On the alignment of cellulose microfibrils by cortical microtubules: A review and a model

Summary The hypothesis that microtubules align microfibrils, termed the alignment hypothesis, states that there is a causal link between the orientation of cortical microtubules and the orientation of nascent microfibrils. I have assessed the generality of this hypothesis by reviewing what is known about the relation between microtubules and microfibrils in a wide group of examples: in algae of the family Characeae,Closterium acerosum, Oocystis solitaria, and certain genera of green coenocytes and in land plant tip-growing cells, xylem, diffusely growing cells, and protoplasts. The salient features about microfibril alignment to emerge are as follows. Cellulose microfibrils can be aligned by cortical microtubules, thus supporting the alignment hypothesis. Alignment of microfibrils can occur independently of microtubules, showing that an alternative to the alignment hypothesis must exist. Microfibril organization is often random, suggesting that self-assembly is insufficient. Microfibril organization differs on different faces of the same cell, suggesting that microfibrils are aligned locally, not with respect to the entire cell. Nascent microfibrils appear to associate tightly with the plasma membrane. To account for these observations, I present a model that posits alignment to be mediated through binding the nascent microfibril. The model, termed templated incorporation, postulates that the nascent microfibril is incorporated into the cell wall by binding to a scaffold that is oriented; further, the scaffold is built and oriented around either already incorporated microfibrils or plasma membrane proteins, or both. The role of cortical microtubules is to bind and orient components of the scaffold at the plasma membrane. In this way, spatial information to align the microfibrils may come from either the cell wall or the cell interior, and microfibril alignment with and without microtubules are subsets of a single mechanism.Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Orientation of cortical microtubules correlates with cell shape and division direction

Summary Cortical microtubules in the epidermis of regeneratingGraptopetalum plants were examined by in situ immunofluorescence. Paradermal slices of tissue were prepared by a method that preserves microtubule arrays and also maintains cell junctions. To test the hypothesis that cortical microtubule arrays align perpendicular to the direction of organ growth, arrays were visualized and their orientation quantified. A majority of microtubules are in transverse orientation with respect to the organ axis early in shoot development when the growth habit is uniform. Later in development, when growth habit is non-uniform and the tissue is contoured, cortical microtubules are increasingly longitudinal and oblique in orientation. Microtubules show only a minor change in orientation at the site of greatest curvature, the transition zone of a developing leaf. To assess the role of the division plane on orientation of arrays, the pattern of microtubules was examined in individual cells of common shape. Cells derived from transverse divisions have predominately transverse cortical arrays, whereas cells derived from oblique and longitudinal divisions have non-transverse arrays. The results show that, regardless of the stage of development, microtubules orient with respect to cell shape and plane of division. The results suggest that cytoskeletal function is best considered in small domains of growth within an organ.Abbrevations DMSO dimethylsulfoxide - EGTA ethylene glycol-bis-(ß-aminoethyl ether)-N, N, N, N-tetra acetic acid - FITC fluorescein isothiocyanate - MTSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Microtubule orientation and dynamics in elongating characean internodal cells following cytosolic acidification, induction of pH bands, or premature growth arrest

Summary Cortical microtubules (MTs) at indifferent zones in immatureNitella internodes were investigated by injection of fluorescently tagged sheep brain tubulin into living cells and by immunofluorescence on fixed material. Nearly identical MT patterns and numbers were detected with the two techniques, indicating that sheep brain tubulin incorporated into all cortical MTs. MTs were aligned transversely to the long axis of the cell and approximately one MT was present every micrometer of longitudinal cell distance. Treatment of internodes with propionic acid to acidify cytosolic pH caused depolymerization of MTs and an increase in the unpolymerized tubulin pool. Transfer of young, vigorously elongating cells to media inducing premature growth cessation resulted in a slight decrease in microtubule numbers but did not significantly alter microtubule orientation patterns or microtubule lifespans. MTs remained transverse for days following growth cessation before finally assuming a more random alignment characteristic of mature, non-growing internodes. No differences in MT numbers, orientation, or dynamics were detected between acid and alkaline bands in internodes incubated in a band-inducing medium. Thus, properties of cortical MT arrays were not closely coupled to growth status or to regional differences in cellular physiology associated with pH banding.Abbrevations BIM band-inducing medium - CCM Chara culture medium - CF carboxyfluorescein - FRAP fluorescence redistribution after photobleaching - MT microtubule     

