Cytokinesis in tobacco BY-2 and root tip cells: a new model of cell plate formation in higher plants

Cell plate formation in tobacco root tips and synchronized dividing suspension cultured tobacco BY-2 cells was examined using cryofixation and immunocytochemical methods. Due to the much improved preservation of the cells, many new structural intermediates have been resolved, which has led to a new model of cell plate formation in higher plants. Our electron micrographs demonstrate that cell plate formation consists of the following stages: (1) the arrival of Golgi-derived vesicles in the equatorial plane, (2) the formation of thin (20 +/- 6 nm) tubes that grow out of individual vesicles and fuse with others giving rise to a continuous, interwoven, tubulo-vesicular network, (3) the consolidation of the tubulo-vesicular network into an interwoven smooth tubular network rich in callose and then into a fenestrated plate-like structure, (4) the formation of hundreds of finger-like projections at the margins of the cell plate that fuse with the parent cell membrane, and (5) cell plate maturation that includes closing of the plate fenestrae and cellulose synthesis. Although this is a temporal chain of events, a developing cell plate may be simultaneously involved in all of these stages because cell plate formation starts in the cell center and then progresses centrifugally towards the cell periphery. The "leading edge" of the expanding cell plate is associated with the phragmoplast microtubule domain that becomes concentrically displaced during this process. Thus, cell plate formation can be summarized into two phases: first the formation of a membrane network in association with the phragmoplast microtubule domain; second, cell wall assembly within this network after displacement of the microtubules. The phragmoplast microtubules end in a filamentous matrix that encompasses the delicate tubulo-vesicular networks but not the tubular networks and fenestrated plates. Clathrin-coated buds/vesicles and multivesicular bodies are also typical features of the network stages of cell plate formation, suggesting that excess membrane material may be recycled in a selective manner. Immunolabeling data indicate that callose is the predominant lumenal component of forming cell plates and that it forms a coat-like structure on the membrane surface. We postulate that callose both helps to mechanically stabilize the early delicate membrane networks of forming cell plates, and to create a spreading force that widens the tubules and converts them into plate-like structures. Cellulose is first detected in the late smooth tubular network stage and its appearance seems to coincide with the flattening and stiffening of the cell plate.     

Ultrastructure of cell division in the fusiform cells of the vascular cambium of Robinia pseudoacacia

 The ultrastructure of periclinally dividing fusiform cells was studied in the vascular cambium of Robinia pseudoacacia. Fusiform cell division begins in April at Madison, Wisconsin, when the cambial cells still have many characteristics of a dormant cambium. Soon afterward, the cambial cells acquire the appearance typical of an active cambium. Sequential phases of the microtubule cycle were documented: cortical microtubules bordering the cell wall during interphase, perinuclear microtubules preceding formation of the mitotic spindle, spindle microtubules, and phragmoplast microtubules. A preprophase band of microtubules was not encountered. An extended phragmosome was not encountered in periclinally dividing fusiform cells. During cytokinesis, the phragmosome is represented by a broad cytoplasmic plate which precedes the developing phragmoplast and cell plate as they migrate toward the ends of the cell.     

AN ULTRASTRUCTURAL STUDY OF CELL DIVISION IN THE CAMBIUM

The fine structure of dividing cambial cells of Ulmus americana and Tilia americana has been studied in material fixed in glutaraldehyde followed by osmium tetroxide. The cambia examined consisted of 7–9 rows of unexpanded fusiform cells, all of which had similar ultrastructural components. The fine structure and sequence of events of mitosis and cytokinesis in the dividing cambial cells apparently are similar to those of dividing cells in root tips and leaves. Of special interest was the observation that during cytokinesis, a broad cytoplasmic plate or phragmosome precedes the developing phragmoplast and cell plate through the dividing cambial cell. Smooth and coated vesicles derived from dictyosomes are associated with cell plate formation in these cells, smooth vesicles primarily with earlier stages of plate formation, and coated vesicles in later stages.     

