Cell wall pectins and xyloglucans are internalized into dividing root cells and accumulate within cell plates during cytokinesis.

Recently, we have reported that cell wall pectins are internalized into apical meristem root cells. In cells exposed to the fungal metabolite brefeldin A, all secretory pathways were inhibited, while endocytic pathways remained intact, resulting in accumulation of internalized cell wall pectins within brefeldin A-induced compartments. Here we report that, in addition to the already published cell wall epitopes, rhamnogalacturonan I and xyloglucans also undergo large-scale internalization into dividing root cells. Interestingly, multilamellar endosomes were identified as compartments internalizing arabinan cell wall pectins reactive to the 6D7 antibody, while large vacuole-like endosomes internalized homogalacturonans reactive to the 2F4 antibody. As all endosomes belong topographically to the exocellular space, cell wall pectins deposited in these "cell wall islands", enclosed by the plasma-membrane-derived membrane, are ideally suited to act as temporary stores for rapid formation of cell wall and generation of new plasma membrane. In accordance with this notion, we report that all cell wall pectins and xyloglucans that internalize into endosomes are highly enriched within cytokinetic cell plates and accumulate within brefeldin A compartments. On the other hand, only small amounts of the pectins reactive to the JIM7 antibody, which are produced in the Golgi apparatus, localize to cell plates and they do not accumulate within brefeldin A compartments. In conclusion, meristematic root cells have developed pathways for internalization and recycling of cell wall molecules which are relevant for plant-specific cytokinesis.

Wall ingrowth architecture in epidermal transfer cells of Vicia faba cotyledons.

We describe the use of scanning electron microscopy to provide novel views of the three-dimensional morphology of the ingrowth wall in epidermal transfer cells of cotyledons of developing Vicia faba seed. Wall ingrowth deposition in these cells amplifies the surface area of plasma membrane available for transport of solutes during cotyledon development. Despite the physiological importance of such amplification, little is known about wall ingrowth morphology and deposition in transfer cells. A detailed morphological analysis of wall deposition in this study clearly established for the first time that wall ingrowths are deposited at scattered, discrete loci as papillate ingrowth projections. The new views of the ingrowth wall revealed that these projections branch and fuse laterally, and fusion occurs by fine connections to form a fenestrated sheet or layer. This sheet of wall material then provides a base for further deposition of ingrowth projections to progressively build many interconnected, fenestrated layers. Consolidations, or filling-in, of the fenestrae in these layers appears to occur from small fingerlike protrusions of wall material which extend laterally from the most recently deposited surface of the fenestrae. We propose that deposition of fenestrated layers may provide a mechanism for maintaining continuous amplification of plasma membrane surface area in the face of turnover of the plasma membrane and transporter proteins associated with it. The techniques reported in this paper will provide new opportunities to investigate wall ingrowth deposition and its regulation in transfer cells.

Actin and actin-binding proteins in higher plants.

The actin cytoskeleton is a complex and dynamic structure that participates in diverse cellular events which contribute to plant morphogenesis and development. Plant actins and associated actin-binding proteins are encoded by large, differentially expressed gene families. The complexity of these gene families is thought to have been conserved to maintain a pool of protein isovariants with unique properties, thus providing a mechanistic basis for the observed diversity of plant actin functions. Plants contain actin-binding proteins which regulate the supramolecular organization and function of the actin cytoskeleton, including monomer-binding proteins (profilin), severing and dynamizing proteins (ADF/cofilin), and side-binding proteins (fimbrin, 135-ABP/villin, 115-ABP). Although significant progress in documenting the biochemical activities of many of these classes of proteins has been made, the precise roles of actin-binding proteins in vivo awaits clarification by detailed mutational analyses.

Fluorescently-labeled fimbrin decorates a dynamic actin filament network in live plant cells.

Recently it has been established, through a detailed biochemical analysis, that recombinant Arabidopsis thaliana fimbrin 1 (AtFim1) is a member of the fimbrin/plastin family of actin filament bundling or cross-linking proteins [D.R. Kovar et al. (2000) Plant J 24:625-636]. To determine whether AtFim1 can function as an F-actin-binding protein in the complex environment of the plant cell cytoplasm, we created a fluorescent protein analog and introduced it by microinjection into live Tradescantia virginiana L. stamen hair cells. AtFim1 derivatized with Oregon Green 488 had biochemical properties similar to unlabeled fimbrin, including the Kd value for binding to plant F-actin and the ability to cross-link filaments into higher-order structures. Fluorescent-fimbrin decorated an array of fine actin filaments in the cortical cytoplasm of stamen hair cells, which were shown with time-course studies to be highly dynamic. These data establish AtFim1 as a bona fide member of the fimbrin/plastin family, and represent the first use of a plant actin-binding protein as a powerful cytological tool for tracking the spatial and temporal redistribution of actin filaments in individual cells.