Cellulose microfibril alignment recovers from DCB-induced disruption despite microtubule disorganization

Cellulose microfibril deposition patterns define the direction of plant cell expansion. To better understand how microfibril alignment is controlled, we examined microfibril orientation during cortical microtubule disruption using the temperature-sensitive mutant of Arabidopsis thaliana, mor1-1. In a previous study, it was shown that at restrictive temperature for mor1-1, cortical microtubules lose transverse orientation and cells lose growth anisotropy without any change in the parallel arrangement of cellulose microfibrils. In this study, we investigated whether a pre-existing template of well-ordered microfibrils or the presence of well-organized cortical microtubules was essential for the cell to resume deposition of parallel microfibrils. We first transiently disrupted the parallel order of microfibrils in mor1-1 using a brief treatment with the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB). We then analysed the alignment of recently deposited cellulose microfibrils (by field emission scanning electron microscopy) as cellulose synthesis recovered and microtubules remained disrupted at the mor1-1 mutant's non-permissive culture temperature. Despite the disordered cortical microtubules and an initially randomized wall texture, new cellulose microfibrils were deposited with parallel, transverse orientation. These results show that transverse cellulose microfibril deposition requires neither accurately transverse cortical microtubules nor a pre-existing template of well-ordered microfibrils. We also demonstrated that DCB treatments reduced the ability of cortical microtubules to form transverse arrays, supporting a role for cellulose microfibrils in influencing cortical microtubule organization.     

Microtubular configurations during the cellularization of coenocytic endosperm in Ranunculus sceleratus L.

Summary Endosperm cellularization in Ranunculus sceleratus was studied in terms of the initiation of cell-wall formation in the coenocytic endosperm. The first endosperm cell walls were in an anticlinal position relative to the cell wall of the embryo sac and originated from the cell plates and not from wall ingrowths from the embryo-sac wall itself. Alveolar endosperm was formed 3 days after pollination. Microtubules were associated with the freely growing wall ends of the anticlinal walls and were observed in various orientations that generally ranged from angles of 45 ° to 90 ° to the plane of the wall. They were absent in the regions where vesicles had already fused. These microtubules may function in maintaining the growth and the direction of growth of the anticlinal wall until cellularization is completed. At the site where three neighbouring alveoli share their freely growing wall ends, remarkable configurations of microtubules were observed: in each alveolus, microtubules ran predominantly parallel to the bisector of the angle formed by the common walls. These microtubules may form a physically stable framework and maintain the direction of growth of the wall edges. It is concluded that the growing edge of the anticlinal endosperm wall and its associated microtubules are a special continuum of the original phragmoplast that gave rise to the anticlinal wall.     

Microtubule organization and cell division in embryogenie protoplast cultures of white spruce (Picea glauca)

Summary Immunofluorescence methods were developed for examining the distribution of microtubules in freshly isolated and cultured protoplasts and regenerated somatic embryos of white spruce (Picea glauca). Freshly isolated protoplasts consisted of both uniand multinucleate types. Uninucleate protoplasts established parallel cortical microtubules during cell wall formation and cell shaping, divided within 24 h and developed into somatic embryos in culture. Dividing cells were characterized by preprophase bands (PPBs) of microtubules, atypical spindle microtubules focused at the poles and a typical phragmoplast at telophase. Multinucleate protoplasts also established parallel arrays of cortical microtubules during cell wall formation. In addition their nuclei divided synchronously within 4 days, then cell walls formed between the daughter nuclei. Individual multinucleate protoplast-derived colonies subsequently gave rise to elongate suspensor cells thereby forming embryo-like structures by 7 days.     

