Nuclear localization of profilin during the cell cycle in Tradescantia virginiana stamen hair cells.

The localization of the actin-monomer-binding protein profilin during the cell cycle of living Tradescantia virginiana stamen hair cells has been studied by microinjection of a fluorescently labeled analog of the protein. In contrast to previously published studies performed on chemically fixed animal cells, we do not find a specific colocalization of profilin with actin filament arrays. Our results show that, besides a general cytoplasmic distribution, profilin specifically accumulates in the nucleus in interphase and prophase cells. This nuclear localization was confirmed by means of electron microscopic immunolocalization of endogenous profilin (in Gibasis scheldiana stamen hair cells). During mitosis, as the nuclear envelope and nuclear matrix break down at the onset of prometaphase, the nuclear profilin redistributes equally into the accessible volume (cytosol) of the cell. During metaphase and anaphase no specific localization of profilin can be observed associated with the mitotic apparatus. However, during telophase, as nuclear envelopes and nuclear matrices re-form and the sister chromatids start to decondense, a subset of the microinjected profilin again localizes to the nucleus. No accumulation of profilin could be observed in the phragmoplast, where a distinct array of actin filaments exists. The function of profilin in the nucleus remains unclear.

Cytoplasmic acidification with butyric acid does not alter the ionic conductivity of plasmodesmata.

The effect of lowering cytoplasmic pH on the ionic conductivity of higher-plant plasmodesmata was investigated with corn (Zea mays L. cv. Black Mexican Sweet) suspension culture cells. Exposure to butyric acid decreased the cytoplasmic pH by 0.8 units. Intercellular communication was monitored by electrophysiological techniques that allowed the measurement of membrane resistances of sister cells and the electrical resistance of the plasmodesmata connecting them. The decrease in cytoplasmic pH did not affect the resistance of plasmodesmata, despite the fact that the butyric acid treatment more than doubled the concentration of cytoplasmic calcium. This is discussed in light of previous findings that increases in cytoplasmic calcium increase the electrical resistance of plasmodesmata.

Actin and pollen tube growth.

Actin microfilaments (MFs) are essential for the growth of the pollen tube. Although it is well known that MFs, together with myosin, deliver the vesicles required for cell elongation, it is becoming evident that the polymerization of new actin MFs, in a process that is independent of actomyosin-dependent vesicle translocation, is also necessary for cell elongation. Herein we review the recent literature that focuses on this subject, including brief discussions of the actin-binding proteins in pollen, and their possible role in regulating actin MF activity. We promote the view that polymerization of new actin MFs polarizes the cytoplasm at the apex of the tube. This process is regulated in part by the apical calcium gradient and by different actin-binding proteins. For example, profilin binds actin monomers and gives the cell control over the initiation of polymerization. A more recently discovered actin-binding protein, villin, stimulates the formation of unipolar bundles of MFs. Villin may also respond to the apical calcium gradient, fragmenting MFs, and thus locally facilitating actin remodeling. While much remains to be discovered, it is nevertheless apparent that actin MFs play a fundamental role in controlling apical cell growth in pollen tubes.

Polarized cell growth in higher plants.

Pollen tubes and root hairs are highly elongated, cylindrically shaped cells whose polarized growth permits them to explore the environment for the benefit of the entire plant. Root hairs create an enormous surface area for the uptake of water and nutrients, whereas pollen tubes deliver the sperm cells to the ovule for fertilization. These cells grow exclusively at the apex and at prodigious rates (in excess of 200 nm/s for pollen tubes). Underlying this rapid growth are polarized ion gradients and fluxes, turnover of cytoskeletal elements (actin microfilaments), and exocytosis and endocytosis of membrane vesicles. Intracellular gradients of calcium and protons are spatially localized at the growing apex; inward fluxes of these ions are apically directed. These gradients and fluxes oscillate with the same frequency as the oscillations in growth rate but not with the same phase. Actin microfilaments, which together with myosin generate reverse fountain streaming, undergo rapid turnover in the apical domain, possibly being regulated by key actin-binding proteins, e.g., profilin, villin, and ADF/cofilin, in concert with the ion gradients. Exocytosis of vesicles at the apex, also dependent on the ion gradients, provides precursor material for the continuously expanding cell wall of the growing cell. Elucidation of the interactions and of the dynamics of these different components is providing unique insight into the mechanisms of polarized growth.