Preprophase band loses its function as a cytokinetic apparatus in mitosis of neck canal mother cell

Summary. Preprophase bands in the neck canal mother cell and the central cell of the archegonium of the fern Dryopteris crassirhizoma are observed with immunofluorescence microscopy. No phragmoplast is found during mitosis of the neck canal mother cell; however, the phragmoplast develops very well in the central cell. The neck canal mother cell undergoes karyokinesis but not cytokinesis and finally produces only one binucleate neck canal cell. However, the central cell undergoes cytokinesis and produces an egg cell and a ventral canal cell. These observations suggest that the preprophase band in the neck canal mother cell loses its function as a cytokinetic apparatus and becomes an evolutionary vestige in the development of the archegonium. Received January 22, 2002; accepted April 26, 2002; published online October 31, 2001 RID="*" ID="*" Correspondence and reprints: College of Life Sciences, Peking University, Beijing 100871, Peoples' Republic of China. E-mail: rlyou@pku.edu.cn     

Caffeine inhibits cell plate formation by disrupting membrane reorganization just after the vesicle fusion step

Summary We have re-examined the effects of caffeine on cell plate formation in synchronized tobacco BY-2 cells by means of cryofixation, immunocytochemistry, and calcium staining techniques. Because cryofixation preserves structural intermediates of cell plates that are not seen in chemically fixed cells, this methodology has enabled us to define not only when caffeine acts but also which assembly steps are inhibited. Caffeine acts at an early stage of cytokinesis, just after the Golgi-derived vesicles have arrived at the cell equator and begun to fuse with each other via thin (20 nm) membrane tubules. This initial round of fusions produces a delicate membrane network which in control cells is rapidly converted in a more substantial tubulo-vesicular network covered by a thick, fuzzy coat on its cytoplasmic surface. Caffeine disrupts the conversion of the fragile, thin, fusion tube-generated membrane network into the more stable tubulo-vesicular network, the assembly of its fuzzy coat, and the budding of clathrin-coated vesicles from its surface. Normally, the tubulo-vesicular network also provides the structural framework for calcium-dependent callose synthases that deposit a callose layer over the lumenal surface of the cell plate membranes. In the presence of caffeine, no stabilizing callose layer is formed, and the thin tubule membrane network fragments into vesicles of variable sizes. Cell plates in caffeine-treated cells stained with chlortetracycline, a fluorescent stain of membrane-associated calcium, also display a significant reduction in fluorescence at the cell plate, suggesting a major decrease in cell plate membrane-associated calcium. However, this latter finding needs to be confirmed by more sophisticated calcium measuring techniques. Current theories of the mechanism of action of caffeine, including its ability to disrupt local calcium gradients, are discussed within the new ultrastructural context that this study provides. Our findings, finally, suggest a new method for isolating just fused but not further matured cell plate forming vesicles for biochemical studies.Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Postmeiotic cytokinesis and pollen aperture number determination in eudicots: effect of the cleavage wall number

Summary.  In eudicot postmeiotic tetrads, apertures are usually joined in pairs in highly conserved areas. These appear to be located at the last points of contact persisting at the end of cytokinesis between the cytoplasm of the future microspores. In order to investigate the relationship between cytokinesis and aperture formation, aperture distribution within postmeiotic tetrads and the progression of meiosis were studied in Nicotiana tabacum cv. Ambalema. This variety (inbred line) produces about 85% tricolporate pollen and 15% tetracolporate pollen grains. In addition, about 7% of tetrads are composed of four equal-sized microspores and a supernumerary pseudomicrospore of small size and an equal proportion of tetrads exhibit unpaired apertures (these apertures are not joined in pairs within tetrads). Observation of cytokinesis indicates that both unpaired apertures and pseudomicrospores could result from the persistence of late communications between microsporocytes. Observations of tetrads indicate that an increase in the number of elements that are separated during cytokinesis is correlated with an increase in microspore aperture number. All data converge to support the hypothesis that aperture site determination is partly controlled by the number of walls formed to separate the different elements of the tetrad. Received May 22, 2002; accepted October 29, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: Laboratoire de Ecologie, Systematique et Evolution, Batiment 362, Université Paris Sud, 91405 Orsay cedex, France.     

Development and disintegration of phragmoplasts in living cultured cells of a GFP::TUA6 transgenic Arabidopsis thaliana plant

