A 34K SNP genotyping array for Populus trichocarpa: design, application to the study of natural populations and transferability to other Populus species.

Genetic mapping of quantitative traits requires genotypic data for large numbers of markers in many individuals. For such studies, the use of large single nucleotide polymorphism (SNP) genotyping arrays still offers the most cost-effective solution. Herein we report on the design and performance of a SNP genotyping array for Populus trichocarpa (black cottonwood). This genotyping array was designed with SNPs pre-ascertained in 34 wild accessions covering most of the species latitudinal range. We adopted a candidate gene approach to the array design that resulted in the selection of 34 131 SNPs, the majority of which are located in, or within 2 kb of, 3543 candidate genes. A subset of the SNPs on the array (539) was selected based on patterns of variation among the SNP discovery accessions. We show that more than 95% of the loci produce high quality genotypes and that the genotyping error rate for these is likely below 2%. We demonstrate that even among small numbers of samples (n = 10) from local populations over 84% of loci are polymorphic. We also tested the applicability of the array to other species in the genus and found that the number of polymorphic loci decreases rapidly with genetic distance, with the largest numbers detected in other species in section Tacamahaca. Finally, we provide evidence for the utility of the array to address evolutionary questions such as intraspecific studies of genetic differentiation, species assignment and the detection of natural hybrids.

The characean internodal cell as a model system for studying wound healing.

This work describes the characean internodal cell as a model system for the study of wound healing and compares wounds induced by certain chemicals and UV irradiation with wounds occurring in the natural environment. We review the existing literature and define three types of wound response: (1) cortical window formation characterised by disassembly of microtubules, transient inhibition of actin-dependent cytoplasmic streaming and chloroplast detachment, (2) fibrillar wound walls characterised by exocytosis of vesicles carrying wall polysaccharides and membrane-bound cellulose synthase complexes coupled with endocytosis of surplus membrane and (3) amorphous, callose- and membrane-containing wound walls characterised by exocytosis of vesicles and endoplasmic reticulum cisternae in the absence of membrane recycling. We hypothesize that these three wound responses reflect the extent of damage, probably Ca(2+) influx, and that the secretion of Ca(2+) -loaded endoplasmic reticulum cisternae is an emergency reaction in case of severe Ca(2+) load. Microtubules are not required for wound healing but their disassembly could have a signalling function. Transient reorganisation of the actin cytoskeleton into a meshwork of randomly oriented filaments is required for the migration of wound wall forming organelles, just as occurs in tip-growing plant cells. New data presented in this study show that during the deposition of an amorphous wound wall numerous actin rings are present, which may indicate specific ion fluxes and/or a storage form for actin. In addition, we present new evidence for the exocytosis of FM1-43-stained organelles, putative endosomes, required for plasma membrane repair during wound healing. Finally, we show that quickly growing fibrillar wound walls, even when deposited in the absence of microtubules, have a highly ordered helical structure of consistent handedness comprised of cellulose microfibrils.

Investigating cytoskeletal function in chloroplast protrusion formation in the arctic-alpine plant Oxyria digyna.

Arctic and alpine plants like Oxyria digyna have to face enhanced environmental stress. This study compared leaves from Oxyria digyna collected in the Arctic at Svalbard (78 degrees N) and in the Austrian Alps (47 degrees N) at cellular, subcellular, and ultrastructural levels. Oxyria digyna plants collected in Svalbard had significantly thicker leaves than the samples collected in the Austrian Alps. This difference was generated by increased thickness of the palisade and spongy mesophyll layers in the arctic plants, while epidermal cells had no significant size differences between the two habitats. A characteristic feature of arctic, alpine, and cultivated samples was the occurrence of broad stroma-filled chloroplast protrusions, 2 - 5 microm broad and up to 5 microm long. Chloroplast protrusions were in close spatial contact with other organelles including mitochondria and microbodies. Mitochondria were also present in invaginations of the chloroplasts. A dense network of cortical microtubules found in the mesophyll cells suggested a potential role for microtubules in the formation and function of chloroplast protrusions. No direct interactions between microtubules and chloroplasts, however, were observed and disruption of the microtubule arrays with the anti-microtubule agent oryzalin at 5 - 10 microM did not alter the appearance or dynamics of chloroplast protrusions. These observations suggest that, in contrast to studies on stromule formation in Nicotiana, microtubules are not involved in the formation and morphology of chloroplast protrusions in Oxyria digyna. The actin microfilament-disrupting drug latrunculin B (5 - 10 microM for 2 h) arrested cytoplasmic streaming and altered the cytoplasmic integrity of mesophyll cells. However, at the ultrastructural level, stroma-containing, thylakoid-free areas were still visible, mostly at the concave sides of the chloroplasts. As chloroplast protrusions were frequently found to be mitochondria-associated in Oxyria digyna, a role in metabolite exchange is possible, which may contribute to an adaptation to alpine and arctic conditions.