Inositol 1,4,5 trisphosphate is inactivated by a 5-phosphatase in stamen hair cells of Tradescantia.

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 polymerization is essential for pollen tube growth.

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.

Cellular oscillations and the regulation of growth: the pollen tube paradigm.

The occurrence of oscillatory behaviours in living cells can be viewed as a visible consequence of stable, regulatory homeostatic cycles. Therefore, they may be used as experimental windows on the underlying physiological mechanisms. Recent studies show that growing pollen tubes are an excellent biological model for these purposes. They unite experimental simplicity with clear oscillatory patterns of both structural and temporal features, most being measurable during real-time in live cells. There is evidence that these cellular oscillators involve an integrated input of plasma membrane ion fluxes, and a cytosolic choreography of protons, calcium and, most likely, potassium and chloride. In turn, these can create positive feedback regulation loops that are able to generate and self-sustain a number of spatial and temporal patterns. Other features, including cell wall assembly and rheology, turgor, and the cytoskeleton, play important roles and are targets or modulators of ion dynamics. Many of these features have similarities with other cell types, notably with apical-growing cells. Pollen tubes may thus serve as a powerful model for exploring the basis of cell growth and morphogenesis. BioEssays 23:86-94, 2001.

The kinesin-like calmodulin binding protein is differentially involved in cell division.

The kinesin-like calmodulin (CaM) binding protein (KCBP), a minus end-directed microtubule motor protein unique to plants, has been implicated in cell division. KCBP is negatively regulated by Ca(2)+ and CaM, and antibodies raised against the CaM binding region inhibit CaM binding to KCBP in vitro; therefore, these antibodies can be used to activate KCBP constitutively. Injection of these antibodies into Tradescantia virginiana stamen hair cells during late prophase induces breakdown of the nuclear envelope within 2 to 10 min and leads the cell into prometaphase. However, mitosis is arrested, and the cell does not progress into anaphase. Injection of antibodies later during cell division has no effect on anaphase transition but causes aberrant phragmoplast formation and delays the completion of cytokinesis by approximately 15 min. These effects are achieved without any apparent degradation of the microtubule cytoskeleton. We propose that during nuclear envelope breakdown and anaphase, activated KCBP promotes the formation of a converging bipolar spindle by sliding and bundling microtubules. During metaphase and telophase, we suggest that its activity is downregulated.

The role of plant villin in the organization of the actin cytoskeleton, cytoplasmic streaming and the architecture of the transvacuolar strand in root hair cells of Hydrocharis.

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.

Plant GTPases: the Rhos in bloom.

In animal cells and in fungi, small GTP-binding proteins of the Rho family have well-established roles in morphogenesis, cell-cycle progression, gene transcription and the generation of superoxide anions. The presence of these proteins in plant cells, however, has been established only recently, and the role of Rho GTPases in plants is now coming into view. Already, it is apparent that there are both striking similarities and fascinating differences in how Rho GTPases are regulated and used in plant versus animal and fungal cells. These new findings define certain core properties that might be common to members of this protein family in all eukaryotes.

Physiological elevations in cytoplasmic free calcium by cold or ion injection result in transient closure of higher plant plasmodesmata.

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).

Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip.

Using both the proton selective vibrating electrode to probe the extracellular currents and ratiometric wide-field fluorescence microscopy with the indicator 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran to image the intracellular pH, we have examined the distribution and activity of protons (H+) associated with pollen tube growth. The intracellular images reveal that lily pollen tubes possess a constitutive alkaline band at the base of the clear zone and an acidic domain at the extreme apex. The extracellular observations, in close agreement, show a proton influx at the extreme apex of the pollen tube and an efflux in the region that corresponds to the position of the alkaline band. The ability to detect the intracellular pH gradient is strongly dependent on the concentration of exogenous buffers in the cytoplasm. Thus, even the indicator dye, if introduced at levels estimated to be of 1.0 microM or greater, will dissipate the gradient, possibly through shuttle buffering. The apical acidic domain correlates closely with the process of growth, and thus may play a direct role, possibly in facilitating vesicle movement and exocytosis. The alkaline band correlates with the position of the reverse fountain streaming at the base of the clear zone, and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins. These studies not only demonstrate that proton gradients exist, but that they may be intimately associated with polarized pollen tube growth.