Regulation of Growth Anisotropy in Well-Watered and Water-Stressed Maize Roots. II. Role of Cortical Microtubules and Cellulose Microfibrils

We tested the hypothesis that the degree of anisotropic expansion of plant tissues is controlled by the degree of alignment of cortical microtubules or cellulose microfibrils. Previously, for the primary root of maize (Zea mays L.), we quantified spatial profiles of expansion rate in length, radius, and circumference and the degree of growth anisotropy separately for the stele and cortex, as roots became thinner with time from germination or in response to low water potential (B.M. Liang, A.M. Dennings, R.E. Sharp, T.I. Baskin [1997] Plant Physiol 115:101–111). Here, for the same material, we quantified microtubule alignment with indirect immunofluorescence microscopy and microfibril alignment throughout the cell wall with polarized-light microscopy and from the innermost cell wall layer with electron microscopy. Throughout much of the growth zone, mean orientations of microtubules and microfibrils were transverse, consistent with their parallel alignment specifying the direction of maximal expansion rate (i.e. elongation). However, where microtubule alignment became helical, microfibrils often made helices of opposite handedness, showing that parallelism between these elements was not required for helical orientations. Finally, contrary to the hypothesis, the degree of growth anisotropy was not correlated with the degree of alignment of either microtubules or microfibrils. The mechanisms plants use to specify radial and tangential expansion rates remain uncharacterized.     

Mutation or drug-dependent microtubule disruption causes radial swelling without altering parallel cellulose microfibril deposition in Arabidopsis root cells

As critical determinants of growth anisotropy in plants, cortical microtubules are thought to constrain the movement of cellulose synthase complexes and thus align newly deposited cellulose microfibrils. We tested this cellulose synthase constraint model using the temperature-sensitive mor1-1 mutant of Arabidopsis. Contrary to predictions, the disruption of cortical microtubules in mor1-1 root epidermal cells led to left-handed root twisting and radial swelling but did not alter the transverse orientation of cellulose microfibrils. We also found that drug-dependent disassembly or hyperstabilization of cortical microtubules did not alter the parallel order of cellulose microfibrils. By measuring cellulose content in mor1-1 seedlings, we verified that cellulose synthesis is not reduced at the restrictive temperature. The independence of cortical microtubule organization and cellulose microfibril alignment was supported by the observation that double mutants of mor1-1 and rsw1-1, the cellulose-deficient mutant with misaligned microfibrils, had additive phenotypes. Our results suggest that cortical microtubules regulate growth anisotropy by some mechanism other than cellulose microfibril alignment or synthesis.     

The change of pattern in microfibril arrangement on the inner surface of the cell wall of Closterium acerosum during cell growth

Closterium acerosum (Schrank) Ehrenberg cells cultured on cycles of 16 h light and 8 h dark, undergo cell division synchronously in the dark period. After cell division, the symmetry of the daughter semicells is restored by controlled expansion, the time required for this restoration, 3.5–4 h, being relatively constant. The restoration of the symmetry is achieved by highly oriented surface expansion occurring along the entire length of the new semicell. During early semicell expansion, for about 2.5 h, microfibrils are deposited parallel to one another and transversely to the cell axis on the inner surface of the new wall. Wall microtubules running parallel to the transversely oriented microfibrils are observed during this period. About 2.5 h after septum formation, preceding the cessation of cell elongation, bundles of 7–11 microfibrils running in various directions begin to overlay the parallel-arranged microfibrils already deposited. In the fully elongated cells, no wall microtubules are observed.     

Microtubules do not control orientation of secondary cell wall microfibril deposition inMicrasterias

Summary The influence of the microtubule disorganizing substances amiprophos-methyl (APM) and colchicine on secondary wall formation inMicrasterias denticulata was investigated by the freezeetch technique. The results reveal that neither microtubule inhibitor changes the pattern of microfibril deposition. The application of APM or colchicine also does not cause any structural alterations of the microfibrils or of the protoplasmic (Pf) and the exoplasmic (Ef) fracture face of the plasma membrane, thus indicating that microtubules are not involved in secondary wall formation inM. denticulata. However, since areas of the plasma membrane which collapsed upon freeze-etching are restricted to the Pf-face of cells treated with microtubule inhibitors, cortical microtubules may function as mechanical support during secondary wall formation. In the cortical cytoplasm filamentous structures are found in close spatial relationship and an almost parallel alignment to rosettes of the plasma membrane.     

