Exogenous Jasmonic and Abscisic Acids Act Differentially in Elongating Tissues from Oat Stem Segments

Segments can be cut from the peducular-1 internode of oat (Avena sativa L.) shoots so as to contain the graviresponsive, auxin-sensitive leaf sheath pulvinus, and the gibberellin-sensitive internodal tissue. These two growth-capable tissues were used to study the effects and interactions of jasmonic acid (JA) and abscisic acid (ABA) in regulating cell elongation. When supplied alone at physiologic concentrations (10⁻⁵, 10⁻⁴ m), JA promoted growth and cell wall synthesis in the internodal tissue, whereas by itself, ABA inhibited internodal elongation and even inhibited JA-promoted growth. When gibberellic acid (GA₃) was used to stimulate internodal elongation, JA and ABA caused similar levels of inhibition and, at certain concentrations, were synergistic. Inhibition by ABA was initiated several hours earlier than inhibition by JA, and only the ABA effect could be partially overcome by 10⁻³ m aminoethoxyvinylglycine. Both JA and ABA inhibited elongation of pulvinar tissue that was induced to grow by gravistimulus or auxin, although here JA was more potent than ABA at equimolar concentrations. When 10⁻⁵ m fusicoccin was used as a general nonphysiologic growth stimulus, JA had no effect on the internode but inhibited the pulvinus, whereas ABA had no effect on the pulvinus but inhibited the internode. These results provide strong physiologic evidence that JA and ABA act by different mechanisms in the regulation of elongation, at least in this representative grass. Received May 28, 1996; accepted November 7, 1996     

STRUCTURE OF GRAVITY-SENSITIVE SHEATH AND INTERNODAL PULVINI IN GRASS SHOOTS

The location and some morphological, anatomical, and functional aspects of the gravity-sensitive pulvini of a selected number of grass shoots are examined. There are two distinct gravity-sensitive regions near the nodal regions of Gramineae. One, the leaf sheath pulvinus, is located at the base of the sheathing leaf bases, and is characteristic of the subfamily Festucoideae. The other, the internodal pulvinus, is located at the base of the internode, a little above the nodal joint. Most members of the Panicoideae possess internodal pulvini, in addition to more or less developed leaf sheath pulvini. Three members of the Oryzoideae examined possess leaf sheath pulvini only, while Phragmites australis (Arundinoideae) possesses both leaf sheath and internodal pulvini. Leaf sheath pulvini of some grasses develop hairs, cork-silica cell pairs or stomatal apparatuses over the epidermis while many others are devoid of any such idioblasts. Both the leaf sheath and internodal pulvini of all grasses examined preferentially exclude, or accumulate very little silica, whereas the regions of the shoot immediately above and below the pulvini in these same grasses accumulate large quantities of silica. Pulvini remain unsilicified or poorly silicified throughout their life and even after several days following geotropic bending. Pulvini are also distinguished from the regions above and below them by the lack of lignin in the bundle cap cells. Lignin is found only in the xylem vascular tissue, and this consists of annular and helical vessel elements only. The bundle cap cells are rich in pectin and are described as collenchymatous. All pulvini possess specialized zones of cells containing starch statoliths. In response to horizontal displacement of the shoots, the lower side of the pulvini grows by cell elongation only. The collenchymatous cells stretch in a manner that results in alternately thin and thick regions of cell wall.     

Internodal elongation and orientation of cellulose microfibrils and microtubules in deepwater rice

