Cortical microtubules mark the mucilage secretion domain of the plasma membrane in Arabidopsis seed coat cells

During their differentiation Arabidopsis thaliana seed coat cells undergo a brief but intense period of secretory activity that leads to dramatic morphological changes. Pectic mucilage is secreted to one domain of the plasma membrane and accumulates under the primary cell wall in a ring-shaped moat around an anticlinal cytoplasmic column. Using cryofixation/transmission electron microscopy and immunofluorescence, the cytoskeletal architecture of seed coat cells was explored, with emphasis on its organization, function and the large amount of pectin secretion at 7 days post-anthesis. The specific domain of the plasma membrane where mucilage secretion is targeted was lined by abundant cortical microtubules while the rest of the cortical cytoplasm contained few microtubules. Actin microfilaments, in contrast, were evenly distributed around the cell. Disruption of the microtubules in the temperature-sensitive mor1-1 mutant affected the eventual release of mucilage from mature seeds but did not appear to alter the targeted secretion of vesicles to the mucilage pocket, the shape of seed coat cells or their secondary cell wall deposition. The concentration of cortical microtubules at the site of high vesicle secretion in the seed coat may utilize the same mechanisms required for the formation of preprophase bands or the bands of microtubules associated with spiral secondary cell wall thickening during protoxylem development.     

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     

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.     

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.     

Ethylene modulates root-wave responses in Arabidopsis

When stimulated to bend downward by being held at 45 degrees off vertical but unable to penetrate into agar-based media, Arabidopsis roots develop waving and looping growth patterns. Here, we demonstrate that ethylene modulates these responses. We determined that agar-containing plates sealed with low-porosity film generate abiotic ethylene concentrations of 0.1 to 0.3 microL L(-1), whereas in plates wrapped with porous tape, ethylene remains at trace levels. We demonstrate that exogenous ethylene at concentrations as low as a few nanoliters per liter modulates root waving, root growth direction, and looping but through partly different mechanisms. Nutrients and Suc modify the effects of ethylene on root waving. Thus, ethylene had little effect on temporal wave frequency when nutrients were omitted but reduced it significantly on nutrient-supplemented agar. Suc masked the ethylene response. Ethylene consistently suppressed the normal tendency for roots of Landsberg erecta to skew to the right as they grow against hard-agar surfaces and also generated righthanded petiole twisting. Furthermore, ethylene suppressed root looping, a gravity-dependent growth response that was enhanced by high nutrient and Suc availability. Our work demonstrates that cell file twisting is not essential for root waving or skewing to occur. Differential flank growth accounted for both the extreme root waving on zero-nutrient plates and for root skewing. Root twisting was nutrient-dependent and was thus strongly associated with the looping response. The possible role of auxin transport in these responses and the involvement of circadian rhythms are discussed.     

Cytoskeletal dynamics in living plant cells

Microtubules and microfilaments have been imaged in living plant cells and their dynamic changes recorded during division, growth and development. Carboxyfluorescein labeled brain tubulin has been injected into cells that are maintained in an active state in a culture chamber on the microscope stage. Subsequent imaging with the confocal microscope reveals microtubules in the preprophase band, the mitotic apparatus, the phragmoplast, and the cortical array. The structural changes of these microtubules have been observed during transitional stages. In addition, their dynamic features are demonstrated by depolymerization in elevated calcium, low temperature, and in the drug oryzalin, and by repolymerization when returned to normal conditions. Examination of living Tradescantia stamen hair cells, which have been injected with fluorescent phalloidin to label the actin microfilaments, reveals hitherto undisclosed aspects of the preparation of the division site and dynamics of the phragmoplast cytoskeleton. During prophase microfilaments occur throughout the cell cortex, with those in the region of the preprophase band becoming transversely aligned. At nuclear envelope breakdown, these specifically disassemble, leaving a circumferential zone from which microfilaments remain absent throughout division. During cytokinesis microfilaments arise within the phragmoplast, oriented parallel to the microtubules, but excluded from the zone where the MTs overlap and where cell plate vesicles aggregate. The phragmoplast microfilaments, in a manner similar to microtubules, shorten in length, expand in girth, and eventually disassemble when the cell plate is complete.     

Gene-based microsatellites for cassava (Manihot esculenta Crantz): prevalence, polymorphisms, and cross-taxa utility

Background  

Cassava (Manihot esculenta Crantz), a starchy root crop grown in tropical and subtropical climates, is the sixth most important crop in the world after wheat, rice, maize, potato and barley. The repertoire of simple sequence repeat (SSR) markers for cassava is limited and warrants a need for a larger number of polymorphic SSRs for germplasm characterization and breeding applications.