Summary.  Cultured suspension cells of Arabidopsis thaliana that stably express a green-fluorescent protein–α-tubulin 6 fusion protein were used to follow the development and disintegration of phragmoplasts. The development and disintegration of phragmoplasts in the living cultured cells could be successively observed by detecting the green-fluorescent protein fluorescence of the microtubules. In the early telophase spindle, where two kinetochore groups and two daughter chromosome groups had completely separated from one another, fluorescence appeared in the interzone between the two chromosome groups. The fluorescent region was gradually condensed at the previous equator and increased in fluorescence intensity, and finally it formed the initial phragmoplast. The initial phragmoplast moved from the cell center towards the cell periphery, and it lost fluorescence at its center and became double rings in shape. The expansion orientation of the phragmoplast was not always the same as that of the future new cell wall before it came in contact with the cell wall. The phragmoplast did not usually come in contact with the cell wall simultaneously with its entire length. A portion of the phragmoplast which was earlier in contact with the cell wall disappeared earlier than other portions of the phragmoplast. The duration of contact between any portions of the phragmoplast and the plasma membrane of the cell wall was 15–30 min. The fluorescence intensity of the cytoplasm did not seem to be elevated by the disintegration of the strongly fluorescent phragmoplast. Received August 8, 2002; accepted September 25, 2002; published online March 11, 2003     

Cell cycle-dependent changes in Golgi stacks, vacuoles, clathrin-coated vesicles and multivesicular bodies in meristematic cells of Arabidopsis thaliana: A quantitative and spatial analysis

Cytokinesis in plants involves both the formation of a new wall and the partitioning of organelles between the daughter cells. To characterize the cellular changes that accompany the latter process, we have quantitatively analyzed the cell cycle-dependent changes in cell architecture of shoot apical meristem cells of Arabidopsis thaliana. For this analysis, the cells were preserved by high-pressure freezing and freeze-substitution techniques, and their Golgi stacks, multivesicular bodies, vacuoles and clathrin-coated vesicles (CCVs) characterized by means of serial thin section reconstructions, stereology and electron tomography techniques. Interphase cells possess ∼35 Golgi stacks, and this number doubles during G2 immediately prior to mitosis. At the onset of cytokinesis, the stacks concentrate around the periphery of the growing cell plate, but do not orient towards the cell plate. Interphase cells contain ∼18 multivesicular bodies, most of which are located close to a Golgi stack. During late cytokinesis, the appearance of a second group of cell plate-associated multivesicular bodies coincides with the onset of CCV formation at the cell plate. During this period a 4× increase in CCVs is paralleled by a doubling in number and a 4× increase in multivesicular bodies volume. The vacuole system also undergoes major changes in organization, size, and volume, with the most notable change seen during early telophase cytokinesis. In particular, the vacuoles form sausage-like tubular compartments with a 50% reduced surface area and an 80% reduced volume compared to prometaphase cells. We postulate that this transient reduction in vacuole volume during early telophase provides a means for increasing the volume of the cytosol to accommodate the forming phragmoplast microtubule array and associated cell plate-forming structures.     

Electron tomographic analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by high-pressure freezing

We have investigated the process of somatic-type cytokinesis in Arabidopsis (Arabidopsis thaliana) meristem cells with a three-dimensional resolution of approximately 7 nm by electron tomography of high-pressure frozen/freeze-substituted samples. Our data demonstrate that this process can be divided into four phases: phragmoplast initials, solid phragmoplast, transitional phragmoplast, and ring-shaped phragmoplast. Phragmoplast initials arise from clusters of polar microtubules (MTs) during late anaphase. At their equatorial planes, cell plate assembly sites are formed, consisting of a filamentous ribosome-excluding cell plate assembly matrix (CPAM) and Golgi-derived vesicles. The CPAM, which is found only around growing cell plate regions, is suggested to be responsible for regulating cell plate growth. Virtually all phragmoplast MTs terminate inside the CPAM. This association directs vesicles to the CPAM and thereby to the growing cell plate. Cell plate formation within the CPAM appears to be initiated by the tethering of vesicles by exocyst-like complexes. After vesicle fusion, hourglass-shaped vesicle intermediates are stretched to dumbbells by a mechanism that appears to involve the expansion of dynamin-like springs. This stretching process reduces vesicle volume by approximately 50%. At the same time, the lateral expansion of the phragmoplast initials and their CPAMs gives rise to the solid phragmoplast. Later arriving vesicles begin to fuse to the bulbous ends of the dumbbells, giving rise to the tubulo-vesicular membrane network (TVN). During the transitional phragmoplast stage, the CPAM and MTs disassemble and then reform in a peripheral ring phragmoplast configuration. This creates the centrifugally expanding peripheral cell plate growth zone, which leads to cell plate fusion with the cell wall. Simultaneously, the central TVN begins to mature into a tubular network, and ultimately into a planar fenestrated sheet (PFS), through the removal of membrane via clathrin-coated vesicles and by callose synthesis. Small secondary CPAMs with attached MTs arise de novo over remaining large fenestrae to focus local growth to these regions. When all of the fenestrae are closed, the new cell wall is complete. Few endoplasmic reticulum (ER) membranes are seen associated with the phragmoplast initials and with the TVN cell plate that is formed within the solid phragmoplast. ER progressively accumulates thereafter, reaching a maximum during the late PFS stage, when most cell plate growth is completed.     