Excised stem sections of deepwater rice (Oryza sativa L.) containing the highest internode were used to study the induction of rapid internodal elongation by gibberellin (GA). It has been shown before that this growth response is based on enhanced cell division in the intercalary meristem and on increased cell elongation. In both GA-treated and control stem sections, the basal 5-mm region of the highest internode grows at the fastest rate. During 24 h of GA treatment, the internodal elongation zone expands from 15 to 35 mm. Gibberellin does not promote elongation of internodes from which the intercalary meristem has been excised. The orientation of cellulose microfibrils (CMFs) is a determining factor in cell growth. Elongation is favored when CMFs are oriented transversely to the direction of growth while elongation is limited when CMFs are oriented in the oblique or longitudinal direction. The orientation of CMFs in parenchymal cells of GA-treated and control internodes is transverse throughout the internode, indicating that CMFs do not restrict elongation of these cells. Changes in CMF orientation were observed in epidermal cells, however. In the basal 5-mm zone of the internode, which includes the intercalary meristem, CMFs of the epidermal cell walls are transversely oriented in both GA-treated and control stem sections. In slowly growing control internodes, CMF orientation changes to the oblique as cells are displaced from this basal 5-mm zone to the region above it. In GA-treated rapidly growing internodes, the reorientation of CMFs from the transverse to the oblique is more gradual and extends over the 35-mm length of the elongation zone. The CMFs of older epidermal cells are obliquely oriented in control and GA-treated internodes. The orientation of the CMFs parallels that of the cortical microtubules. This is consistent with the hypothesis that cortical microtubules determine the direction of CMF deposition. We conclude that GA acts on cells that have transversely oriented CMFs but does not promote growth of cells whose CMFs are already obliquely oriented at the start of GA treatment.     

微管骨架在轮藻节间细胞伸长生长中的作用

利用免疫荧光定位及激光共聚焦扫描显微镜,结合细胞生长曲线的定量测定,对不同生长阶段的轮藻节间细胞微管骨架进行了观察研究,结果如下：轮藻顶端生长活跃的新生细胞中,与细胞长轴垂直的周质微管(cortical microtubules)占绝对优势,随着生长速率的减慢,周质微管由垂直于细胞长轴逐渐转为平行排列;基部生长基本停止的节间细胞中,胞内微管则以平行细胞长轴为主;不同生长阶段节间细胞的微管骨架,对微管特异解聚剂黄草消(oryzalin)处理的敏感性表现不相同。顶端生长活跃的节间细胞经oryzalin处理40min后,绝大多数周质微管发生解聚;而基部生长基本停止的老细胞中,即使延长处理时间,仍残留一些尚未完全解聚的微管片段;10μmol／L微管解聚剂oryzalin处理轮藻顶端新生细胞,在高精度的细胞伸长生长测定装置监测下,发现oryzalin对细胞的伸长生长速率有明显的抑制作用,去掉药剂后,伸长生长又有一定的恢复。并且发现,经oryzalin处理后,微管的解聚(40min左右)与顶端节间细胞伸长生长的停止(100min左右)两者间存在着时间上的差异,即微管解聚在先,细胞伸长停止在后。以上结果均说明微管骨架在轮藻节间细胞生长中具有重要作用。     

Microtubule orientation in globular leaflet cells of <Emphasis Type="Italic">Chara inflata</Emphasis>

Chara inflata has globular leaflet cells and cylindrical internodal cells. The morphology of the leaflet cells is different from that of other Characeae. The orientation of cortical microtubules (MTs) in young leaflet and internodal cells of this species was analyzed by immunofluorescence microscopy. MTs with random orientation were observed in leaflet cells, while those relatively transverse to the cell axis were observed in cylindrical internodal cells. In cylindrical leaflet cells in Chara corallina, transverse MTs were observed. These results imply that C. inflata is a morphological mutant lacking a mechanism for orienting cortical MTs transverse in leaflet cells.     

Microtubules at wound sites of Nitella internodal cells passively co-align with actin bundles when exposed to hydrodynamic forces generated by cytoplasmic streaming