AtFim1 is an actin filament crosslinking protein from Arabidopsis thaliana.

ATFIM1 is a widely expressed gene in Arabidopsis thaliana that encodes a putative actin filament-crosslinking protein, AtFim1, belonging to the fimbrin/plastin class of actin-binding proteins. In this report we have used bacterially expressed AtFim1 and actin isolated from Zea mays pollen to demonstrate that AtFim1 functions as an actin filament-crosslinking protein. AtFim1 binds pollen actin filaments (F-actin) in a calcium-independent manner, with an average dissociation constant (Kd) of 0.55+/-0.21 microM and with a stoichiometry at saturation of 1:4 (mol AtFim1 : mol actin monomer). AtFim1 also crosslinks pollen F-actin by a calcium-independent mechanism, in contrast to crosslinking of plant actin by human T-plastin, a known calcium-sensitive actin-crosslinking protein. When micro-injected at high concentration into living Tradescantia virginiana stamen hair cells, AtFim1 caused cessation of both cytoplasmic streaming and transvacuolar strand dynamics within 2-4 min. Using the 'nuclear displacement assay' as a measure of the integrity of the actin cytoskeleton in living stamen hair cells, we demonstrated that AtFim1 protects actin filaments in these cells from Z. mays profilin (ZmPRO5)-induced depolymerization, in a dose-dependent manner. The apparent ability of AtFim1 to protect actin filaments in vivo from profilin-mediated depolymerization was confirmed by in vitro sedimentation assays. Our results indicate that AtFim1 is a calcium-independent, actin filament-crosslinking protein that interacts with the actin cytoskeleton in living plant cells.

Hexose transporters of tomato: molecular cloning, expression analysis and functional characterization.

A full-length (LeHT2) and two partial (LeHT1 and LeHT3) cDNA clones, encoding hexose transporters, were isolated from tomato (Lycopersicon esculentum) fruit and flower cDNA libraries. Southern blot analysis confirmed the presence of a gene family of hexose transporters in tomato consisting of at least three members. The full-length cDNA (LeHT2) encodes a protein of 523 amino acids, with a calculated molecular mass of 57.6 kDa. The predicted protein has 12 putative membrane-spanning domains and belongs to the Major Facilitator Superfamily of membrane carriers. The three clones encode polypeptides that are homologous to other plant monosaccharide transporters and contain conserved amino acid motifs characteristic of this superfamily. Expression of the three genes in different organs of tomato was investigated by quantitative PCR. LeHT1 and LeHT3 are expressed predominantly in sink tissues, with both genes showing highest expression in young fruit and root tips. LeHT2 is expressed at relatively high levels in source leaves and certain sink tissues such as flowers. LeHT2 was functionally expressed in a hexose transport-deficient mutant (RE700A) of Saccharomyces cerevisiae. LeHT2-dependent transport of glucose in RE700A exhibited properties consistent with the operation of an energy-coupled transporter and probably a H+/hexose symporter. The Km of the symporter for glucose is 45 microM.

Is 2,3-butanedione monoxime an effective inhibitor of myosin-based activities in plant cells?

The effectiveness of 2,3-butanedione monoxime (BDM) as an inhibitor of plant myosins has been investigated. Three myosin-dependent motility phenomena in plants, namely cytoplasmic streaming in Chara corallina, light-dependent chloroplast repositioning in Elodea sp., and brefeldin A(BFA)-induced Golgi membrane dynamics in wheat (Triticum aestivum L. cv. Kite) root-tip cells were investigated. All three processes were inhibited by the sulfhydryl-modifying agent N-ethylmalemide (NEM), indicating the probable involvement of myosin as the motor protein in each case. However, none of these myosin-dependent processes were inhibited by BDM at concentrations as high as 20 mM in some instances. These results therefore question the general usefulness of BDM as an inhibitor of myosin-based activities in plant cells.

Molecular cloning of a novel fimbrin-like cDNA from Arabidopsis thaliana.

Fimbrin is a 68-70 kDa actin-bundling protein in animal cells and lower eukaryotes that participates in diverse morphogenetic processes by cross-linking actin filaments into bundles. Here we report the cloning by degenerate polymerase chain reaction (PCR) of ATFIM1, a 2.3 kb cDNA from Arabidopsis thaliana that codes for a novel 76 kDa fimbrin-like polypeptide (AtFim1). The predicted sequence of AtFim1 shares ca. 40% identity with nonplant fimbrins and contains two tandem repeats, each possessing a 27 amino acid region identified as a putative actin-binding domain in fimbrins and in a larger family of actin cross-linking proteins. Preceding the tandem repeats at the amino terminus of AtFim1 is a single-EF-hand-like domain with moderate homology to calmodulin-like calcium-binding proteins. AtFim1 differs from non-plant fimbrins, however, in that it contains an extended carboxy-terminal tail of ca. 65 amino acids. ATFIM1 is encoded by a single gene, although sequencing of two partial fimbrin-like expressed sequence tag (EST) clones indicates that Arabidopsis contains at least two fimbrin-like proteins. Northern blot analysis and reverse-transcription PCR (RT-PCR) demonstrated that ATFIM1 is expressed in all major organs examined (roots, leaves, stems, flowers and siliques). This is the first report of the cloning of a full length plant gene that encodes a putative actin filament-bundling protein.