Relationship between cyclic AMP, microtubule organization, and mammalian cell shape : Studies on Chinese hamster ovary cells and their variants

Treatment of Chinese hamster ovary cells with N⁶,2′-O-dibutyryl adenosine cyclic 3′,5′-monophosphate (db-cAMP) and hormones converts their shape from a knobbed, epithelial-like morphology to a smooth fibroblast-like form. Ultrastructural studies demonstrate an increased number of microtubules and their arrangement in parallel array along the long axis of the cell after treatment with these agents. Although an epithelial-like variant, when treated with db-cAMP, shows an increase in the number of microtubules; these microtubules remain in a disorganized nonparallel array. The numerous long microtubules which are already present in a fibroblast-like variant become further elongated when the cells are treated with db-cAMP. These experiments establish the relationship between cAMP level, assembly and organization of microtubules, and cell shape in cultured Chinese hamster cells.     

Fine Structure and Taxonomic Position of the Giant Amoeboid Flagellate Pelomyxa palustris1,2

ABSTRACT Specimens of Pelomyxa palustris from five collecting sites had numerous nonmotile flagella. The structures are called flagella because of morphological similarities to flagella and because P. palustris has affinities with amoeboid flagellates. Flagella were photographed on living cells and studied by transmission and scanning electron microscopy. From 64 to 742 flagella per cell were estimated from scanning electron microscopy of ten cells 204 to 1269 μm in length. The nonmotile flagella arise from basal granules which were, in one strain, surrounded by radiating electron-dense microtubules. This strain also had excess axonemal microtubules. Abundant cytoplasmic microtubules were arranged in several different patterns. In about half of the P. palustris cells in which nuclei were studied, microtubules were either apposed to the nuclear membrane in a parallel alignment (with some also radiating) or radiating from the nuclear membrane (with none parallel). Bacteria associated with nuclei were of three characteristic types: Gram-negative rods, Gram-positive rods, and large rods. All nuclei within a given trophozoite had similar perinuclear features. Recent proposals for separation of Pelomyxa to its own phylum (based on its proposed primitive, unique nature) can not be justified. Pelomyxa is a complex, highly specialized organism adapted to live in a specific fresh-water environment. Mastigamoebid amoeboid flagellates of the genera Mastigamoeba, Mastigella, Mastigina, and possibly Dinamoeba are placed with Pelomyxa within the order Pelobiontida Page, 1976, emend., containing two families. Pelomyxidae Schulze, 1877, and Mastigamoebidae Goldschmidt, 1907.     

Independence of two microtubule systems in fertilized frog eggs: the sperm aster and the vegetal parallel array

Two microtubule-containing structures are implicated in dorsoventral polarization of the frog egg, and we examined the relationship between them. The sperm aster provides a directional cue for a cortical rotation specifying polarity, and a vegetal cortical array of parallel microtubules is likely part of the rotational machinery. The growing aster has an accumulation of microtubules marking the path of the sperm pronucleus, and its microtubules extend into the egg cortex as well as the cytoplasm. To test whether the vegetal parallel array was an extension of astral cortical growth, fertilized or activated eggs were bisected into animal and vegetal fragments. The vegetal fragments formed parallel arrays, even when isolated within a few minutes of egg activation. Neither the sperm centrosome nor another microtubule organizing center in the animal half of the egg is required for formation of the parallel array, but some animal half activity is involved in its disappearance. Correspondence to: R.P. Elinson     

BOTERO1 is required for normal orientation of cortical microtubules and anisotropic cell expansion in Arabidopsis