The organization of cortical microtubules at wound sites in Nitella pseudoflabellata(A. Br. & Nordst.) em. R.D.W. and N. flexilis(L.) Ag. internodal cells was examined in relation to the regeneration of actin filament bundles in order to identify the mechanisms by which microtubules are oriented. Actin bundle regrowth occurs prior to that of microtubules, so it was considered possible that microtubule alignment is actin-dependent, perhaps mediated by cross-linking proteins. In all types of wounds investigated, subcortical actin bundles regenerated parallel to the direction of cytoplasmic streaming. Microtubule orientation patterns, however, varied according to the nature of wound formation and the type of wound wall eventually produced. In chloroplast-free windows induced by blue light irradiation, microtubule orientation varied according to the size of the window. Microtubules were randomized in 10- to 30-μm-wide windows where exposure to cytoplasmic flow is minimal, but were aligned more or less parallel to regenerated actin bundles in 80- to 100-μm-wide windows. Where co-alignment between microtubules and actin bundles was obvious after fluorescence labelling, electron micrographs revealed that microtubules and actin bundles were too widely spaced to account for any cross-linkages. Furthermore, treatments that inhibited or reduced cytoplasmic streaming without altering the direction of actin bundles caused randomization of microtubules previously oriented in the streaming direction, even in the presence of taxol. When evenly flat wound walls were induced by 10⁻⁴ M chlortetracycline, microtubules were co-aligned with nearby actin bundles at the surface of the wound wall. At wounds induced by treatment with 5 × 10⁻² M CaCl₂, however, microtubules were randomly oriented and preferentially located in the narrow clefts between the wound-wall protuberances, up to several micrometers away from the actin bundles near the wound-wall tips. These results indicate that microtubules regenerated in wounds are merely co-aligned with actin filament bundles because they are passively aligned by the hydrodynamic forces created by cytoplasmic flow. Received: 4 August 1998 / Accepted: 30 January 1999     

The cyclic reorientation of cortical microtubules in epidermal cells of azuki bean epicotyls: the role of actin filaments in the progression of the cycle

The orientation of microtubules (MTs) was examined in epidermal cells of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls. The orientation of MTs adjacent to the outer tangential wall of the cells, which has a crossed polylamellate structure with lamellae of longitudinal cellulose microfibrils alternating with lamellae of transverse cellulose microfibrils, differed from one cell to another. Treatment with an auxin-free solution caused the accumulation of cells with longitudinal MTs and subsequent treatment with a solution that contained auxin resulted in the accumulation of cells with transverse MTs, showing that sequential treatments with auxin-free and auxin-containing solutions can synchronize the reorientation of MTs. The MTs, once reoriented from longitudinal to transverse, returned to longitudinal and then back to transverse once again, the duration of the cycle being about 6 h. Gibberellic acid, known to increase the percentage of cells with transverse MTs, promoted reorientation of MTs from longitudinal to transverse and inhibited that from transverse to longitudinal. Cytochalasin D, an agent that disrupts actin filaments, speeded up the reorientation from transverse to longitudinal and slowed down that from longitudinal to transverse. It caused an increase in the percentage of cells with MTs in mixed orientation, and the percentage of such cells was highest when the percentage of cells with longitudinal MTs was decreasing and that of cells with transverse MTs was increasing. Received: 27 November 1997 / Accepted: 7 January 1998     

Structural Characteristics of Developing Nitella Internodal Cell Walls

The Nilella intermodal cell is formed by a division of the segment cell, the latter being a direct derivative of the shoot apical cell. The internodal cell is remarkable in that it elongates from an initial length of about 20 microns to a mature length of about 60 millimeters. The structures of the apical and segment cells, and the internodal cells in all stages of development were examined with the techniques of interference, polarization, and electron microscopy. The apical and segment cells were found to be isotropic. The upper part of the segment cell, destined to form a node, shows a curious pitted structure that was characteristic of certain node structures. The lower part of the segment cell, destined to become an internodal cell, shows a vague transverse arrangement of fibrils at the inner wall surface. The internodal cells, from the time they are first formed, show negative birefringence and a transverse arrangement of microfibrils at the inner wall surface. The elongation of the internodal cell is characterized by a rise, dip, and rise in both the optical thickness and retardation of the cell wall. The dip in both these variables coincides with the attainment of the maximum relative elongation rate. After the cessation of elongation, wall deposition continues, but the fibrils at .the inner surface of the wall are now seen to occur in fields of nearly parallel microfibrils. These fields, with varying fibrillar directions, may partly overlap each other or may merge with one another. Unlike the growing wall, this wall which is deposited after the end of elongation is isotropic.     

