Higher Plant Cells Research Articles

Force and compliance: rethinking morphogenesis in walled cells

In the turgid cells of plants, protists, fungi, and bacteria, walls resist swelling; they also confer shape on the cell. These two functions are not unrelated: cell physiologists have generally agreed that morphogenesis turns on the deformation of existing wall and the deposition of new wall, while turgor pressure produces the work of expansion. In 1990, I summed up consensus in a phrase: “localized compliance with the global force of turgor pressure.” My purpose here is to survey the impact of recent discoveries on the traditional conceptual framework. Topics include the recognition of a cytoskeleton in bacteria; the tide of information and insight about budding in yeast; the role of the Spitzenkörper in hyphal extension; calcium ions and actin dynamics in shaping a tip; and the interplay of protons, expansins and cellulose fibrils in cells of higher plants.

Glycerophosphocholine metabolism in higher plant cells. Evidence of a new glyceryl-phosphodiester phosphodiesterase.

Glycerophosphocholine (GroPCho) is a diester that accumulates in different physiological processes leading to phospholipid remodeling. However, very little is known about its metabolism in higher plant cells. (31)P-Nuclear magnetic resonance spectroscopy and biochemical analyses performed on carrot (Daucus carota) cells fed with GroPCho revealed the existence of an extracellular GroPCho phosphodiesterase. This enzymatic activity splits GroPCho into sn-glycerol-3-phosphate and free choline. In vivo, sn-glycerol-3-phosphate is further hydrolyzed into glycerol and inorganic phosphate by acid phosphatase. We visualized the incorporation and the compartmentation of choline and observed that the major choline pool was phosphorylated and accumulated in the cytosol, whereas a minor fraction was incorporated in the vacuole as free choline. Isolation of plasma membranes, culture medium, and cell wall proteins enabled us to localize this phosphodiesterase activity on the cell wall. We also report the existence of an intracellular glycerophosphodiesterase. This second activity is localized in the vacuole and hydrolyzes GroPCho in a similar fashion to the cell wall phosphodiesterase. Both extra- and intracellular phosphodiesterases are widespread among different plant species and are often enhanced during phosphate deprivation. Finally, competition experiments on the extracellular phosphodiesterase suggested a specificity for glycerophosphodiesters (apparent K(m) of 50 microM), which distinguishes it from other phosphodiesterases previously described in the literature.

Endopolygalacturonase of Saccharomyces cerevisiae: involvement in pseudohyphae development of haploids and in pathogenicity on Vitis vinifera

We have cloned the PGL1 gene from Saccharomyces cerevisiae, an poly-α-1,4 galacturonide glycanohydrolase (EC 3.2.1.15) commonly named endopolygalacturonase (endoPG), with a cis-acting regulatory region. This enzyme is known as pectin hydrolase, pectin being one of the main constituents of primary cell walls and middle lamella of higher plant cells. These pectins constitute a structural barrier to microbial invasion. The construct pRPGL1-2 has been introduced into the haploid receiver strain of S. cerevisiae, devoided of endoPG activity, to study the potential role of polygalacturonases in pathogenicity towards Vitis vinifera and in pseudohyphae formation. The latter corresponds to a switch from a single-cell form to the formation of long-branched chains of elongated cells, a phenomenon mainly reported for diploid yeast. We observed in the isogenic S. cerevisiae strains that endoPG, encoded by PGL1 gene, is required for pseudohyphae development and is involved in plant pathogenesis. The direct penetration of the host plant has been confirmed histologically for the transformed haploid receiver.

The plant Spc98p homologue colocalizes with gamma-tubulin at microtubule nucleation sites and is required for microtubule nucleation.

