Elongating Plant Cells Research Articles

Mechanistic Insights into Plant Chiral Growth

The latent left–right asymmetry (chirality) of vascular plants is best witnessed as a helical elongation of cylindrical organs in climbing plants. Interestingly, helical handedness is usually fixed in given species, suggesting genetic control of chirality. Arabidopsis thaliana, a small mustard plant, normally does not twist but can be mutated to exhibit helical growth in elongating organs. Genetic, molecular and cell biological analyses of these twisting mutants are providing mechanistic insights into the left–right handedness as well as how potential organ skewing is suppressed in most plants. Growth direction of elongating plant cells is determined by alignment of cellulose microfibrils in cell walls, which is guided by cortical microtubules localized just beneath the plasma membrane. Mutations in tubulins and regulators of microtubule assembly or organization give rise to helical arrangements of cortical microtubule arrays in Arabidopsis cells and cause helical growth of fixed handedness in axial organs such as roots and stems. Whether tubulins are assembled into a microtubule composed of straight or tilted protofilaments might determine straight or twisting growth. Mechanistic understanding of helical plant growth will provide a paradigm for connecting protein filament structure to cellular organization.

Ascorbate in the apoplast of elongating plant cells

It was shown that basipetal retardation of cell elongation in the growth zone of etiolated maize mesocotyls correlates with a steep decrease in the apoplastic ascorbic acid (ASC) concentration (50 μM → 10 μM) and ascorbate redox state (17 % → 5 %). Exogenous ASC (0.3 mM) not only inhibits peroxidase-dependent oxidation of phenols in vitro. It also exerts a highly specific effect on the secretion of peroxidases by stimulating the release of some isoforms while inhibiting the release of others to the cell walls. This effect points to the hypothetic signaling function of apoplastic ascorbate. Previously, we described a basipetal decrease in hydrogen peroxide concentration in the apoplast (from 5.1 to 2.0 μM) and a two-times increase in cell wall peroxidase potential activity (Sharova et al., 2012). Summarizing found gradients, we can assume that the conditions in the apoplast of the upper mesocotyl segment are favorable for the occurrence of the Fenton reaction (high ASC and hydrogen peroxide concentrations) and unfavorable for the oxidation of phenols (high ASC concentration and low potential peroxidase activity), which contributes to cell wall extensibility and rapid cell elongation.

A new kymogram-based method reveals unexpected effects of marker protein expression and spatial anisotropy of cytoskeletal dynamics in plant cell cortex

BackgroundCytoskeleton can be observed in live plant cells in situ with high spatial and temporal resolution using a combination of specific fluorescent protein tag expression and advanced microscopy methods such as spinning disc confocal microscopy (SDCM) or variable angle epifluorescence microscopy (VAEM). Existing methods for quantifying cytoskeletal dynamics are often either based on laborious manual structure tracking, or depend on costly commercial software. Current automated methods also do not readily allow separate measurements of structure lifetime, lateral mobility, and spatial anisotropy of these parameters.ResultsWe developed a new freeware-based, operational system-independent semi-manual technique for analyzing VAEM or SDCM data, QuACK (Quantitative Analysis of Cytoskeletal Kymograms), and validated it on data from Arabidopsis thaliana fh1 formin mutants, previously shown by conventional methods to exhibit altered actin and microtubule dynamics compared to the wild type. Besides of confirming the published mutant phenotype, QuACK was used to characterize surprising differential effects of various fluorescent protein tags fused to the Lifeact actin probe on actin dynamics in A. thaliana cotyledon epidermis. In particular, Lifeact-YFP slowed down actin dynamics compared to Lifeact-GFP at marker expression levels causing no macroscopically noticeable phenotypic alterations, although the two fluorophores are nearly identical. We could also demonstrate the expected, but previously undocumented, anisotropy of cytoskeletal dynamics in elongated epidermal cells of A. thaliana petioles and hypocotyls.ConclusionsOur new method for evaluating plant cytoskeletal dynamics has several advantages over existing techniques. It is intuitive, rapid compared to fully manual approaches, based on the free ImageJ software (including macros we provide here for download), and allows measurement of multiple parameters. Our approach was already used to document unexpected differences in actin mobility in transgenic A. thaliana expressing Lifeact fusion proteins with different fluorophores, highlighting the need for cautious interpretation of experimental results, as well as to reveal hitherto uncharacterized anisotropy of cytoskeletal mobility in elongated plant cells.

