx has been studied in pollen tubes of Lilium longliflorum in which the processes of cell elongation and exocytosis have been uncoupled by use of Yariv phenylglycoside ((beta-D-Glc)3). Growing pollen tubes were pressure injected with the ratio dye fura-2 dextran and imaged after application of (beta-D-Glc)3, which binds arabinogalactan proteins (AGPs). Application of (beta-D-Glc)3 inhibited growth but not secretion. Ratiometric imaging of [Ca2+]i revealed an initial spread in the locus of the apical [Ca2+]i gradient and substantial elevations in basal [Ca2+]i followed by the establishment of new regions of elevated [Ca2+]i on the flanks of the tip region. Areas of elevated [Ca2+]i corresponded to sites of pronounced exocytosis, as evidenced by the formation of wall ingrowths adjacent to the plasma membrane. Ca2+o influx at the tip of (beta-D-Glc)3-treated pollen tubes was not significantly different to that of control tubes. Taken together these data indicate that regions of elevated [Ca2+]i, probably resulting from Ca2+o influx across the plasma membrane, stimulate exocytosis in pollen tubes independent of cell elongation.

The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads.

Ratio images of cytosolic Ca2+ (Ca2+i) in growing, fura-2-dextran-loaded Lilium longiflorum pollen tubes taken at 3- to 5-sec intervals showed that the tip-focused [Ca2+]i gradient oscillates with the same period as growth. Similarly, measurement of the extracellular inward current, using a noninvasive ion-selective vibrating probe, indicated that the tip-directed extracellular Ca2+ (Ca2+o) current also oscillates with the same period as growth. Cross-correlation analysis revealed that whereas the [Ca2+]i gradient oscillates in phase with growth, the influx of Ca2+o lags by ~11 sec. Ion influx thus appears to follow growth, with the effect that the rate of growth at a given point determines the magnitude of the ion influx ~11 sec later. To explain the phase delay in the extracellular inward current, there must be a storage of Ca2+ for which we consider two possibilities: either the inward current represents the refilling of intracellular stores (capacitative calcium entry), or it represents the binding of the ion within the cell wall domain.

The ability to maintain the cytoplasmic Ca2+ concentration ([Ca2+]cyt) at homeostatic levels has been examined during leaf senescence in detached parsley (Petroselinum crispum) leaves. Fluorescence ratiometric imaging of mesophyll cells isolated from parsley leaves at various senescence stages and loaded with the Ca2+ indicator fura-2 has revealed a distinct elevation of [Ca2+]cyt, which was positively correlated with the progress of leaf senescence. This initial increase of [Ca2+]cyt, which was first observed in cells isolated from 3-d-senescent leaves, occurred 1 d before or in parallel with changes in two established senescence parameters, chlorophyll loss and lipid peroxidation. However, the [Ca2+]cyt elevation followed by 2 d the initial increase in the senescence-associated proteolysis. Whereas the [Ca2+]cyt of nonsenescent cells remained at the basal level, the elevated [Ca2+]cyt of the senescent cells was a long-lasting effect. Experimental retardation of senescence processes, achieved by pretreatment of detached leaves with the cytokinin benzyladenine, resulted in maintenance of homeostatic levels of [Ca2+]cyt in cells isolated from 3-d-senescent leaves. These observations demonstrate for the first time to our knowledge a correlation between elevated [Ca2+]cyt and the process of senescence in parsley leaves. Such senescence-associated elevation of [Ca2+]cyt, which presumably results from a loss of the cell's capability to extrude Ca2+, may serve as a signal inducing subsequent deteriorative processes.

Cytoplasmic MTs are recognized as important elements in plant development. They are categorized in four principal arrays: the PPB, the MA, the phragmoplast, and the cortical array, and they appear to participate in fundamental aspects of cell division, growth, and differentiation. Thus, such diverse activities as prediction of the alignment of the cell plate, the movement of chromosomes and transport of cell plate vesicles, and the orientation of the cellulose wall microfibrils are all mediated by MTs to varying degrees. Our knowledge about the distribution and function of MTs has been gleaned largely from studies at the electron microscope level, and more recently at the light microscope level using immunofluorescence methods. Because most of these observations have been based on cells that have been fixed, it has not been possible to examine directly the dynamic properties and behavior of MTs. Transitional states have been inferred from looking at numerous isolated examples, which have then been placed into a sequence. Other aspects, such as the sensitivity of MTs to drugs, ions, or cold temperature, have also been approximated through studies in which cells have been fixed at periodic intervals following application of an agent. Some important aspects of MT behavior, including, notably, their turnover, have been beyond the reach of these indirect methods. To enlarge our understanding of the dynamic properties of MTs, we have introduced the technique of fluorescent analog cytochemistry as applied to plant cells (Zhang et al., 1990b). In this procedure fluorescently labeled exogenous tubulin, which is injected into the cell, incorporates into the endogenous pool and acts as a reporter. MTs, which are observed with the CLSM, can be examined under any experimental condition to which the cells are exposed. The results have produced new information about MT transitions and dynamic behavior in live cells, which we address in this Update. Other aspects of plant MTs, including the identification of MT organizing centers, tubulin isoforms, and MAPs have been considered in other recent topical reviews (Lambert, 1993; Cyr, 1994; Goddard et al., 1994; Shibaoka, 1994; Cyr and Palevitz, 1995; Joshi and Palevitz, 1996), so they will not be covered here. INCORPORATION OF HETEROLOGOUS ANIMAL TUBULIN INTO PLANT MTS; OBSERVATIONS BY CLSM