Actin dynamics during the cell cycle in Chlamydomonas reinhardtii.

We have used two monoclonal antibodies to demonstrate the presence and localization of actin in interphase and mitotic vegetative cells of the green alga Chlamydomonas reinhardtii. Commercially available monoclonal antibodies raised against smooth muscle actin (Lessard: Cell Motil. Cytoskeleton 10:349-362, 1988; Lin: Proc. Natl. Acad. Sci. USA 78:2335-2339, 1981) identify Chlamydomonas actin as a approximately 43,000-M(r) protein by Western immunoblot procedures. In an earlier study, Detmers and coworkers (Cell Motil. 5:415-430, 1985) first identified Chlamydomonas actin using NBD-phallacidin and an antibody raised against Dictyostelium actin; they demonstrated that F-actin is localized in the fertilization tubule of mating gametes. Here, we show by immunofluorescence that vegetative Chlamydomonas cells have an array of actin that surrounds the nucleus in interphase cells and undergoes dramatic reorganization during mitosis and cytokinesis. This includes the following: reorganization of actin to the anterior of the cell during preprophase; the formation of a cruciate actin band in prophase; reorganization to a single anterior actin band in metaphase; rearrangement forming a focus of actin anterior to the metaphase plate; reextension of the actin band in anaphase; presence of actin in the forming cleavage furrow during telophase and cytokinesis; and finally reestablishment of the interphase actin array. The studies presented here do not allow us to discriminate between G and F-actin. None the less, our observations, demonstrating dynamic reorganization of actin during the cell cycle, suggest a role for actin that may include the movement of basal bodies toward the spindle poles in mitosis and the formation of the cleavage furrow during cytokinesis.

Calcium-dependent protein kinase in the green alga Chara.

Cytoplasmic streaming in the characean algae is inhibited by micromolar rises in the level of cytosolic free Ca(2+), but both the mechanism of action and the molecular components involved in this process are unknown. We have used monoclonal antibodies against soybean Ca(2+)-dependent protein kinase (CDPK), a kinase that is activated by micromolar Ca(2+) and co-localizes with actin filaments in higher-plant cells (Putnam-Evans et al., 1989, Cell Motil. Cytoskel. 12, 12-22) to identify and localize its characean homologue. Immunoblot analysis revealed that CDPK in Chara corralina Klein ex. Wild shares the same relative molecular mass (51-55 kDa) as the kinase purified from soybean, and after electrophoresis in denaturing gels is capable of phosphorylating histone III-S in a Ca(2+)-dependent manner. Immunofluorescence microscopy localized CDPK in Chara to the subcortical actin bundles and the surface of small organelles and other membrane components of the streaming endoplasm. The endoplasmic sites carrying CDPK were extracted from internodal cells by vacuolar perfusion with 1 mM ATP or 10(-4) M Ca(2+). Both the localization of CDPK and its extraction from internodal cells by perfusion with ATP or high Ca(2+) are properties similar to that reported for the heavy chain of myosin in Chara (Grolig et al., 1988, Eur. J. Cell Biol. 47, 22-31). Based on its endoplasmic location and inferred enzymatic properties, we suggest that CDPK may be a putative element of the signal-transduction pathway that mediates the rapid Ca(2+)-induced inhibition of streaming that occurs in the characean algae.

p34cdc2 homologue level, cell division, phytohormone responsiveness and cell differentiation in wheat leaves.

Formation of a plant involves generation of new cells by the division cycle and development in these of specialised structure and metabolism. Specialisation is accompanied by a decreasing capacity for division, which declines with particular rapidity in cells of monocotyledonous plants such as the cereals. Here we report that in wheat leaves a homologue of the cell cycle control protein p34(cdc2) participates in the control of these developmental programmes. Accumulation of p34(cdc2) to a maximum level in dividing cells and the cessation of its accumulation during subsequent cell growth and expansion indicate that it contributes specifically to division. There is a decline in p34(cdc2) level as cell differentiation proceeds, in close parallel with the previously established decline of cell division in response to auxin hormones. A basal level of p34(cdc2) in fully differentiated cells that is one-sixteenth of that in dividing cells correlates with their loss of capacity to divide. We conclude that p34(cdc2) level is controlled in diverse multicellular eukaryotes and suggest that it is an important element in the switch from cell division to differentiation.