Indole-3-acetic acid-induced oxidative burst and an increase in cytosolic calcium ion concentration in rice suspension culture.

Indole-3-acetic acid (IAA) is the major natural auxin involved in the regulation of a variety of growth and developmental processes such as division, elongation, and polarity determination in growing plant cells. It has been shown that dividing and/or elongating plant cells accompanies the generation of reactive oxygen species (ROS) and a number of reports have suggested that hormonal actions can be mediated by ROS through ROS-mediated opening of ion channels. Here, we surveyed the link between the action of IAA, oxidative burst, and calcium channel activation in a transgenic cells of rice expressing aequorin in the cytosol. Application of IAA to the cells induced a rapid and transient generation of superoxide which was followed by a transient increase in cytosolic Ca(2+) concentration ([Ca(2+)]c). The IAA-induced [Ca(2+)]c elevation was inhibited by Ca(2+) channel blockers and a Ca(2+) chelator. Furthermore, ROS scavengers effectively blocked the action of IAA on [Ca(2+)]c elevation.

Cytoplasmic Streaming in the Drosophila Oocyte.

Objects are commonly moved within the cell by either passive diffusion or active directed transport. A third possibility is advection, in which objects within the cytoplasm are moved with the flow of the cytoplasm. Bulk movement of the cytoplasm, or streaming, as required for advection, is more common in large cells than in small cells. For example, streaming is observed in elongated plant cells and the oocytes of several species. In the Drosophila oocyte, two stages of streaming are observed: relatively slow streaming during mid-oogenesis and streaming that is approximately ten times faster during late oogenesis. These flows are implicated in two processes: polarity establishment and mixing. In this review, I discuss the underlying mechanism of streaming, how slow and fast streaming are differentiated, and what we know about the physiological roles of the two types of streaming.

The binding of cellulase variants to dislocations: a semi-quantitative analysis based on CLSM (confocal laser scanning microscopy) images.

Binding of enzymes to the substrate is the first step in enzymatic hydrolysis of lignocellulose, a key process within biorefining. During this process elongated plant cells such as fibers and tracheids have been found to break into segments at irregular cell wall regions known as dislocations or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method was developed to assess and quantify the abundance of the binding of cellulases to dislocations as compared to the surrounding cell wall. Only Humicola insolens EGV was found to have stronger binding preference to dislocations than to the surrounding cell wall, while no difference in binding affinity was seen for any of the other cellulose variants included in the study (H. insolens EGV variants, Trichoderma reesei CBHI, CBHII and EGII). This result favours the hypothesis that fibers break at dislocations during the initial phase of hydrolysis mostly due to mechanical failure rather than as a result of faster degradation at these locations.

Mechanisms of Self-Organization of Cortical Microtubules in Plants Revealed by Computational Simulations

Microtubules confined to the two-dimensional cortex of elongating plant cells must form a parallel yet dispersed array transverse to the elongation axis for proper cell wall expansion. Some of these microtubules exhibit free minus-ends, leading to migration at the cortex by hybrid treadmilling. Collisions between microtubules can result in plus-end entrainment ("zippering") or rapid depolymerization. Here, we present a computational model of cortical microtubule organization. We find that plus-end entrainment leads to self-organization of microtubules into parallel arrays, whereas catastrophe-inducing collisions do not. Catastrophe-inducing boundaries (e.g., upper and lower cross-walls) can tune the orientation of an ordered array to a direction transverse to elongation. We also find that changes in dynamic instability parameters, such as in mor1-1 mutants, can impede self-organization, in agreement with experimental data. Increased entrainment, as seen in clasp-1 mutants, conserves self-organization, but delays its onset and fails to demonstrate increased ordering. We find that branched nucleation at acute angles off existing microtubules results in distinctive sparse arrays and infer either that microtubule-independent or coparallel nucleation must dominate. Our simulations lead to several testable predictions, including the effects of reduced microtubule severing in katanin mutants.