Wall architecture in the cellulose-deficient rsw1 mutant of Arabidopsis thaliana: microfibrils but not microtubules lose their transverse alignment before microfibrils become unrecognizable in the mitotic and elongation zones of roots.

The rsw1 mutant of Arabidopsis thaliana has a single amino acid substitution in a putative glycosyl transferase that causes a temperature-dependent reduction in cellulose production. We used recently described methods to examine root growth by surface marker particles, cell wall structure by field emission scanning electron microscopy and microtubule alignment by immunofluorescence after the mutant is transferred to its restrictive temperature. We find that raising the temperature quickly accelerates root elongation in both wild type and mutant, presumably as a result of general metabolic stimulation, but that in the mutant, the rate declines within 7-8 h and elongation almost ceases after 24 h. Radial swelling begins at about 6 h in the mutant and root diameter continues to increase until about 24 h. The normal transverse alignment of microfibrils is severely impaired in the mutant after 8 h, and chemical inhibition of cellulose synthesis by 2,6-dichlorobenzonitrile causes a similar loss of orientation. After 24 h, microfibrils are not clearly visible in the walls of cells that would have been in the mitotic and early-elongation zone of wild-type roots. Changes in older cells are less marked; loss of transverse microfibril orientation occurs without disruption to the transverse orientation of cortical microtubules. The wild type shows none of the changes except for acceleration of elongation, which in its case is sustained. We conclude that microfibril alignment requires the normal functioning of RSW1 and that, in view of the effects of dichlorobenzonitrile, there may be a more general linkage between the rate of cellulose production and its proper alignment.

MOR1 is essential for organizing cortical microtubules in plants.

Microtubules orchestrate cell division and morphogenesis, but how they disassemble and reappear at different subcellular locations is unknown. Microtubule organizing centres are thought to have an important role, but in higher plants microtubules assemble in ordered configurations even though microtubule organizing centres are inconspicuous or absent. Plant cells generate highly organized microtubule arrays that coordinate mitosis, cytokinesis and expansion. Inhibiting microtubule assembly prevents chromosome separation, blocks cell division and impairs growth polarity. Microtubules are essential for the formation of cell walls, through an array of plasma-membrane-associated cortical microtubules whose control mechanisms are unknown. Using a genetic strategy to identify microtubule organizing factors in Arabidopsis thaliana, we isolated temperature-sensitive mutant alleles of the MICROTUBULE ORGANIZATION 1 (MOR1) gene. Here we show that MOR1 is the plant version of an ancient family of microtubule-associated proteins. Point mutations that substitute single amino-acid residues in an amino-terminal HEAT repeat impart reversible temperature-dependent cortical microtubule disruption, showing that MOR1 is essential for cortical microtubule organization.

New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis.

This article explores root epidermal cell elongation and its dependence on two structural elements of cells, cortical microtubules and cellulose microfibrils. The recent identification of Arabidopsis morphology mutants with putative cell wall or cytoskeletal defects demands a procedure for examining and comparing wall architecture and microtubule organization patterns in this species. We developed methods to examine cellulose microfibrils by field emission scanning electron microscopy and microtubules by immunofluorescence in essentially intact roots. We were able to compare cellulose microfibril and microtubule alignment patterns at equivalent stages of cell expansion. Field emission scanning electron microscopy revealed that Arabidopsis root epidermal cells have typical dicot primary cell wall structure with prominent transverse cellulose microfibrils embedded in pectic substances. Our analysis showed that microtubules and microfibrils have similar orientation only during the initial phase of elongation growth. Microtubule patterns deviate from a predominantly transverse orientation while cells are still expanding, whereas cellulose microfibrils remain transverse until well after expansion finishes. We also observed microtubule-microfibril alignment discord before cells enter their elongation phase. This study and the new technology it presents provide a starting point for further investigations on the physical properties of cell walls and their mechanisms of assembly.