The molecular basis of microtubule nucleation is still not known in higher plant cells. This process is better understood in yeast and animals cells. In the yeast spindle pole body and the centrosome in animal cells, gamma-tubulin small complexes and gamma-tubulin ring complexes, respectively, nucleate all microtubules. In addition to gamma-tubulin, Spc98p or its homologues plays an essential role. We report here the characterization of rice and Arabidopsis homologues of SPC98. Spc98p colocalizes with gamma-tubulin at the nuclear surface where microtubules are nucleated on isolated tobacco nuclei and in living cells. AtSpc98p-GFP also localizes at the cell cortex. Spc98p is not associated with gamma-tubulin along microtubules. These data suggest that multiple microtubule-nucleating sites are active in plant cells. Microtubule nucleation involving Spc98p-containing gamma-tubulin complexes could then be conserved among all eukaryotes, despite differences in structure and spatial distribution of microtubule organizing centers.

Replacement of midgut epithelium in the greater wax moth, Galleria mellonela, during larval-pupal moult.

The epithelium of larval midgut of the greater wax moth, Galleria mellonela, was replaced during the larval-pupal moult. The development of this moth was tentatively divided into 11 stages, from the full-grown larva of last instar to the 4-day-old pupa. The midgut at each stage was observed for (1) overall structure, (2) the position of goblet cells, and (3) the appearance of the yellow body. Light microscopy revealed that cell death in the midgut began in a cocoon-spinning larva (stage II), when pigments in the stemmata started to migrate. Before drastic remodeling started to occur, cytoplasmic projections in the goblet cavities were transformed. The larval midgut changed markedly at stage III, when the pigments left the stemmata. The epithelium of the larval midgut dropped as a whole into the lumen, transforming into the yellow body. Simultaneously, a pupal midgut epithelium developed. Electron microscopy of the columnar cells of a stage III larva showed that microvilli and mitochondria looked normal even though the nucleus with condensed heterochromatin resembled an apoptotic nucleus of vertebrate and higher plant cells. Caspase-3-like protease activity was restricted to the larval midgut and increased in parallel with the formation of the yellow body. The results indicate that the replacement of the larval midgut is facilitated by a typical apoptotic process.

Measurement of ferrochelatase activity using a novel assay suggests that plastids are the major site of haem biosynthesis in both photosynthetic and non-photosynthetic cells of pea (Pisum sativum L.)

Ferrochelatase is the terminal enzyme of haem biosynthesis, catalysing the insertion of ferrous iron into the macrocycle of protoporphyrin IX, the last common intermediate of haem and chlorophyll synthesis. Its activity has been reported in both plastids and mitochondria of higher plants, but the relative amounts of the enzyme in the two organelles are unknown. Ferrochelatase is difficult to assay since ferrous iron requires strict anaerobic conditions to prevent oxidation, and in photosynthetic tissues chlorophyll interferes with the quantification of the product. Accordingly, we developed a sensitive fluorimetric assay for ferrochelatase that employs Co2+ and deuteroporphyrin in place of the natural substrates, and measures the decrease in deuteroporphyrin fluorescence. A hexane-extraction step to remove chlorophyll is included for green tissue. The assay is linear over a range of chloroplast protein concentrations, with an average specific activity of 0.68nmol·min−1·mg of protein−1, the highest yet reported. The corresponding value for mitochondria is 0.19nmol·min−1·mg of protein−1. The enzyme is inhibited by N-methylprotoporphyrin, with an estimated IC50 value of ≈ 1nM. Using this assay we have quantified ferrochelatase activity in plastids and mitochondria from green pea leaves, etiolated pea leaves and pea roots to determine the relative amounts in the two organelles. We found that, in all three tissues, greater than 90% of the activity was associated with plastids, but ferrochelatase was reproducibly detected in mitochondria, at levels greater than the contaminating plastid marker enzyme, and was latent. Our results indicate that plastids are the major site of haem biosynthesis in higher plant cells, but that mitochondria also have the capacity for haem production.

Ped3p is a peroxisomal ATP-binding cassette transporter that might supply substrates for fatty acid beta-oxidation.