Cambial reactivation in locally heated stems of the evergreen conifer Abies sachalinensis (Schmidt) Masters

A study was made of cambial activity, the localization of storage starch around the cambium, and the localization and occurrence of microtubules in cambial cells from dormancy to reactivation in locally heated (22–26 °C) stems of the evergreen conifer Abies sachalinensis. Heating induced localized reactivation of the cambium in the heated portions of the stem. Erect ray cambial cells resumed cell division 1 d prior to the reactivation of fusiform cambial cells and procumbent ray cambial cells. The re-initiation of the division of fusiform cambial cells occurred first on the phloem side. During the heat treatment, the amount of storage starch decreased in procumbent ray cambial cells and in the phloem parenchyma adjacent to the cambium but increased in fusiform cambial cells. Preprophase bands of microtubules, spindle microtubules and phragmoplast microtubules were observed both in erect ray cambial cells and in procumbent ray cambial cells. By contrast, no evidence of the presence of such preprophase bands of microtubules was detected in fusiform cambial cells. The results suggest that the localized heating of stems of evergreen conifers might provide a useful experimental model system for studies of the dynamics of cambial reactivation in intact trees. Received: 25 May 2000 / Accepted: 12 July 2000     

Cellular changes associated with rest and quiescence in winter-dormant vascular cambium of<Emphasis Type="Italic"> Pinus contorta</Emphasis>

In winter, dormant cambial cells contain many small vacuoles interspersed throughout the cytoplasm. This differs dramatically from actively growing cambial cells whose structure is dominated by large central vacuoles. Structure reported in studies using conventional chemical fixation and transmission electron microscopy (TEM) conflicts with that described earlier for live cambial cells using light microscopy. In this study, cryofixation (high-pressure freezing/freeze substitution) was used to preserve dormant Pinus contorta fusiform cambial cells, revealing structure more consistent with that in early micrographs of live cambial cells. At the ultrastructural level, the plasmalemma was consistently smooth and tightly associated with the cell wall, contrary to the highly in-folded plasmalemma seen in chemically fixed cambial cells. In addition, both TEM and live-cell confocal microscopy demonstrated that, in some places, dormant cells were partitioned into more numerous, smaller vacuoles than were observed after chemical fixation. Populations of different vacuoles were apparent based on size, shape and membrane staining. Larger vacuoles had prominent tonoplasts and were often present as axially elongated, interconnecting networks with associated microfilament bundles. Endoplasmic reticulum fragmented during rest into numerous vesicular structures similar to small vacuoles, then with the transition to quiescence reformed into the smooth cisternal form.     

Cytokinesis and nodal anatomy in the charophycean green algaChara zeylanica

Summary Cell plate formation inChara zeylanica was compared with recent models of cytokinesis in higher plants in order to gain insight into the evolutionary origin of plant cytokinetic processes. Transmission electron microscopy (TEM) reveals that while cytokinesis inC. zeylanica bears many features in common with that in higher plants, there are significant differences. Unlike that in higher plants, cytokinesis inC. zeylanica begins with a congregation of smooth membrane tubules that are closely associated with endoplasmic reticulum (ER) and Golgi membranes. Mitochondria and other organelles excluded by the phragmoplast in higher plants are present as well. Unlike in higher plants, phragmoplast microtubules persist throughout cytokinesis inC. zeylanica, and the cell plate generally forms across the whole cell at once, though development is patchy, due to small regions developing at different rates; the ends of the plate form last. By identifying aspects of cytokinesis that are different inC. zeylanica and plants, our study indicates which cytokinetic features are more likely to be derived, and which are more likely to be ancestral. In addition, we demonstrated that all nodal cells ofC. zeylanica are interconnected via plasmodesmata, lending support to the idea that, whileChara spp. are generally considered to be filamentous organisms, nodal regions may be thought of as meristemlike tissues.Abbreviations HPF high-pressure freezing - KFe potassium ferricyanide - SCF stepwise chemical fixation - TEM transmission electron microscopy     