The cytoskeleton and growth polarity.

We are currently witnessing the discovery of many novel proteins that are associated with cytoskeletal activity. Integrated analyses of growth, cytoskeletal and cell-wall patterns are yielding surprising results, which demand reflection on the current model for wall construction. Meanwhile, research on actin filament and microtubule activity during gravitropic bending and trichome morphogenesis is stimulating new ideas about the establishment and maintenance of polarity.

Gibberellin-induced changes in growth anisotropy precede gibberellin-dependent changes in cortical microtubule orientation in developing epidermal cells of barley leaves. Kinematic and cytological studies on a gibberellin-responsive dwarf mutant, M489.

We conducted kinematic and cytological studies on "between vein" epidermal cells of the gibberellin (GA)-deficient M489 dwarf mutant of barley (Hordeum vulgare L. Himalaya). GAs affect radial and axial components of cell expansion and cortical microtubule orientation. Adaxial cells in particular expand radially after leaving the elongation zone (EZ), probably as part of leaf unrolling. Exogenous gibberellic acid corrects the mutant's short, wide blades, short EZ, and slow elongation rate. Cell production rates increase more on the adaxial than on the abaxial surface. Cells spend equal periods of time elongating in dwarf and tall plants, but relative elemental growth rates start to decline sooner in the dwarf. GA increased the rate at which longitudinal wall area increased because the increased axial growth more than compensated for reduced radial growth. In dwarf leaves, increased radial expansion was detected in basal parts of the EZ before cortical microtubules lost transverse orientation in the distal elongation zone. We conclude that loss of microtubule orientation is not required for low GA levels to reduce growth anisotropy.

The C-terminal dilysine motif confers endoplasmic reticulum localization to type I membrane proteins in plants.

The tomato Cf-9 disease resistance gene encodes a type I membrane protein carrying a cytosolic dilysine motif. In mammals and yeast, this motif promotes the retrieval of type I membrane proteins from the Golgi apparatus to the endoplasmic reticulum (ER). To test whether the C-terminal KKXX signal of Cf-9 is functional as a retrieval motif and to investigate its role in plants, green fluorescent protein (GFP) was fused to the transmembrane domain of Cf-9 and expressed in yeast, Arabidopsis, and tobacco cells. The fusion protein was targeted to the ER in each of these expression systems, and mutation of the KKXX motif to NNXX led to secretion of the fusion protein. In yeast, the mutant protein reached the vacuole, but plants secreted it as a soluble protein after proteolytic removal of the transmembrane domain. Triple hemagglutinin (HA)-tagged full-length Cf-9 was also targeted to the ER in tobacco cells, and cleavage was also observed for the NNXX mutant protein, suggesting an endoprotease recognition site located within the Cf-9 lumenal sequence common to both the GFP- and the HA-tagged fusions. Our results indicate that the KKXX motif confers ER localization in plants as well as mammals and yeast and that Cf-9 is a resident protein of the ER.

Growth conditions modulate root-wave phenotypes in Arabidopsis.

The cause for the wave-like growth of Arabidopsis thaliana roots on semi-solid medium remains unclear. Researchers have hypothesized a gravity-induced touch-response, circumnutation, or combinations thereof act as the major stimuli. Our data demonstrate that the gaseous environment within the Petri dish can override gravitational effects. Furthermore, we show that medium ion concentrations and gelling polymers modify the wave response. Although the mechanisms driving our wide-ranging wildtype phenotypes are currently unknown, these results are of immediate significance for interpreting genetic and physiological modifications of environmentally and genetically induced characteristics.

Distribution and dynamics of the cytoskeleton in graviresponding protonemata and rhizoids of characean algae: exclusion of microtubules and a convergence of actin filaments in the apex suggest an actin-mediated gravitropism.