Mutants at the BOTERO1 locus are affected in anisotropic growth in all non-tip-growing cell types examined. Mutant cells are shorter and broader than those of the wild type. Mutant inflorescence stems show a dramatically reduced bending modulus and maximum stress at yield. Our observations of root epidermis cells show that the cell expansion defect in bot1 is correlated with a defect in the orientation of the cortical microtubules. We found that in cells within the apical portion of the root, which roughly corresponds to the meristem, microtubules were loosely organized and became much more highly aligned in transverse arrays with increasing distance from the tip. Such a transition was not observed in bot1. No defect in microtubule organization was observed in kor-1, another mutant with a radial cell expansion defect. We also found that in wild-type root epidermal cells, cessation of radial expansion precedes the increased alignment of cortical microtubules into transverse arrays. Bot1 roots still show a gravitropic response, which indicates that ordered cortical microtubules are not required for differential growth during gravitropism. Interestingly, the fact that in the mutant, these major changes in microtubule organization cause relatively subtle changes in cell morphology, suggest that other levels of control of growth anisotropy remain to be discovered. Together, these observations suggest that BOT1 is required for organizing cortical microtubules into transverse arrays in interphase cells, and that this organization is required for consolidating, rather than initiating, changes in the direction of cell expansion.     

Reorientation of Microfibrils and Microtubules at the Outer Epidermal Wall of Maize Coleoptiles During Auxin-Mediated Growth

Auxin-mediated elongation growth of isolated subapical coleoptile segments of maize (Zea mays L.) is controlled by the extensibility of the outer cell wall of the outer epidermis (Kutschera et al., 1987). Here we investigate the hypothesis that auxin controls the extensibility of this wall by changing the orientation of newly deposited microfibrils through a corresponding change in the orientation of cortical microtubules. On the basis of electron micrographs it is shown that cessation of growth after removal of the endogenous source of auxin is correlated with a relative increase of longitudinally orientated microfibrils and microtubules at the inner wall surface. Conversely, reinduction of growth by exogenous auxin is correlated with a relative increase of transversely orientated microfibrils and microtubules at the inner wall surface. These changes can be detected 30–60 min after the removal and addition of auxin, respectively. The functional significance of directional changes of newly desposited wall microfibrils for the control of elongation growth is discussed.     

Microtubule solutions display nematic liquid crystalline structure

We report a study of the spontaneous formation of ordered arrays of microtubules in solution. Form birefringence and anisotropic light-scattering appear rapidly and spontaneously when tubulin, initially present in homogeneous solution, self-assembles into microtubules. This phenomenon is reversible and occurs at protein concentrations of a few milligrams per ml, in the presence or absence of microtubule-associated proteins. Light and electron microscopic examination reveals that extensive regions of these birefringent solutions consist of nearly parallel microtubules. Measurement of the order parameter, S, yields a value of 0.81 +/- 0.05, indicating a high degree of alignment. Comparison of these observations to qualitative predictions developed from the theory of Onsager ((1949) Ann. N.Y. Acad. Sci. 51, 627-659) leads to the conclusion that microtubules form a nematic liquid crystalline phase in vitro under ordinary conditions. Simultaneous spectrophotometric observation of turbidity (a measure of microtubule assembly) and birefringence shows that the parallel ordering lags only slightly behind assembly, thus demonstrating that much microtubule growth must occur by addition of tubulin to the ends of microtubules that are already aligned. These observations of anisotropy are important to the understanding of microtubule dynamics in vitro.     

ELECTRON MICROSCOPIC OBSERVATIONS ON TUBULAR STRUCTURES INVOLVED IN CRYSTAL FORMATION IN THE RED ALGA PLEONOSPORIUM SQUARRULOSUM (HARV.) ABBOTT1

Hexagonal or angular crystalline inclusions in Pleonosporium (Naeg.) Hauck vegetative cells were examined using electron microscopy. Ultrastructural analysis reveals that the inclusions initially contain tubular elements resembling microtubules but, with continued differentiation, are transformed into rod containing crystals. The tubular structures initially measure 25 nm in diameter. Scattered tubules become arranged in a parallel and alternate pattern and undergo subsequent enlargement to approximately 29 nm. Following enlargement, each tubule apparently disaggregates into rods that form a crystal having hexagonally arranged rod-like subunits. It is suggested that these tubules may represent microtubules and the resultant crystals are composed of tubulin.     