Cytokinesis in Coleochaete orbicularis (Charophyceae): an ancestral mechanism inherited by plants

Recently, highly vacuolate cells of Arabidopsis were shown to exhibit "polarized" cytokinesis, in which the phragmoplast and cell plate contact the mother cell wall and then progress from one side of the cell to the other, rather than forming uniformly outward from the cell center (Cutler and Ehrhardt, 2002, Proceedings of the National Academy of Sciences, USA 99: 2812-2817). It was not known if such a mechanism was unique to flowering plants or whether it occurred more broadly in the plant clade. To determine if a polar mechanism of cell division might have been characteristic of the first plants, differential interference contrast optics were used to examine living cells of the charophycean green alga Coleochaete orbicularis, a close relative of plants, with cytokinesis involving a phragmoplast. By recording images in different focal planes over time, such "polarized" cytokinesis was found in cells dividing either parallel or perpendicular to the edge of this radially symmetrical organism. Previously reported differences between these two types of division in Coleochaete were clarified. Polarized cytokinesis appears to be an ancestral mechanism of plant cell division inherited from the highly vacuolate cells of the charophycean algal ancestors of plants.     

The microtubule‐associated kinase‐like protein RUNKEL functions in somatic and syncytial cytokinesis

The microtubule (MT)‐associated putative kinase RUNKEL (RUK) is an important component of the phragmoplast machinery involved in cell plate formation in Arabidopsis somatic cytokinesis. Since loss‐of‐function ruk mutants display seedling lethality, it was previously not known whether RUK functions in mature sporophytes or during gametophyte development. In this study we utilized RUK proteins that lack the N‐terminal kinase domain to further examine biological processes related to RUK function. Truncated RUK proteins when expressed in wild‐type Arabidopsis plants cause cellularization defects not only in seedlings and adult tissues but also during male meiocyte development, resulting in abnormal pollen and reduced fertility. Ultrastructural analysis of male tetrads revealed irregular and incomplete or absent intersporal cell walls, caused by disorganized radial MT arrays. Moreover, in ruk mutants endosperm cellularization defects were also caused by disorganized radial MT arrays. Intriguingly, in seedlings expressing truncated RUK proteins, the kinesin HINKEL, which is required for the activation of a mitogen‐activated protein kinase signaling pathway regulating phragmoplast expansion, was mislocalized. Together, these observations support a common role for RUK in both phragmoplast‐based cytokinesis in somatic cells and syncytial cytokinesis in reproductive cells.     

Existence of traveling wave solutions in a diffusive predator-prey model

 We establish the existence of traveling front solutions and small amplitude traveling wave train solutions for a reaction-diffusion system based on a predator-prey model with Holling type-II functional response. The traveling front solutions are equivalent to heteroclinic orbits in R ⁴ and the small amplitude traveling wave train solutions are equivalent to small amplitude periodic orbits in R ⁴ . The methods used to prove the results are the shooting argument and the Hopf bifurcation theorem. Received: 25 May 2001 / Revised version: 5 August 2002 / Published online: 19 November 2002 RID="*" ID="*" Research was supported by the National Natural Science Foundations (NNSF) of China. RID="*" ID="*" Research was partially supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada. On leave from the Department of Mathematics and Statistics, Dalhousie University, Halifax, Nova Scotia B3H 3J5, Canada. Mathematics Subject Classification (2000): 34C35, 35K57 Key words or phrases: Traveling wave solution – Wazewski set – Shooting argument – Hopf bifurcation Acknowledgements. We would like to thank the two referees for their careful reading and helpful comments.     

Expansion of the phragmoplast during plant cytokinesis: a MAPK pathway may MAP it out

Plant cytokinesis involves the formation of a cell plate. This is accomplished with the help of the phragmoplast, a plant-specific cytokinetic apparatus that consists of microtubules and microfilaments. During centrifugal growth of the cell plate, the phragmoplast expands to keep its microtubules at the leading edge of the cell plate. Recent studies have revealed potential regulators of phragmoplast microtubule dynamics and the involvement of a mitogen-activated protein kinase cascade in the control of phragmoplast expansion. These studies provide new insights into the molecular mechanisms of plant cytokinesis.     

Analysis of formin functions during cytokinesis using specific inhibitor SMIFH2

The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing Small Molecular Inhibitor of Formin Homology 2 (SMIFH2), a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule-Associated protein of 215 kDa (XMAP215) homolog MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane.