The presence and localization of actin and myosin have been examined in pollen tubes of Lilium longiflorum and Nicotiana alata. Immunoblot analysis of pollen tube extracts with antibodies to actin, myosins IA and IB, myosin II, and myosin V reveals the presence of these contractile proteins. Immunofluorescence microscopy using various methods to preserve the pollen tubes; chemical fixation, rapid freeze fixation and freeze substitution (RF-FS) followed by rehydration or by embeddment in a methacrylate mixture, was performed to optimize preservation. Immunocytochemistry reaffirmed that actin is localized longitudinally in the active streaming lanes and near the cortical surface of the pollen tube. Myosin I was localized to the plasma membrane, larger organelles, the surface of the generative cell and the vegetative nucleus, whereas, myosin V was found in the vegetative cytoplasm in a punctate fashion representing smaller organelles. Myosin II subfragment 1 and light meromyosin were localized in a punctate fashion on the larger organelles throughout the vegetative cytoplasm. In addition, isolated generative cells and vegetative nuclei labeled only with the myosin I antibody. Competition studies indicated the specificity of the heterologous antibodies utilized in this study suggesting the presence of three classes of myosins in pollen. These results lead to the following hypothesis: Myosin I may move the generative cell and vegetative nucleus unidirectionally through the pollen tube to the tip, while myosin V moves the smaller organelles and myosins I and II move the larger organelles (bidirectionally) that are involved in growth.

Rhodamine-phalloidin or FITC-phalloidin has been injected in small amounts into living, developing cells of Micrasterias denticulata and the stained microfilaments visualized by confocal laser scanning microscopy. The results reveal that two different actin filament systems are present in a growing cell: a cortical actin network that covers the inner surface of the cell and is extended far into the tips of the lobes in both the growing and the nongrowing semicell; it is also associated with the surface of the chloroplast. The second actin system ensheathes the nucleus at the isthmus-facing side during nuclear migration. Its arrangement corresponds to that of the microtubule system that has been described in earlier electron microscopic investigations. The spatial correspondence between the distribution of actin filaments and microtubules suggests a cooperation between both cytoskeleton elements in generating the motive force for nuclear migration. The function of the cortical actin network is not yet clear. It may be involved in processes like transport and fusion of secretory vesicles and may also function in shaping and anchoring the chloroplast.

The frequency of red mangrove trees, Rhizophora mangle L., that were monohybrid heterozygotes for chlorophyll-deficient alleles was determined for populations along the southwest coast of Puerto Rico. Segregation ratios for the offspring of these trees were also calculated. Although nuclear, the different mutant homozygous genotypes had profound effects on chloroplast ultrastructure. In comparison to Florida and San Salvador Island R. mangle populations, Puerto Rican mangroves were more outcrossed (28.8% vs. 4.8%) and had lower per generation per genome mutation rates for chlorophyll-deficient mutations (2.1 x 10-3 vs. 5.8 x 10-3).

Stamen hair cells of Tradescantia virginiana have been microinjected with p13suc1 labeled with carboxyfluorescein (CF) and studied throughout the division cycle in living cells by using the confocal laser scanning microscope. The protein, p13suc1, is essential for the rapid inactivation of the key mitotic catalyst, p34cdc2 kinase, at anaphase and for completion of nuclear division. During interphase or prophase, CF-p13suc1 concentrates quickly (< 2 min) in nuclei, reaching levels that are approximately 2-fold greater than those in the cytoplasm. At nuclear envelope breakdown, CF-p13suc1 permeates throughout the entire spindle and nonspindle cytoplasm. The protein is excluded from the tightly condensed chromosomes but otherwise no regions accumulate or exclude the protein. It remains evenly distributed throughout metaphase, anaphase, and well into cytokinesis; however, during telophase CF-p13suc1 reconcentrates in the daughter nuclei.