Microtubule patterning in apical epidermal cells ofVinca minor preceding leaf emergence

Summary Serial thin sectioning for electron microscopy was carried out on the cortical cytoplasm of surface cells of the apical dome ofVinca minor. The cellulose reinforcement pattern in the outer epidermal walls forming this surface is known to correlate well with the decussate phyllotaxis pattern. The purpose of this study was to determine the location of microtubules immediately under these epidermal walls as a first step toward finding out how the cellulose pattern arises. First, correspondence between the patterns of microtubules and cellulose was checked. Second, the role of potential orienting cues for the alignment of microtubule arrays in specific cells was evaluated.Microtubule arrays which were well or moderately ordered (70% of the total interphase cells) generally had alignment parallel to the adjacent leaf base, as has been seen for cellulose. The aligned features or cues potentially correlating with a given array were: (1) orientation and length of the previous anticlinal cross-wall, (2) alignment of microtubules in adjacent cells, and (3) direction of inferred stretch, parallel to the nearby leaf bases. All three features were found to agree with the microtubule alignment in 17 of 34 cells with ordered arrays. At least two features agreed in 33 of the 34 cases. All 34 cells with ordered arrays had at least one feature parallel to the array. Random association between microtubule orientation and these features would lead to such correlations less than 0.01% of the time. Thirty percent of the interphase cells showed no obvious order. Most of these cells were located in the central linear corridor region of the apex. The unordered cells were more likely than the ordered cells to have more than one orientation specified by the potential cues; i.e., no single orientation parallel to all of the cues existed. This indicates that uniformity of the orientation cues may be as important as their direction.     

Apical Growth Cessation and Shoot Tip Abscission in Salix

Time course of apical shoot growth and shoot tip abortion in northern ecotypes (lat. 69°39′N, long. 18°37′E) of Salix pentandra and S. caprea have been investigated. In trees more than 15 years old growing under natural climatic conditions apical growth cessation and shoot tip abortion normally occurred in June-July when the day length still was 24 h. Application of GA₃, in spring to the apex effectively delayed growth cessation and shoot tip abortion. Application of kinetin was without effect. First-year seedlings of both species grew continuously at temperatue of 9 to 24°C in 24 h photoperiod. Short days induced apical growth cessation, but two to four (S. pentandra) or three to five (S. caprea) weeks of 12 h photoperiod were required to stop the elongation growth. The results indicated that the critical photoperiod for apical growth cessation in the used ecotype of S. pentandra was 16 to 18 h at 18°C. Short days had a minor effect only on the formation of apical leaf primordia in small seedlings. Development of axillary buds and radial growth were stimulated by short days when compared with long days. Small seedlings of both species (3 to 8 cm high at the start) formed terminal buds in short days, but in large seedlings (more than about 15 cm high) apical growth cessation was accompanied by shoot tip abortion. Abscisic acid applied to the apex or through a leaf did not induce growth cessation in S. pentandra seedlings grown in continuous light. The growth retardants CCC, B-9 and Phosphon D reduced growth rate under continuous light and induced shoot tip abortion in some plants. The effect of CCC was counteracted by GA₃. Apical growth cessation in short days was significantly delayed by a single GA₁ application.     

Plant cell growth responds to external forces and the response requires intact microtubules

Microfibril deposition in most plant cells is influenced by cortical microtubules. Thus, cortical microtubules are templates that provide spatial information to the cell wall. How cortical microtubules acquire their spatial information and are positioned is unknown. There are indications that plant cells respond to mechanical stresses by using microtubules as sensing elements. Regenerating protoplasts from tobacco (Nicotiana tabacum) were used to determine whether cells can be induced to expand in a preferential direction in response to an externally applied unidirectional force. Additionally, an anti-microtubule herbicide was used to investigate the role of microtubules in the response to this force. Protoplasts were embedded in agarose, briefly centrifuged at 28 to 34g, and either cultured or immediately prepared for immunolocalization of their microtubules. The microtubules within many centrifuged protoplasts were found to be oriented parallel to the centrifugal force vector. Most protoplasts elongated with a preferential axis that was oriented 60 to 90 degrees to the applied force vector. Protoplasts treated transiently with the reversible microtubule-disrupting agent amiprophos-methyl (applied before and during centrifugation) elongated but without a preferential growth axis. These results indicate that brief biophysical forces may influence the alignment of cortical microtubules and that microtubules themselves act as biophysical responding elements.