Formins regulate phragmoplast expansion, microtubule turnover rate, actin nucleation, and cell plate membrane remodeling during cytokinesis.     

A discrete,size-structured model of phytoplankton growth in the chemostat: introduction of inhomogeneous cell division size

 We introduce inhomogeneous, substrate dependent cell division in a time discrete, nonlinear matrix model of size-structured population growth in the chemostat, first introduced by Gage et al. [8] and later analysed by Smith [13]. We show that mass conservation is verified, and conclude that our system admits one non zero globally stable equilibrium, which we express explicitly. Then we run numerical simulations of the system, and compare the predictions of the model to data related to phytoplankton growth, whose obtention we discuss. We end with the identification of several parameters of the system. Received: 9 February 2000 / Revised version: 10 October 2001 / Published online: 23 August 2002 RID="*" ID="*" Present address: Department of Mathematics and Statistics, University of Victoria, B.C., Canada. e-mail: jarino@math.uvic.ca Key words or phrases: Chemostat – Structured population models – Discrete model – Inhomogeneous division size     

O-4-Linked coniferyl and sinapyl aldehydes in lignifying cell walls are the main targets of the Wiesner (phloroglucinol-HCl) reaction

Summary.  The nature and specificity of the Wiesner test (phloroglucinol-HCl reagent) for the aromatic aldehyde fraction contained in lignins is studied. Phloroglucinol reacted in ethanol-hydrochloric acid with coniferyl aldehyde, sinapyl aldehyde, vanillin, and syringaldehyde to yield either pink pigments (in the case of hydroxycinnamyl aldehydes) or red-brown pigments (in the case of hydroxybenzaldehydes). However, coniferyl alcohol, sinapyl alcohol, and highly condensed dehydrogenation polymers derived from these cinnamyl alcohols and aldehydes did not react with phloroglucinol in ethanol-hydrochloric acid. The differences in the reactivity of phloroglucinol with hydroxycinnamyl aldehydes and their dehydrogenation polymers may be explained by the fact that, in the latter, the unsubstituted (α,β-unsaturated) cinnamaldehyde functional group, which is responsible for the dye reaction, is lost due to lateral chain cross-linking reactions involving the β carbon. Fourier transform infrared spectroscopy and thioacidolysis analyses of phloroglucinol-positive lignifying plant cell walls belonging to the plant species Zinnia elegans L., Capsicum annuum var. annuum, Populus alba L., and Pinus halepensis L. demonstrated the presence of 4-O-linked hydroxycinnamyl aldehyde end groups and 4-O-linked 4-hydroxy-3-methoxy-benzaldehyde (vanillin) end groups in lignins. However, given the relatively low abundance of 4-O-linked vanillin in lignifying cell walls and the low extinction coefficient of its red-brown phloroglucinol adduct, it is unlikely that vanillin contributes to a great extent to the phloroglucinol-positive stain reaction. These results suggest that the phloroglucinol-HCl pink stain of lignifying xylem cell walls actually reveals the 4-O-linked hydroxycinnamyl aldehyde structures contained in lignins. Histochemical studies showed that these aldehyde structures are assembled, as in the case of coniferyl aldehyde, during the early stages of xylem cell wall lignification. Received April 17, 2002; accepted May 21, 2002; published online October 31, 2002 RID="*" ID="*" Correspondence and reprints: Department of Plant Biology, University of Murcia, 30100 Murcia, Spain. Abbreviations: DHP dehydrogenation polymers; FT-IR spectroscopy Fourier transform infrared spectroscopy.     

Endoxyloglucan transferase is localized both in the cell plate and in the secretory pathway destined for the apoplast in tobacco cells

Intracellular trafficking of enzymes responsible for constructing and modifying the cell wall architecture in plants is mostly unknown. To examine their translocation pathways, we employed an endoxyloglucan transferase (EXGT), a key enzyme responsible for forming and rearranging the cellulose/xyloglucan network of the cell wall. We traced its intracellular localization in suspension-cultured cells of tobacco bright yellow-2 by means of green fluorescent protein-fusion gene procedures as well as by indirect immunofluorescence. During interphase the protein was extensively secreted into the apoplast via the endoplasmic reticulum-Golgi apparatus network, whereas during cytokinesis, the protein was exclusively located in the phragmoplast and eventually transported to the cell plate. These results clearly indicate commitment of EXGT protein to the construction of both the cell plate and the cell wall. This study also visualized the process of phragmoplast development at a level of vesicle translocation in the living cell.