Glyoxysomes, a group of specialized peroxisomes, are organelles that degrade fatty acids by the combination of fatty acid beta-oxidation and glyoxylate cycle. However, the mechanism underlying the transport of the fatty acids across the peroxisomal membrane is still obscure in higher plant cells. We identified and analyzed the PED3 gene and its gene product, Ped3p. The phenotype of the Arabidopsis ped3 mutant indicated that the mutation in the PED3 gene inhibits the activity of fatty acid beta-oxidation. Ped3p is a 149-kDa protein that exists in peroxisomal membranes. The amino acid sequence of Ped3p had a typical characteristic for "full-size" ATP-binding cassette (ABC) transporter consisting of two transmembrane regions and two ATP-binding regions. This protein was divided into two parts, that had 32% identical amino acid sequences. Each part showed a significant sequence similarity with peroxisomal "half" ABC transporters so far identified in mammals and yeast. Ped3p may contribute to the transport of fatty acids and their derivatives across the peroxisomal membrane.

Direct coupling of action potential generation in cells of a higher plant (Cucurbita pepo) with the operation of an electrogenic pump

Changes in membrane potential (Em) were investigated during action potential (AP) generation by excitable cells in bundle parenchyma of 2-week-old pumpkin (Cucurbita pepo L.) seedlings. APs were evoked by gradual cooling from 21 to 6°C at a rate of 1.3–1.5°C/min. Changes in Em were measured with standard microelectrode technique. The plots of the function dEm/dt = f (Em) were obtained from experimental data by numerical differentiation of Em records. The depolarization proceeded as a one-stage process. Conversely, AP repolarization comprised two stages, distinguished by specific dynamics of dEm/dt. The second stage of repolarization occurred on a larger scale of Em and was more sensitive to temperature than the first one. It is supposed that the second stage of repolarization during AP is related to the operation of electrogenic H+-pump in the excitable membrane. The scheme of AP generation in higher plant cells is suggested; it foresees the involvement in AP generation of both passive and active mechanisms of electrogenesis.

Regulation of callose metabolism in higher plant cells in vitro

Temporary accumulation of callose in suspension-cultured wheat (Triticum timopheevii Zhuk.) cells at the exponential growth phase was correlated with the mitotic index due to the formation of the cell plates in dividing cells. Callose disappeared in expanding cells owing to enhanced activities of endo- and exoglucanases. The exogluconase activity was reduced when the cells were treated with cycloheximide, an inhibitor of protein synthesis. A similar pattern was observed when elicitors experimentally enhanced callose synthesis. Apparently, in such cases, callose behaves as a temporary component repairing the cell wall. We presume that plant cells comprise a universal mechanism for regulating callose synthesis.