Microtubule Organization in Three-Dimensional Confined Geometries: Evaluating the Role of Elasticity Through a Combined In Vitro and Modeling Approach

Microtubules or microtubule bundles in cells often grow longer than the size of the cell, which causes their shape and organization to adapt to constraints imposed by the cell geometry. We test the reciprocal role of elasticity and confinement in the organization of growing microtubules in a confining box-like geometry, in the absence of other (active) microtubule organizing processes. This is inspired, for example, by the cortical microtubule array of elongating plant cells, where microtubules are typically organized in an aligned array transverse to the cell elongation axis. The method we adopt is a combination of analytical calculations, in which the polymers are modeled as inextensible filaments with bending elasticity confined to a two-dimensional surface that defines the limits of a three-dimensional space, and in vitro experiments, in which microtubules are polymerized from nucleation seeds in microfabricated chambers. We show that these features are sufficient to organize the polymers in aligned, coiling configurations as for example observed in plant cells. Though elasticity can account for the regularity of these arrays, it cannot account for a transverse orientation of microtubules to the cell’s long axis. We therefore conclude that an additional active, force-generating process is necessary to create a coiling configuration perpendicular to the long axis of the cell.

A new structural element containing glycine-rich proteins and rhamnogalacturonan I in the protoxylem of seed plants.

The water pipes of elongating plant organs are the result of programmed cell death and are formed by the walls of dead and empty protoxylem elements. These protoxylem elements are passively elongated many times by the surrounding tissue before they are replaced and collapse. Well-known adaptations for this unique task include the characteristic secondary wall thickenings, forming rings and helices. A new, clearly distinct structural element containing glycine-rich proteins is now visualized for the first time, using confocal laser scanning microscopy in the mature protoxylem of elongating organs of seed plants. This structural element is arranged along the longitudinal axis of the protoxylem elements. It interconnects the secondary wall thickenings within and between protoxylem elements, as well as the protoxylem with other cell types such as xylem parenchyma cells and metaxylem elements. The structural element is stable against detergent extractions, proteinase, pectinase and cellulase hydrolysis, and is closely associated with rhamnogalacturonan-I, a pectic polysaccharide. The results clearly demonstrate that the cell wall of protoxylem cells is a highly dynamic and complex structure. The typical polysaccharide-rich primary wall of living and elongating plant cells is progressively modified and finally replaced by a protein-rich wall in the dead and passively stretched protoxylem elements. These glycine-rich walls originated early in the evolution of the seed plants as confirmed by the analysis of genomic information.

The architecture of polarized cell growth: the unique status of elongating plant cells.

Polarity is an inherent feature of almost all prokaryotic and eukaryotic cells. In most eukaryotic cells, growth polarity is due to the assembly of actin-based growing domains at particular locations on the cell periphery. A contrasting scenario is that growth polarity results from the establishment of non-growing domains, which are actively maintained at opposite end-poles of the cell. This latter mode of growth is common in rod-shaped bacteria and, surprisingly, also in the majority of plant cells, which elongate along the apical-basal axes of plant organs. The available data indicate that the non-growing end-pole domains of plant cells are sites of intense endocytosis and recycling. These actin-enriched end-poles serve also as signaling platforms, allowing bidirectional exchange of diverse signals along the supracellular domains of longitudinal cell files. It is proposed that these actively remodeled end-poles of elongating plant cells remotely resemble neuronal synapses.

A Unified Nomenclature for Plant Fibres for Industrial Use

Cellulose in the cell wall occurs in microfibrils of various sizes. “Fibre” should refer only to single elongated plant cells, “fibre bundle” to assemblages of fibres. Fibres and bundles can be of various cell types (most sclerenchyma), sizes, volume fractions of cellulose and degree of lignification. Usage of any other terms is liable to lead to confusion. Fibre bundles for industrial use should be defined only by their dimensions, botanical origin and the extraction procedures used.