Actin of chara giant internodal cells: a single isoform in the subcortical filament bundles and a larger, immunologically related protein in the chloroplasts.

Internodal cells of Chara corallina Klein ex. Wild have been studied to determine the number of actin isoforms they contain and whether actin occurs at locations in the cortical cytoplasm outside the filament bundles. A monoclonal antibody to chicken actin is specific for actin in numerous animal cells but binds to two Chara proteins after their separation by two-dimensional polyacrylamide gel electrophoresis. One protein resembles known actins in relative molecular mass (43,000-M(r)) and isoelectric point (5.5) while the other is distinctly different (58,000-M(r), isoelectric point = 4.8). Because it is indetectable in cells whose actin bundles have been extracted, the 43,000-M(r) protein is assigned to the bundles and concluded to be rare or absent in the remaining cortical cytoplasm. The 58,000-M(r) protein, in contrast, does not extract with the actin bundles. It was localized within the chloroplasts by immunofluorescence and by the dependence of proteolysis on the permeabilization of the chloroplast envelope.

An actin-related protein inside pea chloroplasts.

A pea chloroplast protein resembles vertebrate and algal actins by several chemical and immunological criteria. On two-dimensional polyacrylamide gels it migrated with a slightly lower relative molecular mass (Mr = 41,000) and slightly more basic isoelectric point than rabbit skeletal muscle actin. A monoclonal antibody to chicken gizzard actin reacted on immunoblots with rabbit skeletal actin, with Chara actin and with a 41,000 Mr band from pea chloroplasts. Pea and Chara bands of approximately 58,000 Mr were also stained. A DNase I-affinity column that bound muscle actin also bound 41,000 and 58,000 Mr chloroplast polypeptides. Similarities existed between enzymically and chemically generated fragments of the 41,000 Mr chloroplast polypeptide and rabbit muscle actin. The 41,000 Mr protein was protected from degradation by thermolysin only in preparations of intact, but not ruptured, isolated chloroplasts, indicating that this protein resides within the outer envelope membrane of these organelles. It is concluded that a 41,000 Mr protein with major similarities to actin occurs inside pea chloroplasts, and that a 58,000 Mr protein with some similarities to actin also probably exists within chloroplasts.

Immunogold electron microscopy of phytochrome in Avena: identification of intracellular sites responsible for phytochrome sequestering and enhanced pelletability.

Using monoclonal antibodies to the plant photoreceptor, phytochrome, we have investigated by immunogold electron microscopy the rapid, red light-induced, intracellular redistribution (termed "sequestering") of phytochrome in dark-grown Avena coleoptiles. Pre-embedding immunolabeling of 5-micron-thick cryosections reveals that sequestered phytochrome is associated with numerous, discrete structures of similar morphology. Specific labeling of these structures was also achieved by post-embedding ("on-grid") immunostaining of LR-White-embedded tissue, regardless of whether the tissue had been fixed chemically or by freeze substitution. The phytochrome-associated structures are globular to oval in shape, 200-400 nm in size, and are composed of amorphous, granular material. No morphologically identifiable membranes are present either surrounding or within these structures, which are often present as apparent aggregates that approach several micrometers in size. An immunogold labeling procedure has also been developed to identify the particulate, subcellular component with which phytochrome is associated in vitro as a consequence of irradiation of Avena coleoptiles before their homogenization. Structures with appearance similar to those identified in situ are the only components of the pelletable material that are specifically labeled with gold. We conclude that the association of phytochrome with these structures in Avena represents the underlying molecular event that ultimately is expressed both as red light-induced sequestering in vivo and enhanced pelletability of phytochrome detected in vitro.

Kinetics of intracellular redistribution of phytochrome in Avena coleoptiles after its photoconversion to the active, far-red-absorbing form.

The kinetics of the intracellular redistribution of phytochrome (sequestering) in Avena sativa L. coleoptiles following a brief, saturating actinic pulse of red (R) light have been determined. Immunocytochemical labelling of phytochrome with monoclonal antibodies showed that at 22°C sequestering can occur within 1-2 s from the onset of R irradiation and is dependent upon the continued presence of the far-red-absorbing form of phytochrome (Pfr). The initial rate, but not the final extent, of sequestering is reduced by lowering the temperature of the tissue to 1°C. Sequestering at 22°C appears to involve two distinct stages: (1) a rapid association of Pfr with putative binding sites initiates the sequestered condition, following which (2) these sites of sequestered phytochrome appear to aggregate. Neither of these two processes was affected by the cytoskeletal inhibitors colchicine or cytochalasin B. Phytochrome sequestering therefore resembles R-light-induced phytochrome pelletability with respect to kinetics, temperature sensitivity, and dependence upon the continued presence of Pfr in the cell.