Arabidopsis Variegation Mutants

Variegation mutants have been defined as “any plant that develops patches of different colors in its vegetative parts” (Kirk and Tilney-Bassett, 1978). Some of the most common variegations have green and white (or yellow) sectors in normally-green tissues and organs of the plant. Whereas cells in the green sectors typically contain normal-appearing chloroplasts, cells in the white (or yellow) sectors contain plastids that are deficient in chlorophyll and/or carotenoid pigments. These plastids appear to be blocked at various steps of chloroplast biogenesis because they frequently lack organized internal membrane structures and/or contain only rudimentary lamellae. Despite their widespread occurrence, relatively few variegations have been characterized at the molecular level. Variegation mutants have played a prominent role in the history of genetics (reviewed by Granick, 1955; Kirk and Tilney-Bassett, 1978). As a notable example, the finding that transmission of the variegation trait does not always obey Mendel's laws led to the discovery of non-Mendelian inheritance in the early 1900's. Whereas variegations have long been associated with differences in plastid form and function, it was not possible to gain insight into the molecular basis of this phenomenon until the 1960's and early 1970's, when compelling molecular evidence was presented that mitochondria and chloroplasts contain their own DNA and protein synthesis systems, and that organellar proteins are the products of genes in the chloroplast and mitochondrion, as well as the nucleus (reviewed by Bogorad, 1981). Nuclear-organelle interactions Nuclear DNA-encoded proteins that are destined for chloroplasts and mitochondria are translated as precursors on 80S ribosomes in the cytosol and transported into the organelle post-translationally, whereas proteins encoded in the mitochondrial and plastid genomes are translated on prokaryotic-like 70S ribosomes in the organelle itself (reviewed by Goldschmidt-Clermont, 1998; Leon et al., 1998). Mitochondria and plastids are derived from prokaryotic endosymbionts, and during the process of symbiogenesis most of the genes of the symbiont were lost or transferred to the host genome (Gray, 1992; Doolittle, 1998; Cavalier-Smith, 2000). For instance, current-day plastid genomes in higher plants code for less than 100 of the estimated 1900 to 2500 proteins in a typical chloroplast (Abdallah et al., 2000; Martin and Herrmann, 1998). Plastid and mitochondrial genomes are polyploid, and in higher plant cells they are dispersed among many plastids and mitochondria. For example, a typical mesophyll cell contains from 1,000 to 10,000 identical plastid DNAs distributed among 100 or more chloroplasts (Bendich, 1987). It is thought that the dispersal of genes for plastid and mitochondrial proteins between two different compartments was the central driving force that led to the evolution of mechanisms that integrate nuclear and organellar gene expression (reviewed by Bogorad, 1981). A contributing factor might also have been the vast disparity in copy number between genes in the nuclear and organellar compartments. Much of the regulatory traffic between the nucleus and the organelle is anterograde, i.e., from the nucleus to the organelle, in the form of nuclear gene products that control the transcription and translation of mitochondrial and plastid genes (reviewed by Goldschmidt-Clermont, 1998; Leon et al., 1998). Yet, much of this traffic is also retrograde, i.e., from the organelle to the nucleus (reviewed by Oelmuller, 1989; Taylor, 1989; Mayfield, 1990; Susek and Chory, 1992; Gray et al., 1995; Hess et al., 1997; Rodermel, 2001). Perhaps the best understood examples of retrograde trafficking in plants involve the transcriptional regulation of nuclear genes for photosynthetic proteins by plastid-to-nucleus signaling mechanisms initiated by a variety of “plastid signals” (reviewed by Rodermel, 2001). The plastid signals identified to date are intermediates or by-products of photosynthetic metabolism. Yet, poorly understood retrograde signals also control the expression of nuclear genes for a number of non-plastid proteins (reviewed by Oelmuller, 1989; Barak et al., 2001), as well as the transcription of mitochondrial genes, cell differentiation and leaf morphogenesis (reviewed by Hess et al., 1997; Hedtke et al., 1999; Rodermel, 2001). Consequently, plastid-to-nucleus signaling plays a central role in coordinating gene expression in the nucleus, plastid and mitochondrion, and in integrating pathways of cellular metabolism and development. Presumably, there is crosstalk between retrograde signaling and other signal transduction pathways (e.g., light, hormones, sugars, developmental factors) that also play a role in coordinating nuclear and organelle gene expression (e.g., Neff et al., 2000; Oswald et al., 2001).

Hydroperoxides of fatty acids induce programmed cell death in tomato protoplasts

Arachidonic acid (AA) is a fatty acid elicitor abundant in the glycerolipids of the late blight pathogen Phytophthora infestans and related Oomycete species. Lipoxygenases (LOX), which catalyze the addition of molecular oxygen to the 1 or 5 position of a cis, cis -1,4- Z, Z -pentadiene system in polyunsaturated fatty acids, is induced in host plants such as tomato and potato during infection by P. infestans. Here it is reported that AA, the LOX metabolites of AA, 5- and 15-hydroperoxyeicosatetraenioc acid (5- and 15-HpETE), and the LOX metabolite of linoleic acid, 9-hydroperoxyoctadecadienoic acid (9-HpODE), are potent inducers of programmed cell death (PCD) in tomato protoplasts. 5- and 15-HpETE increased DNA fragmentation as detected by t erminal deoxynucleotidyl transferase-mediated d U TP-X n ick e nd l abeling (TUNEL) and increased DNA laddering as visualized by ligation-mediated PCR. Background levels of DNA laddering were decreased in intensity by Zn2+ and increased by Ca2+, effects that are consistent with the reported action of these cations on PCD-associated endonucleases in other systems. H2O2, methyl jasmonate, and linoleic and linolenic acids were not toxic to tomato protoplasts at concentrations up to 350μM, and lipid peroxides (LD100=80μM) were far more potent inducers of death than free AA within this same concentration range. These results indicate the potential of fatty acid peroxides and LOX-related metabolism to engage an apoptotic type of PCD in higher plant cells.