The geometry of elongating plant cells

Shape and size of elongating cells were examined in three plant tissues: the adaxial epidermis of the petiole ofZebrina pendula L., the abaxial epidermis ofAnacharis densa L. leaves and the abaxial epidermis of the scale leaf ofAllium cepa L. Based on a few simple assumptions, the expected probability distribution frequencies (pdf) for cell length and number of adjacent walls were calculated. Actual data of cell lengths closely approximated those expected with the pdfs being asymmetrical since there are more younger, shorter cells than older, longer cells. Data for number of lateral walls of real cells were similar to that expected and these walls increase in compensating mechanism exists to maintain a constant range of cell lengths through many cell generations. It is expressed by longer than average new daughter cells dividing relatively soon while shorter than average new daughter cells divide after a relatively long cycle.

The geometry of elongating plant cells

The geometry of elongating plant cells

Microtubule arrays in regeneratingMougeotia protoplasts may be oriented by electric fields

Initially non-polar protoplasts of the green algaMougeotia will regenerate to re-establish their original cylindrical cell shape. The orientation of the growth axis of regenerating protoplasts held in agarose was independent of both the direction of incident white light and gravity. Protoplasts elongated parallel to applied DC electric fields of approx. 0.2 Vcm−1 (1 mV/protoplast) and greater, with an increasing percentage oriented with increasing field strength. At the maximum field strength used (10 mV/cell), 53% of protoplasts were oriented within +- 10° of the 0/180° axis of the field. In untreated controls, the orientation of elongation was random. Protoplast survival was unaffected by field treatment. Some protoplasts (up to 37% in 10 mV/cell fields) formed outgrowths towards the cathode and occasionally towards the anode. Regenerating protoplasts in fields displayed the normal sequence of microtubule reorganization. This means that the positioning of the ordered symmetrical array of microtubules centred on two foci that appears within 3 to 4 h, and the subsequent organization of microtubules by 8 to 12 h into a band that intersects both foci and which is transverse to the axis of elongation (Galway and Hardham 1986), may be controlled by externally applied electric fields. In the region of this microtubule band, the applied field causes the plasma membrane to be stretched parallel to the field (Bryant and Wolfe 1987). We suggest that microtubules may become oriented perpendicular to the direction of field-induced membrane stretching, and that membrane stretching may be one of the orienting mechanisms for membrane-linked microtubules in elongating plant cells.

The effects of taxol and colchicine on microtubule and microfibril arrays in elongating plant cells in culture

Treatment with 10 μM taxol or 3 mM colchicine results in the development of similar aberrant morphotypes in Vicia hajastana (vetch) suspension cultures. After 2 days exposure to either drug, cells exhibit a disruption in polarity (i.e., swelling or spherical morphology). In taxol-treated cells, microtubules are abundant and brightly stained. In the presence of colchicine, microtubules are absent. Calcofluor staining illustrates that in the presence of taxol the microfibrils appear as thick ordered bundles, while in the presence of colchicine, microfibril bundles are smaller and exhibit less detectable order. The presence of thick bundles of microfibrils is confirmed in taxol-treated cells by the analysis of birefringence patterns. Walls of colchicine-treated cells exhibit a patchy birefringence pattern in polarized light, with adjacent patches exhibiting different order. Although taxol-treated cells contain abundant microtubules, their morphology and wall deposition resemble those of cells that lack microtubules. These observations indicate that the dynamics of microtubules may be important to their proper functioning in cell wall deposition. The differences in wall morphology between taxol- and colchicine-treated cells may reflect the different mechanisms of action of these two drugs.

Spatial relationship between microtubules and plasma-membrane rosettes during the deposition of primary wall microfibrils in Closterium sp.