Cell Elongation and Microtubule Behavior in the Arabidopsis Hypocotyl: Responses to Ethylene and Auxin

During elongation of the Arabidopsis hypocotyl, each cell reacts to light and hormones in a time- and position-dependent manner. Growth in darkness results in the maximal length a wild-type cell can reach. Elongation starts at the base and proceeds in the acropetal direction. Cells in the upper half of the hypocotyl can become the longest of the whole organ. Light strongly inhibits cell elongation all along the hypocotyl, but proportionally more in the upper half. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is known to stimulate hypocotyl elongation in the light. Here we show that this stimulation only occurs in cells of the apical half of the hypocotyl. Moreover, ACC application can partially overcome light inhibition, whereas indole-3-acetic acid (IAA) cannot. On low-nutrient medium (LNM) in the light, elongation is severely reduced as compared to growth on rich medium, and both ACC and IAA can stimulate elongation to the levels reached on a nutrient-rich medium. Furthermore, microtubule orientation was studied in vivo. During elongation in darkness, transverse and longitudinal patterns are clearly related with rates of elongation. In other conditions, except for the association of longitudinally orientated microtubules with growth arrest, microtubule orientation is merely an indicator of developmental age, not of elongation activity. A hypothesis on the relation between microtubules and elongation rate is discussed.     

Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. II. Effects of brassinosteroids on microtubules and cell elongation in the bul1 mutant

In order to elucidate the involvement of brassinosteroids in the cell elongation process leading to normal plant morphology, indirect immunofluorescence and molecular techniques were use to study the expression of tubulin genes in the bul1-1 dwarf mutant of Arabidopsis thaliana (L.) Heynh., the characteristics of which are reported in this issue (M. Catterou et al., 2001). Microtubules were studied specifically in the regions of the mutant plant where the elongation zone is suppressed (hypocotyls and petioles), making the reduction in cell elongation evident. Indirect immunofluorescence of α-tubulin revealed that very few microtubules were present in mutant cells, resulting in the total lack of the parallel microtubule organization that is typical of elongating cells in the wild type. After brassinosteroid treatment, microtubules reorganized and became correctly oriented, suggesting the involvement of brassinosteroids in microtubule organization. Molecular analyses showed that the microtubule reorganization observed in brassinosteroid-treated bul1-1 plants did not result either from an activation of tubulin gene expression, or from an increase in tubulin content, suggesting that a brassinosteroid-responsive pathway exists which allows microtubule nucleation/organization and cell elongation without activation of tubulin gene expression. Received: 28 April 2000 / Accepted: 6 October 2000     

Levels of {beta}-Glucan and Lignin in Elongating Internodes of Deepwater Rice

Partial submergence or treatment with either ethylene or gibberellicacid (GA₃ induces rapid growth in deepwater rice (Oryza sativaL.). We correlated the synthesis of two cell wall componentswith two phases of internodal elongation, namely (13,14)-ß-glucanformation with cell elongation and lignification with differentiationof the secondary cell wall and cessation of growth. The contentof ß-glucan was highest in the zone of cell elongationin internodes of air-grown plants and plants that were inducedto grow rapidly by submergence. In the intercalary meristemand in the differentiation zone of the internode, ß-glucanlevels were ca. 70% lower than in the zone of cell elongation.The outer cell layers, enriched in epidermis, contained moreß-glucan in submerged, rapidly growing internodesthan in air-grown, control internodes. The ß-glucancontent of the inner, parenchymal tissue was unaffected or slightlylowered by submergence. The epidermis appears to be the growth-limitingstructure of rapidly growing rice internodes. We hypothesizethat elevated levels of ß-glucan contribute to elongationgrowth by increasing the extensibility of the cell wall. Lignificationwas monitored by measuring the content of lignin and the activitiesof two enzymes of the lignin biosynthetic pathway, coniferylalcohol dehydrogenase (CAD) and phenylalanine ammonia-lyase(PAL), in growing and non-growing regions of the internode.Using submerged whole plants and GA₃-treated excised stem segments,we showed that lignin content and CAD activity were up to sixfoldlower in newly formed internodal tissue of rapidly growing ricethan in slowly growing tissue. No differences were observedin parts of the internode that had been formed prior to inductionof growth. PAL activity was reduced throughout the internodeof submerged plants. We conclude that lignification is one ofthe processes that is suppressed to permit rapid growth. ¹ This work was supported by the National Science Foundationthrough grants No. DCB-8718873 and DCB-9103747 and by the Departmentof Energy through grant No. DE-FGO2-90ER20021. M.S. was therecipient of a fellowship from the Max Kade Foundation.     