Actomyosin promotes cell plate alignment and late lateral expansion in Tradescantia stamen hair cells.

Cytokinesis in higher-plant cells involves the formation of a cell plate in the interzone between the separating chromatids. The process is directed by the phragmoplast, an array of microtubules, actin filaments, and membranous elements. To determine if the role of actin in cytokinesis is dependent on myosin, we treated stamen hair cells of Tradescantia virginiana L. with 2,3-butanedione monoxime (BDM), an inhibitor of myosin ATPase and ML-7, a specific inhibitor of myosin light-chain kinase. Treatment with BDM resulted in a tilted cytokinetic apparatus during early initiation and a wavy cell plate with curved phragmoplasts during late lateral expansion. Treatment with ML-7 also resulted in inefficient late lateral expansion of the cell plate, with effects ranging from slower expansion to complete inhibition. Taken together, these results implicate myosin in the control of cell plate expansion and alignment.

Distribution and transcription activity of nucleolar DNA in higher plant cells.

By using the NAMA-Ur DNA selective staining method, we have observed in situ the location of nucleolar DNA in onion cells and found it at the boundary between fibrillar centres (FC) and dense fibrillar component (DFC) in transcriptionally active nucleolus. We have also used anti-NOR serum, which is identified as the RNA Polymerase I transcription factor (UBF) antibody, to study its reactivity with higher plant cells and demonstrated this factor associated to the DFC but not present at the interior of FC. Finally, by employing anti-DNA/RNA hybrid antibodies, we labeled the transcriptionally active rRNA genes in active nucleolus and testified that at the boundary between FC and DFC. The results provide the evidence that the boundary between FC and DFC is the genuine transcription site of rRNA genes in nucleolus.

Improvement of Salt Tolerance in Transgenic Potato Plants by Glyceraldehyde-3 Phosphate Dehydrogenase Gene Transfer

In the previous experiment, we isolated and characterized glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of the oyster mushroom, Pleurotus sajor-caju. Expression levels of the GPD gene in the mycelia of P sajor-caju was significantly increased by exposing the mycelia to abiotic stresses, such as salt, cold, heat, and drought. We also showed that GPD confers abiotic stress resistance when introduced into yeast cells. The survival rate of the transgenic yeast cell that harbored the GPD gene was significantly higher when the yeast cells were subjected to salt, cold, heat, and drought stresses, compared with the yeast that was transformed with the pYES2 vector alone. In order to investigate the functional role of the P. sajor-caju GPD gene in higher plant cells, the complete P. sajor-caju GPD cDNA was fused into the CaMV35S promoter and then introduced into potato plants. Putative potato transformants were screened by using PCR. Twenty-one transformants were further analyzed with RT-PCR to confirm the expression of P. sajor-caju GPD. A RT-PCR Southern blot analysis revealed that 12 transgenics induced the P. sajor-caju GPD gene expression. A bioassay of these transformants revealed that the P. sajor-caju GPD gene was enough to confer salt stress resistance in the potato plant cell system. Results showed that P. sajor-caju GPD, which was continuously expressed in transgenic potato plants under normal growing conditions, resulted in improved tolerance against salt loading.

Cellulose synthesis: mutational analysis and genomic perspectives using Arabidopsis thaliana.