Three-dimensional organization and dynamic changes of the actin cytoskeleton in embryo sacs of Zea mays and Torenia fournieri.

Actin organization was observed in m-maleimidobenzoic acid N-hydroxysuccinimide ester(MBS)-treated maize embryo sacs by confocal laser scanning microscopy. The results revealed that dynamic changes of actin occur not only in the degenerating synergid, but also in the egg during fertilization. The actin filaments distribute randomly in the chalazal part of the synergid before fertilization; they later become organized into numerous aggregates in the chalazal end after pollination. The accumulation of actin at this region is intensified after the pollen tube discharges its contents. Concurrently, actin patches have also been found in the cytoplasm of the egg cell and later they accumulate in the cortical region. To compare with MBS-treated maize embryo sacs, we have performed phalloidin microinjection to label the actin cytoskeleton in living embryo sacs of Torenia fournieri. The results have extended the previous observations on the three-dimensional organization of the actin arrays in the cells of the female germ unit and confirm the occurrence of the actin coronas in the embryo sac during fertilization. We have found that there is an actin cap occurring near the filiform apparatus after anthesis. In addition, phalloidin microinjection into the Torenia embryo sac has proved the presence of intercellular actin between the cells of the female germ unit and thus confirms the occurrence of the actin coronas in the embryo sac during fertilization. Moreover, actin dynamic changes also take place in the egg and the central cell, accomplished with the interaction between the male and female gametes. The actin filaments initially organize into a distinct actin network in the cortex of the central cell after anthesis; they become fragmented in the micropylar end of the cell after pollination. Similar to maize, actin patches have also been observed in the egg cortex after pollination. This is the first report of actin dynamics in the living embryo sac. The results suggest that the actin cytoskeleton may play an essential role in the reception of the pollen tube, migration of the male gametes, and even gametic fusion.

Effects of Yariv phenylglycoside on cell wall assembly in the lily pollen tube.

Arabinogalactan-proteins (AGPs) are proteoglycans with a high level of galactose and arabinose. Their current functions in plant development remain speculative. In this study, (beta-D-glucosyl)3 Yariv phenyl-glycoside [(beta-D-Glc)3] was used to perturb AGPs at the plasmalemma-cell wall interface in order to understand their functional significance in cell wall assembly during pollen tube growth. Lily (Lilium longiflorum Thunb.) pollen tubes, in which AGPs are deposited at the tip, were used as a model. Yariv phenylglycoside destabilizes the normal intercalation of new cell wall subunits, while exocytosis of the secretory vesicles still occurs. The accumulated components at the tip are segregated between fibrillar areas of homogalacturonans and translucent domains containing callose and AGPs. We propose that the formation of AGP/(beta-D-Glc)3 complexes is responsible for the lack of proper cell wall assembly. Pectin accumulation and callose synthesis at the tip may also change the molecular architecture of the cell wall and explain the lack of proper cell wall assembly. The data confirm the importance of AGPs in pollen tube growth and emphasize their role in the deposition of cell wall subunits within the previously synthesized cell wall.

The Cytosolic Ca2+ Concentration Gradient of Sinapis alba Root Hairs as Revealed by Ca2+-Selective Microelectrode Tests and Fura-Dextran Ratio Imaging.