The mechanism by which cortical microtubules (MTs) control the orientation of cellulose microfibril deposition in elongating plant cells was investigated in cells of the green alga, Closterium sp., preserved by ultrarapid freezing. Cellulose microfibrils deposited during formation of the primary cell wall are oriented circumferentially, parallel to cortical MTs underlying the plasma membrane. Some of the microfibrils curve away from the prevailing circumferential orientation but then return to it. Freeze-fracture electron microscopy shows short rows of particle rosettes on the P-face of the plasma membrane, also oriented perpendicular to the long axis of the cell. Previous studies of algae and higher plants have provided evidence that such rosettes are involved in the deposition of cellulose microfibrils. The position of the rosettes relative to the underlying MTs was visualized by deep etching, which caused much of the plasma membrane to collapse. Membrane supported by the MTs and small areas around the rosettes resisted collapse. The rosettes were found between, or adjacent to, MTs, not directly on top of them. Rows of rosettes were often at a slight angle to the MTs. Some evidence of a periodic structure connecting the MTs to the plasma membrane was apparent in freeze-etch micrographs. We propose that rosettes are not actively or directly guided by MTs, but instead move within membrane channels delimited by cortical MTs attached to the plasma membrane, propelled by forces derived from the polymerization and crystallization of cellulose microfibrils. More widely spaced MTs presumably allow greater lateral freedom of movement of the rosette complexes and result in a more meandering pattern of deposition of the cellulose fibrils in the cell wall.

Plant cell extension: structural implications for the origin of the plasma membrane

Abstract. The proposal that rapidly elongating plant cells cannot maintain plasma membrane synthesis by means of the normal endomembrane system has been examined in elongating segments of Avena sativa coleoptiles. Segments were sampled and fixed for electron microscopy, before and after elongation on auxin solutions. Mean cell extensions, cytoplasmic volumes, dictyosomc numbers, and vesicle sizes and numbers were determined. It was shown that there are sufficient dictyosomes present to sustain the vesicle production necessary for the observed plasma membrane extension.

Observations with cytochemistry and ultracryotomy on the fine structure of the expanding walls in actively elongating plant cells.

Ultracryotomy with negative staining and cytochemistry (periodic acid - thiocarbohydrazide - silver proteinate test for polysaccharides, in conjunction with mild extractions) were used to study the architecture of the cell wall and its modifications during expansion. Those techniques were applied to the study in situ of the walls of actively elongating parenchyma of mung bean (Phaseolus aureus), and pea (Pisum sativum) root and of collenchyma of celery (Apium graveolens) petioles. These complementary techniques provide information on the 3-dimensional disposition and fine structure of the subunits of the wall. In all the examples examined, the bulk of growing primary wall appears well-ordered and no progressive evolution from a transverse texture near the plasmalemma to a scattered texture near the middle lamella was observed. It seems unlikely that the development of the wall structure in relation to growth could be explained mechanically by a passive shift of the fibrillar elements in response to cellular stress. There is no evidence for an inert change in fibrillar orientation in the major part of the wall. If such occurs the process is limited to the outermost and senescent part of the wall. Thus, the texture observed does not agree with the classical multinet growth hypothesis but rather with the idea of an ordered structure of the primary wall. With the latter, the components should be able to respond in different ways to specific growth regulators and other environmental signals and thus exert a more positive control over the processes of oriented cell growth.

Changes of the Dna Content of Nuclei During the Process of Cell Elongation in Plants: II. Variations, Other Than Doublings, of the Amount of Feulgenstain in Maturing Plant Cells

Using the histophotometric method for estimation of DNA in elongating plant cells, changes of the DNA content are found of approximately 20 %. The simplest explanation would be that the variations of the amount of Feulgenstain represent true changes of the DNA content.It is suggested that, when a young plant cell is coming in the zone of cell elongation the amount of DNA is decreased by 20 %. After exactly two doublings of the amount of DNA there is a new increase of the DNA content by 20 % during the real cell elongation phase.

Deposition of wall material in thickened primary walls of elongating plant cells

Deposition of wall material in thickened primary walls of elongating plant cells