Cellulose microfibrils containing crystalline beta-1,4-glucan provide the major structural framework in higher-plant cell walls. Genetic analyses of Arabidopsis thaliana now link specific genes to plant cellulose production just as was achieved some years earlier with bacteria. Cellulose-deficient mutants have defects in several members of one family within a complex glycosyltransferase superfamily and in one member of a small family of membrane-bound endo-1,4-beta-glucanases. The mutants also accumulate a readily extractable beta-1,4-glucan that has short chains which, in at least one case, are lipid linked. Cellulose could be made by direct extension of the glucan chain by the glycosyltransferase or, as the mutant suggests, by an indirect route which makes lipid-linked oligosaccharides. Models discussed incorporate the known enzymes and lipo-glucan and raise the possibility that different CesA glycosyltransferases may catalyse different steps.

Kinesin-Related Proteins in Plant Cytokinesis

Cytokinesis in higher plant cells is mediated by the phragmoplast. The framework of the phragmoplast consists of two anti-parallel sets of microtubules with their plus ends facing each other at or near the division site. During cytokinesis, the phragmoplast microtubule array undergoes dynamic reorganization from a solid cylinder to a hollow array. Concomitant with the microtubule reorganization, Golgi-originated vesicles are rapidly transported along microtubules toward their plus ends to give rise to the centrifugally growing cell plate. Kinesin-related motor proteins play crucial roles in microtubule reorganization and vesicle transport. To date, a number of plant proteins in the kinesin superfamily have been localized to the phragmoplast, and possibly exert roles in cytokinesis. Plus end-directed motors in the BIMC subfamily play a role in sliding anti-parallel microtubules apart to establish the phragmoplast microtubule array, while microtubule minus end-directed Ncd/Kar3-like motors in the C-terminal motor subfamily likely act antagonistically to balance the force generated by the BIMC-like kinesins. The novel C-terminal motor kinesin KCBP probably regulates microtubule organization and cross-linkage of the microtubule minus ends in a Ca ++ /calmodulin-dependent manner. By acting at or near the plus ends of the microtubules, the Arabidopsis phragmoplast-associated kinesin-related protein AtPAKRP1 likely plays a role in establishing and/or maintaining the organization of phragmoplast microtubules. We anticipate more phragmoplast-associated motors to be revealed in the next few years as the completed Arabidopsis genome contains more than 45 genes encoding kinesin-related proteins. Orchestrated forces generated by different motors are required for microtubule-dependent activities to take place in the phragmoplast.

Origin of the cytoplasmic pH changes during anaerobic stress in higher plant cells. Carbon-13 and phosphorous-31 nuclear magnetic resonance studies.

We tested the contribution of nucleoside triphosphate (NTP) hydrolysis, ethanol, and organic acid syntheses, and H(+)-pump ATPases activity in the acidosis of anoxic sycamore (Acer pseudoplatanus) plant cells. Culture cells were chosen to alter NTP pools and fermentation with specific nutrient media (phosphate [Pi]-deprived and adenine- or glycerol-supplied). In vivo (31)P- and (13)C-nuclear magnetic resonance (NMR) spectroscopy was utilized to noninvasively measure intracellular pHs, Pi, phosphomonoesters, nucleotides, lactate, and ethanol. Following the onset of anoxia, cytoplasmic (cyt) pH (7.5) decreased to 6.8 within 4 to 5 min, whereas vacuolar pH (5.7) and external pH (6.5) remained stable. The NTP pool simultaneously decreased from 210 to <20 nmol g(-1) cell wet weight, whereas nuceloside diphosphate, nucleoside monophosphate, and cyt pH increased correspondingly. The initial cytoplasmic acidification was at a minimum in Pi-deprived cells containing little NTP, and at a maximum in adenine-incubated cells showing the highest NTP concentration. Our data show that the release of H(+) ions accompanying the Pi-liberating hydrolysis of NTP was the principal cause of the initial cyt pH drop and that this cytoplasmic acidosis was not overcome by H(+) extrusion. After 15 min of anoxia, a partial cyt-pH recovery observed in cells supplied with Glc, but not with glycerol, was attributed to the H(+)-consuming ATP synthesis accompanying ethanolic fermentation. Following re-oxygenation, the cyt pH recovered its initial value (7.5) within 2 to 3 min, whereas external pH decreased abruptly. We suggest that the H(+)-pumping ATPase located in the plasma membrane was blocked in anoxia and quickly reactivated after re-oxygenation.