This review covers historical and recent progress in understanding how respiration, fermentation and mitochondria contribute to pollen tube growth. It also summarizes what is known about the energetic requirements of this growth. Molecular mechanisms are viewed in the context of pollen tube physiology, a necessary perspective to understanding pollen tube growth., Background Pollen tubes grow by transferring chemical energy from stored cellular starch and newly assimilated sugars into ATP. This drives myriad processes essential for cell elongation, directly or through the creation of ion gradients. Respiration plays a central role in generating and regulating this energy flow and thus in the success of plant reproduction. Pollen tubes are easily grown in vitro and have become an excellent model for investigating the contributions of respiration to plant cellular growth and morphogenesis at the molecular, biochemical and physiological levels. Scope In recent decades, pollen tube research has become increasingly focused on the molecular mechanisms involved in cellular processes. Yet, effective growth and development requires an intact, integrated set of cellular processes, all supplied with a constant flow of energy. Here we bring together information from the current and historical literature concerning respiration, fermentation and mitochondrial physiology in pollen tubes, and assess the significance of more recent molecular and genetic investigations in a physiological context. Conclusions The rapid growth of the pollen tube down the style has led to the evolution of high rates of pollen tube respiration. Respiration rates in lily predict a total energy turnover of 40–50 fmol ATP s−1 per pollen grain. Within this context we examine the energetic requirements of cell wall synthesis, osmoregulation, actin dynamics and cyclosis. At present, we can only estimate the amount of energy required, because data from growing pollen tubes are not available. In addition to respiration, we discuss fermentation and mitochondrial localization. We argue that the molecular pathways need to be examined within the physiological context to understand better the mechanisms that control tip growth in pollen tubes.

Significant controversy still swirls around the regulation of extension by tip-growing cells, particularly during stable, oscillatory growth of pollen tubes. One explanation proposes that turgor pressure is both the controlling and driving force. We refute this hypothesis on theoretical and evidentiary grounds. Direct measurement of intracellular pressure reveals constant turgor even as growth rates change. Measured ion fluxes, notably potassium, are insufficient to account for the requisite osmotic changes. Water movement, and hence pressure gradients, occur throughout the cell, unrestricted to local domains. Increases in hydrostatic pressure alone would force water out of the cell rather than cause increased growth. We have recently demonstrated concomitant changes in the apical cell wall that account fully for observed changes in growth rate.

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.

We have previously shown that changes in the intracellular free calcium concentration ([Ca2+]i) modulate the rate of anaphase chromosome motion in stamen hair cells of Tradescantia. Elevations between 0.8-1.0-mu-M accelerate motion, while increases above 2.0-mu-M or decreases below resting level inhibit motion. The related signaling agent GTP-gamma-S also accelerates motion, however, by a mechanism that does not appear to involve changes in [Ca2+]i. To explore further the mechanism by which Ca2+ and GTP-gamma-S regulate chromosome motion we have analyzed the direct effect of these agents on the structure of the spindle microtubules (MTs). First, we injected carboxyfluorescein-derivatized brain tubulin and allowed it to incorporate into spindle MTs. Then, during appropriate times of anaphase, we injected Ca2+ or related agents and monitored their effect on spindle MT fluorescence using a confocal laser scanning microscope. A high level of Ca2+ (10-mu-M), known to inhibit motion, causes extensive degradation of spindle MT structure. An intermediate level (2-mu-M), which slows but does not stop movement, produces a distinct decay of fluorescence. A level of Ca2+ (0.8-1-mu-M) known to accelerate motion, however, generates only a small change in which the kinetochore fibers appear less distinct, and the overall spindle fluorescence is more diffuse. The Ca2+ buffer EGTA, which transiently blocks motion, has no detectable effect on spindle structure. GTP-gamma-S, which enhances motion also has no discernible effect on spindle structure. The results with Ca2+ support the idea that the ion facilitates anaphase motion through depolymerization of kinetochore MTs. However, if the depolymerization is extensive then inhibition of motion ensues. GTP-gamma-S, on the other hand, accelerates motion without inducing a detectable effect on spindle MT structure.

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.