The organization of the microtubule (MT) and actin microfilament (MF) cytoskeleton of tip-growing rhizoids and protonemata of characean green algae was examined by confocal laser scanning microscopy. This analysis included microinjection of fluorescent tubulin and phallotoxins into living cells, as well as immunofluorescence labeling of fixed material and fluorescent phallotoxin labeling of unfixed material. Although the morphologically very similar positively gravitropic (downward growing) rhizoids and negatively gravitropic (upward growing) protonemata show opposite gravitropic responses, no differences were detected in the extensive three-dimensional distribution of actin MFs and MTs in both cell types. Tubulin microinjection revealed that in contrast to internodal cells, fluorescent tubulin incorporated very slowly into the MT arrays of rhizoids, suggesting that MT dynamics are very different in tip-growing and diffusely expanding cells. Microtubules assembled from multiple sites at the plasma membrane in the basal zone, and a dense subapical array emerged from a diffuse nucleation centre on the basal side of the nuclear envelope. Immunofluorescence confirmed these distribution patterns but revealed more extensive MT arrays. In the basal zone, short branching clusters of MTs form two cortical hemicylinders. Subapical, axially oriented MTs are distributed in equal density throughout the peripheral and inner cytoplasm and are closely associated with subapical organelles. Microtubules, however, are completely absent from the apical zones of rhizoids and protonemata. Actin MFs were found in all zones of rhizoids and protonemata including the apex. Two files of axially oriented bundles of subcortical actin MFs and ring-like actin structures in the streaming endoplasm of rhizoids were detected in the basal zones by microinjection or rhodamine-phalloidin labeling. The subapical zone contains a dense array of mainly axially oriented actin MFs that co-distribute with the subapical MT array. In the apex, actin MFs form thicker bundles that converge into a remarkably distinct actin patch in the apical dome, whose position coincides with the position of the endoplasmic reticulum aggregate in the centre of the Spitzenkörper. Actin MFs radiate from the actin patch towards the apical membrane. Together with results from previous inhibitor studies (Braun and Sievers, 1994, Eur J Cell Biol 63: 289-298), these results suggest that MTs have a stabilizing function in maintaining the polar cytoplasmic and cytoskeletal organization. The motile processes, however, are mediated by actin. In particular, the actin cytoskeleton appears to be involved in the structural and functional organization of the Spitzenkörper and thus is responsible for controlling cell shape and growth direction. Despite the similar structural arrangements of the actin cytoskeleton, major differences in the function of actin MFs have been observed in rhizoids and protonemata. Since actin MFs are more directly involved in the gravitropic response of protonemata than of rhizoids, the opposite gravitropsim in the two cell types seems to be based mainly on different properties and activities of the actin cytoskeleton.

Freeze shattering: a simple and effective method for permeabilizing higher plant cell walls.

This article describes a practical technique for permeabilization of higher plant cell walls, which is usually one of the first steps required for immunolocalization of cellular components (and other cytological methods) in plant cell studies. Our strategy involves shattering the walls of cells while the tissues are frozen in liquid nitrogen. It replaces the use of wall degrading enzymes or the need to employ laborious sectioning or other mechanical means for providing access of probes to cells. Freeze-shattering retains the integrity of whole tissues and cells surprisingly well and thus is especially useful when used in conjunction with confocal laser scanning microscopy for recording the three-dimensional arrangement of cytoskeletal elements in relation to cell shape. In this article, we demonstrate the effectiveness of this technique for anti-tubulin and anti-actin immunofluorescence and for rhodamine phalloidin labelling of the cytoskeleton in various higher plant tissues including onion root tip and bulb scale epidermis, Tradescantia stamen hairs and Arabidopsis leaf epidermis and mesophyll cells.

Actin-based vesicle dynamics and exocytosis during wound wall formation in characean internodal cells.

Characean internodal cells readily form wound walls upon local membrane damage. In the present study we documented the dynamics of vesicles involved in wound wall secretion and compared them with actin organization in equivalent cells using immunofluorescence. Single exocytotic events (spreading of vesicle contents) could be visualized using image enhancement by video microscopy. In control unwounded cells vesicles moved unidirectionally along parallel actin bundles and rarely contacted the plasma membrane. The wound response started with (1) local inhibition of active cytoplasmic streaming (unidirectional movements) due to inactivation, depolymerization, or mechanical displacement of the subcortical actin bundles. Accordingly, vesicles performed only oscillating motions and moved slowly with the same velocity and direction as passive endoplasmic flow. (2) Several minutes after wounding, vesicles started to perform random saltatory movements with frequently changing velocities, punctuated by oscillating motion and periods of immobility (docking) at the plasma membrane. Vesicle trajectories correlated with a fine-meshed actin network at the wound site. (3) Several hours after wounding, vesicles moved again unidirectionally along regenerated subcortical actin bundles. Spreading of vesicles (vesicle contents) was observed during wound wall formation, i.e., during the period of saltatory movements when vesicles had access to the plasma membrane. Dependent on the type of wound wall being secreted, three variants could be distinguished: (1) slow and continuous spreading over a time period of several seconds up to 30 min near the plasma membrane, (2) fast spreading within 80 ms inside an already formed wound wall, and/or (3) fast spreading at the plasma membrane. We conclude from our study that wounding-induced changes in vesicle dynamics are due to transient reorganization of the actin cytoskeleton from parallel bundles to a fine-meshed network. Furthermore, our results indicate that spreading of vesicle contents varies considerably with time and may be delayed by vesicle docking and/or discharge.