Functions of the Vacuole in Higher Plant Cells

Current views of the activities inherent in the vacuole as a multifunctional compartment in higher plant cells are outlined. The available data indicate that the vacuole is involved in ion homeostasis of the cytosol, storing products of the primary and secondary metabolism, osmoregulation, generation of defense responses of plant cells under biotic and abiotic stress, and programmed cell death. Transport of diverse molecules and ions across the vacuolar membrane, i.e., the tonoplast, plays the major role in all these functional activities of the vacuole. Much progress toward the identification of the transport systems located in this membrane and the elucidation of the mechanisms underlying their functioning has been achieved in the last 10–15 years and has given new insights into the role of the vacuole in the integration and regulation of plant cell metabolism.

Dynamic Organization of Microtubules and Microfilaments during Cell Cycle Progression in Higher Plant Cells

Abstract: The cytoskeleton, which mainly consists of microtubules (MTs) and actin microfilaments (MFs), plays various significant roles that are indispensable for eukaryotic viability, including determination of cell shape, cell movement, nuclear division, and cytokinesis. In animal cells, MFs appear to be of more importance than MTs, except for spindle formation in nuclear division. In contrast, higher plants have a rigid cell wall around their cells, and have thus evolved elegant systems of MTs to control the direction of cellulose microfibrils (CMFs) deposited in the cell wall, and to divide centrifugally in a physically limited space. Dynamic changes in MTs during cell cycle progression in higher plant cells have been observed over several decades, including cortical MTs (CMTs) during interphase, preprophase bands (PPBs) from late G2 phase to prophase, spindles from prometaphase to anaphase, and phragmoplasts at telophase. The MFs also show some changes not as obvious as MT dynamics. However, questions regarding the process of formation of these arrays, and the precise mechanisms by which they fulfill their roles, remain unsolved. In this article, we present an outline of the changes in the cytoskeleton based on our studies with highly‐synchronized tobacco BY‐2 cells. Some candidate molecules that could play roles in cytoskeletal dynamics are discussed. We also hope to draw attention to recent attempts at visualization of cytoskeletons with molecular techniques, and to some examples of genetic approaches in this field.

Plasmodesmata. A not so open-and-shut case.

In 1897, Eduard Tangl observed striations between cells in cotyledons of Strychnos nuxvomica and hypothesized that the protoplasmic bodies. . . are united by thin strands passing through connect- ing ducts in walls, which put cells into con- nection with each other and so unite them to an entity of higher order (for review, see 2). This was a prophetic view of symplasm, a term coined much later by Munch to describe cytoplasmic contin- uum that occurs between higher plant cells. Tangl's work challenged then-held view that plant cells functioned as autonomous units and stimulated sev- eral studies on other species. However, it was Stras- burger in 1901 who finally named these delicate structures plasmodesmata (2). Space constraints do not permit us to do justice to several pioneers of plamodesmal research. However, we have attempted to select some notable milestones in development of field. Although concept of symplasm became ac- cepted by many plant physiologists, it was not until advent of electron microscopy that fine struc- ture of plasmodesmata was resolved. Following introduction of glutaraldehyde as a fixative, several studies showed plasmodesmata to be plasma mem- brane-lined channels containing a central endoplas- mic reticulum (ER)-derived structure termed des- motubule (16). The demonstration of ER continuity between adjacent cells clearly distinguished plas- modesma from its functional counterpart in animal cells, gap junction. Although some studies sug- gested that there may be homologies between plas- modesmata and gap junctions, consensus today is that these structures differ considerably in both form and function. With introduction of immunologi- cal techniques, plasmodesmata have been shown to be extremely complex structures containing several unique proteins, including cytoskeletal elements (13), and recent studies suggest that there may be impor- tant differences between simple and branched plas- modesmal architectures with respect to molecular trafficking (11).