The cellular pattern and distribution of membranes have been analyzed during cytokinesis in pollen mother cells of Tradescantia and compared with those of actin microfilaments (MFs) and microtubules (MTs). Membranes have been stained with DiOC6(3) and MFs with rhodamine-labeled phalloidin (RP); analysis has been carried out on the confocal laser scanning microscope. MTs have been visualized as birefringent elements in the polarized light microscope. The results show that when the interzone first appears in mid anaphase it contains an even distribution of membranes. However, by late anaphase these elements have been cleared away, leaving the interzone largely devoid of DiOC6(3)-positive material. MTs are found throughout this zone, while MFs appear in two non-overlapping sets on both sides of the cell equator. Thereafter membrane elements reappear in the interzone, but only along the equatorial line of the forming cell plate. Presumably these equatorial elements are composed of endoplasmic reticulum and Golgi vesicles, since the larger organelles, including amyloplasts and mitochondria, are excluded from the phragmoplast. MFs, like MTs, arrange preferentially normal to the cell plate, forming a dense array on both sides, but being absent from the zone occupied by the membranes. By contrast, the parallel set of MTs, while excluding larger organelles from the phragmoplast, intermingle with the membrane elements in the cell equator. As cytokinesis proceeds membranes continue to concentrate on the cell plate as indicated by its marked increase in staining with DiOC6(3). From a consideration of spatial and temporal organization of the phragmoplast components it is reasonable to suggest that both cytoskeletal components participate in the aggregation of vesicles that give rise to the cell plate. Membranes, on the other hand, through the provision of surface binding sites and/or through the regulation of the cytoplasmic calcium ion concentration, might be involved in the assembly and stabilization of the cytoskeleton.

The delivery of cell wall material and membrane to growing plant cell surfaces requires the spatial and temporal coordination of secretory vesicle trafficking. Given the small size of vesicles, their dynamics is difficult to quantify. To quantitatively analyze vesicle dynamics in growing pollen tubes labeled with the styryl dye FM1-43, we applied spatiotemporal correlation spectroscopy on time-lapse series obtained with high-speed confocal laser scanning microscopy recordings. The resulting vector maps revealed that vesicles migrate toward the apex in the cell cortex and that they accumulate in an annulus-shaped region adjacent to the extreme tip and then turn back to flow rearward in the center of the tube. Fluorescence recovery after photobleaching confirmed vesicle accumulation in the shoulder of the apex, and it revealed that the extreme apex never recovers full fluorescence intensity. This is consistent with endocytotic activity occurring in this region. Fluorescence recovery after photobleaching analysis also allowed us to measure the turnover rate of the apical vesicle population, which was significantly more rapid than the theoretical rate computed based on requirements for new cell wall material. This may indicate that a significant portion of the vesicles delivered to the apex does not succeed in contacting the plasma membrane for delivery of their contents. Therefore, we propose that more than one passage into the apex may be needed for many vesicles before they fuse to the plasma membrane and deliver their contents.

Prevention of actin polymerization with low concentrations of latrunculin B (Lat-B; 2 nm) exerts a profound inhibitory effect on pollen tube growth. Using flow-through chambers, we show that growth retardation starts after 10 min treatment with 2 nm Lat-B, and by 15 to 20 min reaches a basal rate of 0.1 to 0.2 microm/s, during which the pollen tube exhibits relatively few oscillations. If treated for 30 min, complete stoppage of growth can occur. Studies on the intracellular Ca(2+) concentration indicate that the tip-focused gradient declines in parallel with the inhibition of growth. Tubes exhibiting nonoscillating growth display a similarly reduced and nonoscillating Ca(2+) gradient. Studies on the pH gradient indicate that Lat-B eliminates the acidic domain at the extreme apex, and causes the alkaline band to move more closely to the tip. Removing Lat-B and returning the cells to control medium reverses these effects. Phalloidin staining of F-actin reveals that 2 nm Lat-B degrades the cortical fringe; it also disorganizes the microfilaments in the shank causing the longitudinally oriented elements to be disposed in swirls. Cytoplasmic streaming continues under these conditions, however the clear zone is obliterated with all organelles moving into and through the extreme apex of the tube. We suggest that actin polymerization promotes pollen tube growth through extension of the cortical actin fringe, which serves as a track to target cell wall vesicles to preferred exocytotic sites on the plasma membrane.

Caulonemata of the moss Funaria hygrometrica were vitally stained with the fluorescent, lipophilic carbocyanine dye DiOC6(3) and examined via confocal laser scanning microscopy. Although DiOC6 (3) stained nearly all of the organelles, cortical endoplasmic reticulum (ER) could be resolved under favorable conditions and appeared as a network of irregular polygons, interspersed with lamellar cisternae in some cell types. The pattern of cortical ER was examined first during side initial formation and then in young branches and buds. The ER network extends into the outgrowth of a developing side initial, keeping pace with elongation of the outgrowth. Prior to the cell division that cuts off the outgrowth from the underlying cell, the network in the outgrowth becomes tighter, i.e. the polygons become smaller. If the side initial develops as a branch, this somewhat tighter ER network is maintained in the tip-growing side branch. If the side initial develops as a bud, dramatic changes in both the configuration and the quantity of the ER network occur. Coincident with the apical swelling that marks the first visible sign of bud formation, the network becomes increasingly tighter until eventually the polygonal configuration is barely discernible. The increased coverage of the bud cortex by the ER network demonstrates that a significant increase in the quantity of membranes also takes place during bud formation in Funaria.