Using Ca2+-selective microelectrodes and fura 2-dextran ratio imaging, the cytosolic free [Ca2+] was measured in Sinapis alba root hair cells. Both methods yielded comparable results, i.e. values between 158 to 251 nM for the basal [Ca2+] of the cells and an elevated [Ca2+] of 446 to 707 nM in the tip region. The zone of elevated [Ca2+] reaches 40 to 60 [mu]m into the cell and is congruent with the region of inwardly directed Ca2+ net currents measured with an external Ca2+- selective vibrating electrode. The channel-blocker La3+ eliminates these currents, stops growth, and almost completely eliminates the cytosolic [Ca2+] gradient without affecting the basal level of the ion. Growth is also inhibited by pressure-injected dibromo-1,2-bis(o-aminophenoxy)ethane-N,N,N[prime],N[prime]-tetraacetic acid, which causes a decrease in the [Ca2+] in the tip in a concentration-dependent manner. Indole-3-acetic acid, used as a model stimulus, decreases cytosolic free [Ca2+] by 0.2 to 0.3 pCa units in the tip, but only by about 0.1 pCa unit in the shank. Nongrowing root hairs may or may not display a [Ca2+] gradient, but still reversibly respond to external stimuli such as La3+, Ca2+, or indole-3-acetic acid with changes in cytosolic free [Ca2+]. During short time periods, dicyclohexylcarbodiimide inhibition of the plasma membrane H+-ATPase, which stops growth, does not abolish the [Ca2+] gradient, nor does it change significantly the basal [Ca2+] level. We conclude that the cytosolic [Ca2+] gradient and an elevated [Ca2+] in the tip, as in other tip-growing cells, is essential for tip growth in root hairs; however, its presence does not indicate growth under all circumstances. We argue that with respect to Ca2+, tip growth regulation and responses to external signals may not interfere with each other. Finally, we suggest that the combination of the methods applied adds considerably to our understanding of the role of cytosolic free [Ca2+] in signal transduction and cellular growth.

Characterization and localization of profilin in pollen grains and tubes of Lilium longiflorum.

Pollen tubes show a rapid and dramatically polarized growth in which the actin cytoskeleton appears to play a central role. In order to understand the regulation of actin we characterized its associated protein, profilin, in pollen tubes of Lilium longiflorum. By using purified polyclonal antibodies prepared against bean root profilin [Vidali et al., 1995: Plant Physiol. 108:115-123] we detected in pollen grains and tubes two profilin polypeptides with molecular masses of 14.4 and 13.4 KDa, and an identical isoelectric point of 5.05. Profilin comprises approximately 0.47% of the total grain protein, with actin being approximately 1.4%. We were unable to detect a statistically significant profilin increase after germination, while the actin increased approximately 68%. We also spatially localized the distribution of profilin using immunocytochemistry of fixed cells at both the light and electron microscope level, and by fluorescent analog cytochemistry on live cells. The results show that profilin is evenly distributed throughout the cytoplasm and does not specifically associate with any cellular structure.

Increases in Cytosolic Ca2+ in Parsley Mesophyll Cells Correlate with Leaf Senescence.

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.

Probing the Plant Actin Cytoskeleton during Cytokinesis and Interphase by Profilin Microinjection.

We have examined the cytological effects of microinjecting recombinant birch profilin in dividing and interphase stamen hair cells of Tradescantia virginiana. Microinjection of profilin at anaphase and telophase led to a marked effect on cytokinesis; cell plate formation was often delayed, blocked, or completely inhibited. In addition, the initial appearance of the cell plate was wrinkled, thin, and sometimes fragmented. Injection of profilin at interphase caused a thinning or the collapse of cytoplasmic strands and a retardation or inhibition of cytoplasmic streaming in a dose-dependent manner. Confocal laser scanning microscopy of rhodamine-phalloidin staining in vivo revealed that high levels of microinjected profilin induced a degradation of the actin cytoskeleton in the phragmoplast, the perinuclear zone, and the cytoplasmic strands. However, some cortical actin filaments remained intact. The data demonstrate that profilin has the ability to act as a regulator of actin-dependent events and that centrally located actin filaments are more sensitive to microinjected profilin than are cortical actin filaments. These results add new evidence supporting the hypothesis that actin filaments play a crucial role in the formation of the cell plate and provide mechanical support for the cytoplasmic strands in interphase cells.