A γ-TIP cross-reacting protein is abundant in the cortex of soybean N2-fixing nodules

The distribution and abundance of tonoplast intrinsic protein (γ-TIP), a putative aquaporin which is abundant in the tonoplast of osmoregulated pulvinus motor cells, were determined in nodules of Glycine max (L.) Merr. using chemical fixation and immunolocalization. This protein was highly expressed in the tonoplast of the inner cortical cells of the nodules but poorly expressed in the vascular transfer cells and in infected cells. It is concluded that the differentiation of the inner cortical cells of the nodules like that of pulvinus motor cells, is accompanied by an increased expression of γ-TIP. This result is consistent with our previous hypothesis that a reversible exchange of intercellular water by the inner cortical cells plays a role in the regulation of nodule conductance to O₂ diffusion, and hence in subsequent N₂-fixing activity. Received: 7 February 1998 / Accepted: 22 May 1998     

Differential Responsiveness of Cortical Microtubule Orientation to Suppression of Cell Expansion among the Developmental Zones of Arabidopsis thaliana Root Apex

Τhe bidirectional relationship between cortical microtubule orientation and cell wall structure has been extensively studied in elongating cells. Nevertheless, the possible interplay between microtubules and cell wall elements in meristematic cells still remains elusive. Herein, the impact of cellulose synthesis inhibition and suppressed cell elongation on cortical microtubule orientation was assessed throughout the developmental zones of Arabidopsis thaliana root apex by whole-mount tubulin immunolabeling and confocal microscopy. Apart from the wild-type, thanatos and pom2-4 mutants of Cellulose SynthaseA3 and Cellulose Synthase Interacting1, respectively, were studied. Pharmacological and mechanical approaches inhibiting cell expansion were also applied. Cortical microtubules of untreated wild-type roots were predominantly transverse in the meristematic, transition and elongation root zones. Cellulose-deficient mutants, chemical inhibition of cell expansion, or growth in soil resulted in microtubule reorientation in the elongation zone, wherein cell length was significantly decreased. Combinatorial genetic and chemical suppression of cell expansion extended microtubule reorientation to the transition zone. According to the results, transverse cortical microtubule orientation is established in the meristematic root zone, persisting upon inhibition of cell expansion. Microtubule reorientation in the elongation zone could be attributed to conditional suppression of cell elongation. The differential responsiveness of microtubule orientation to genetic and environmental cues is most likely associated with distinct biophysical traits of the cells among each developmental root zone.     

Microtubule orientation in pea stem cells: a change in orientation follows the initiation of growth rate decline

The orientation of microtubules in cells of redlight-grown pea plants (Pisum sativum L.) was examined by means of immunofluorescence. Microtubules (MTs) in rapidly elongating, subepidermal cells commonly form multiple, parallel strands that run transverse to the cell's axis of elongation. By contrast, MTs in nonelongating subepidermal cells form steeply pitched helical arrays; MTs in non-elongating epidermal cells are oriented parallel to the axis of elongation. This change in orientation occurs during the time interval in which growth rate is declining. The transition is abrupt rather than gradual and occurs in both epidermal and subepidermal cells at the same time. Plants irradiated for 2 h with a growth-inhibiting fluence of blue light did not undergo the same transition, indicating that factors other than changing elongation rates must be responsible for triggering the reorganization of MT arrays.     

Sperm cell surface characteristics of Plumbago zeylanica L. in relation to transport in the embryo sac

Myosin associated with the male germ cells of angiosperms interacts with actin, promoting transport of the non-motile generative and later sperm cells in the pollen tube. Myosin localizing on the sperm cell plasma membrane seems negligible in Plumbago, as reflected by the absence of: (i) anti-myosin labeling using immunoelectron microscopy, (ii) sperm motility on actin matrices, and (iii) electrophoretic movement changes after addition of antibody. Sperm cells injected directly into actively streaming Nitella internodal cells, however, follow actin bundles and their movement is sensitive to ATP and Mg²⁺. This may be based on simple charge binding since negatively charged latex beads also migrate on actin, whereas neutral or positively-charged latex beads do not. Sperm cells are negatively charged according to capillary microelectrophoresis, whereas killed sperm cells, which are positively charged do not migrate. The sperm cell that normally fertilizes the egg has a higher calculated charge (8.277 × 10³ esu/cm²) compared with the sperm cell that fuses with the central cell (6.120 × 10³ esu/cm²). Received: 15 December 1998 / Accepted: 21 January 1999     

Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion

Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2–4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2–4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone''s expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation.     