Elongation factor 1 alpha is a component of the subcortical actin bundles of characean algae.

Antibodies to elongation factor 1 alpha (EF1 alpha), a known 50 kDa actin-bundling protein in Dictyostelium, identified a protein in a whole cell extract of the characean alga Nitella pseudoflabellata that had an apparent molecular weight of 51 kDa. Indirect immunofluorescence microscopy revealed labelling by the EF1 alpha antibodies of the subcortical actin bundles, even after the motile organelles of the endoplasm were removed by perfusion with ATP-containing solutions.

Microtubule organization differs between acid and alkaline bands in internodal cells of Chara but bands can develop in the absence of microtubules.

We have studied the relationship between pH banding and the organization of cortical microtubules in the alga Chara corallina Klein ex Willd. Microtubules were visualized by immunofluorescence and also by imunogold-silver enhancement to allow immediate comparison of microtubule arrangement with visible structural cell features. In cells that are nearing growth completion, microtubule number and alignment change between acidic and alkaline bands over a distance of a few micrometres. Thus, it appears that the still unknown mechanisms for microtubule organization respond to the localized differences in membrane properties. Band formation was not prevented when microtubules were depolymerized with the herbicide oryzalin, demonstrating that microtubules are not necessary for pH bands to develop in these cells.

The effects of methylmercury on the cytoskeleton of murine embryonal carcinoma cells.

Immunofluorescence staining with antibodies to tubulin and vimentin and staining with phalloidin have been used to examine the effects of methylmercury on the cytoskeleton of embryonal carcinoma cells in culture. Exposure of embryonal carcinoma cells to methylmercury (0.01 to 10 microns) resulted in concentration- and time-dependent disassembly of microtubules in interphase and mitotic cells. These effects were reversible when cultures were washed free of methylmercury. Spindle microtubules were more sensitive than those of interphase cells. Spindle damage resulted in an accumulation of cells in prometaphase/metaphase, which correlated with a temporary delay in the resumption of normal proliferation rate upon removal of methylmercury. Of the interphase cytoskeletal components, microtubules were the first affected by methylmercury. Vimentin intermediate filaments appeared relatively insensitive to methylmercury, but showed a reorganization secondary to the microtubule disassembly. Actin microfilaments appeared unchanged in cells showing complete absence of microtubules. Our results 1) support previous reports suggesting that microtubules are a primary target of methylmercury, 2) document a differential sensitivity of mitotic and interphase microtubule systems and 3) demonstrate the relative insensitivities of other cytoskeletal components.

Effects of methylmercury on retinoic acid-induced neuroectodermal derivatives of embryonal carcinoma cells.

Immunofluorescence staining with antibodies to tubulin, neurofilaments and glial filaments was used to study the effects of methylmercury on the differentiation of retinoic acid-induced embryonal carcinoma cells into neurons and astroglia and on the cytoskeleton of these neuroectodermal derivatives. Methylmercury did not prevent undifferentiated embryonal carcinoma cells from developing into neurons and glia. Treatment of committed embryonal carcinoma cells with methylmercury doses exceeding 1 microM resulted in the formation of neurons with abnormal morphologies. In differentiated cultures, microtubules were the first cytoskeletal element to be affected. Their disassembly was time- and concentration-dependent. Microtubules in glial cells and in neuronal perikarya were more sensitive than those in neuronal processes. Neurofilaments and glial filaments appeared relatively insensitive to methylmercury treatment but showed reorganization after complete disassembly of the microtubules. The data demonstrate 1) the sensitivity of microtubules of both neurons and glia to methylmercury-induced depolymerization, and 2) the heterogeneous response of neuronal microtubules to methylmercury, presumably reflecting posttranslational modifications of different subpopulations of microtubules in the perikarya and neurite.