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     

Control of leaf cell elongation in barley. Generation rates of osmotic pressure and turgor, and growth-associated water potential gradients

In a previous study on the effects of N-supply on leaf cell elongation, the spatial distribution of relative cell elongation rates (RCER), epidermal cell turgor, osmotic pressure (OP) and water potential (Ψ) along the elongation zone of the third leaf of barley was determined (W. Fricke et al. 1997, Planta 202: 522–530). The results suggested that in plants receiving N at fixed relative addition rates (N-supply limitation of growth), cell elongation was rate-limited by the rate of solute provision, whereas in plants growing on complete nutrient solution containing excessive amounts of N (N-demand limitation), cell elongation was rate-limited by the rate of water supply or wall yielding. In the present paper, these suggestions were tested further. The generation rates of cell OP, turgor and Ψ along the elongation zone were calculated by applying the continuity equation of fluid dynamics to the previous data. To allow a more conclusive interpretation of results, anatomical data were collected and bulk solute concentrations determined. The rate of OP generation generally exceeded the rate of turgor generation. As a result, negative values of cell Ψ were created, particularly in demand-limited plants. These plants showed highest RCER along the elongation zone and a Ψ gradient of at least −0.15 MPa between water source (xylem) and expanding epidermal cells. The latter was similar to a theoretically predicted value (−0.18 MPa). Highest rates of OP generation were observed in demand-limited plants, with a maximum rate of 0.112 MPa · h⁻¹ at 16–20 mm from the leaf base. This was almost twice the rate in N-supply-limited plants and implied that the cells in the leaf elongation zone were capable of importing (or synthesising) every minute almost 1 mM of osmolytes. Potassium, Cl⁻ and NO₃ ⁻ were the main inorganic osmolytes (only determined for demand-limited plants). Their concentrations suggest that, unlike the situation in fully expanded epidermal cells, sugars are used to generate OP and turgor. Anatomical data revealed that the zone of lateral cell expansion extended distally beyond the zone of cell elongation. It is concluded that leaf cell expansion in barley relies on high rates of water and solute supply, rates that may not be sustainable during periods of sufficient N-supply (limitation by water supply: Ψ gradients) or limiting N-supply (limitation by solute provision: reduced OP-generation rates). To minimise the possibility of growth limitation by water and osmolyte provision, longitudinal and lateral cell expansion peak at different locations along the growth zone. Received: 15 October 1997 / Accepted: 12 March 1998     

Microtubule reorganization,cell wall synthesis and establishment of the axis of elongation in regenerating protoplasts of the algaMougeotia

Summary Microtubule reorganization and cell wall deposition have been monitored during the first 30 hours of regeneration of protoplasts of the filamentous green algaMougeotia, using immunofluorescence microscopy to detect microtubules, and the cell-wall stain Tinopal LPW to detect the orientation of cell wall microfibrils. In the cylindrical cells of the alga, cortical microtubules lie in an ordered array, transverse to the long axis of the cells. In newly formed protoplasts, cortical microtubules exhibit some localized order, but within 1 hour microtubules become disordered. However, within 3 to 4 hours, microtubules are reorganized into a highly ordered, symmetrical array centered on two cortical foci. Cell wall synthesis is first detected during early microtubule reorganization. Oriented cell wall microfibrils, co-aligned with the microtubule array, appear subsequent to microtubule reorganization but before cell elongation begins. Most cells elongate in the period between 20 to 30 hours. Elongation is preceded by the aggregation of microtubules into a band intersecting both foci, and transverse to the incipient axis of elongation. The foci subsequently disappear, the microtubule band widens, and microfibrils are deposited in a band which is co-aligned with the band of microtubules. It is proposed that this band of microfibrils restricts lateral expansion of the cells and promotes elongation. Throughout the entire regeneration process inMougeotia, changes in microtubule organization precede and are paralleled by changes in cell wall organization. Protoplast regeneration inMougeotia is therefore a highly ordered process in which the orientation of the rapidly reorganized array of cortical microtubules establishes the future axis of elongation.