Thierry Desprez

Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana

In all land plants, cellulose is synthesized from hexameric plasma membrane complexes. Indirect evidence suggests that in vascular plants the complexes involved in primary wall synthesis contain three distinct cellulose synthase catalytic subunits (CESAs). In this study, we show that CESA3 and CESA6 fused to GFP are expressed in the same cells and at the same time in the hypocotyl of etiolated seedlings and migrate with comparable velocities along linear trajectories at the cell surface. We also show that CESA3 and CESA6 can be coimmunoprecipitated from detergent-solubilized extracts, their protein levels decrease in mutants for either CESA3, CESA6, or CESA1 and CESA3, CESA6 and also CESA1 can physically interact in vivo as shown by bimolecular fluorescence complementation. We also demonstrate that CESA6-related CESA5 and CESA2 are partially, but not completely, redundant with CESA6 and most likely compete with CESA6 for the same position in the cellulose synthesis complex. Using promoter-β-glucuronidase fusions we show that CESA5, CESA6, and CESA2 have distinct overlapping expression patterns in hypocotyl and root corresponding to different stages of cellular development. Together, these data provide evidence for the existence of binding sites for three distinct CESA subunits in primary wall cellulose synthase complexes, with two positions being invariably occupied by CESA1 and CESA3, whereas at least three isoforms compete for the third position. Participation of the latter three isoforms might fine-tune the CESA complexes for the deposition of microfibrils at distinct cellular growth stages.

PROCUSTE1 Encodes a Cellulose Synthase Required for Normal Cell Elongation Specifically in Roots and Dark-Grown Hypocotyls of Arabidopsis

Mutants at the PROCUSTE1 (PRC1) locus show decreased cell elongation, specifically in roots and dark-grown hypocotyls. Cell elongation defects are correlated with a cellulose deficiency and the presence of gapped walls. Map-based cloning of PRC1 reveals that it encodes a member (CesA6) of the cellulose synthase catalytic subunit family, of which at least nine other members exist in Arabidopsis. Mutations in another family member, RSW1 (CesA1), cause similar cell wall defects in all cell types, including those in hypocotyls and roots, suggesting that cellulose synthesis in these organs requires the coordinated expression of at least two distinct cellulose synthase isoforms.

Resistance against Herbicide Isoxaben and Cellulose Deficiency Caused by Distinct Mutations in Same Cellulose Synthase Isoform CESA6

Isoxaben is a pre-emergence herbicide that inhibits cellulose biosynthesis in higher plants. Two loci identified by isoxaben-resistant mutants (ixr1-1,ixr1-2, and ixr2-1) in Arabidopsis have been reported previously. IXR1 was recently shown to encode the cellulose synthase catalytic subunit CESA3 (W.-R. Scheible, R. Eshed, T. Richmond, D. Delmer, and C. Somerville [2001] Proc Natl Acad Sci USA 98: 10079–10084). Here, we report on the cloning of IXR2, and show that it encodes another cellulose synthase isoform, CESA6. ixr2-1 carries a mutation substituting an amino acid close to the C terminus of CESA6 that is highly conserved among CESA family members. Transformation of wild-type plants with the mutated gene and not with the wild-type gene conferred increased resistance against the herbicide. The simplest interpretation for the existence of these two isoxaben-resistant loci is that CESA3 and CESA6 have redundant functions. However, loss of functionprocuste1 alleles of CESA6 were previously shown to have a strong growth defect and reduced cellulose content in roots and dark-grown hypocotyls. This indicates that in these mutants, the presence of CESA3 does not compensate for the absence of CESA6 in roots and dark-grown hypocotyls, which argues against redundant functions for CESA3 and CESA6. Together, these observations are compatible with a model in which CESA6 and CESA3 are active as a protein complex.

KOBITO1 Encodes a Novel Plasma Membrane Protein Necessary for Normal Synthesis of Cellulose during Cell Expansion in Arabidopsis

The cell wall is the major limiting factor for plant growth. Wall extension is thought to result from the loosening of its structure. However, it is not known how this is coordinated with wall synthesis. We have identified two novel allelic cellulose-deficient dwarf mutants, kobito1-1 and kobito1-2 (kob1-1 and kob1-2). The cellulose deficiency was confirmed by the direct observation of microfibrils in most recent wall layers of elongating root cells. In contrast to the wild type, which showed transversely oriented parallel microfibrils, kob1 microfibrils were randomized and occluded by a layer of pectic material. No such changes were observed in another dwarf mutant, pom1, suggesting that the cellulose defect in kob1 is not an indirect result of the reduced cell elongation. Interestingly, in the meristematic zone of kob1 roots, microfibrils appeared unaltered compared with the wild type, suggesting a role for KOB1 preferentially in rapidly elongating cells. KOB1 was cloned and encodes a novel, highly conserved, plant-specific protein that is plasma membrane bound, as shown with a green fluorescent protein–KOB1 fusion protein. KOB1 mRNA was present in all organs investigated, and its overexpression did not cause visible phenotypic changes. KOB1 may be part of the cellulose synthesis machinery in elongating cells, or it may play a role in the coordination between cell elongation and cellulose synthesis.

Differential Regulation of Cellulose Orientation at the Inner and Outer Face of Epidermal Cells in the Arabidopsis Hypocotyl

Whereas microtubules and cellulose synthase trajectories are transversely oriented on the outer epidermal surface of Arabidopsis hypocotyl cells only for part of the growth cycle, microtubules and microfibrils at the inner surface remain transverse throughout growth. The inner face of the epidermis is thus established as a regulator of growth anisotropy in the hypocotyl. It is generally believed that cell elongation is regulated by cortical microtubules, which guide the movement of cellulose synthase complexes as they secrete cellulose microfibrils into the periplasmic space. Transversely oriented microtubules are predicted to direct the deposition of a parallel array of microfibrils, thus generating a mechanically anisotropic cell wall that will favor elongation and prevent radial swelling. Thus far, support for this model has been most convincingly demonstrated in filamentous algae. We found that in etiolated Arabidopsis thaliana hypocotyls, microtubules and cellulose synthase trajectories are transversely oriented on the outer surface of the epidermis for only a short period during growth and that anisotropic growth continues after this transverse organization is lost. Our data support previous findings that the outer epidermal wall is polylamellate in structure, with little or no anisotropy. By contrast, we observed perfectly transverse microtubules and microfibrils at the inner face of the epidermis during all stages of cell expansion. Experimental perturbation of cortical microtubule organization preferentially at the inner face led to increased radial swelling. Our study highlights the previously underestimated complexity of cortical microtubule organization in the shoot epidermis and underscores a role for the inner tissues in the regulation of growth anisotropy.

The Cellulase KORRIGAN Is Part of the Cellulose Synthase Complex

An endo-1,4-b-D-glucanase is part of the primary cell wall cellulose synthase complex (CSC) in the plasma membrane and plays a role in the trafficking of the CSC. Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by a large relative molecular weight cellulose synthase complex (CSC), which comprises at least three distinct cellulose synthases. Cellulose synthesis in plants or bacteria also requires the activity of an endo-1,4-β-d-glucanase, the exact function of which in the synthesis process is not known. Here, we show, to our knowledge for the first time, that a leaky mutation in the Arabidopsis (Arabidopsis thaliana) membrane-bound endo-1,4-β-d-glucanase KORRIGAN1 (KOR1) not only caused reduced CSC movement in the plasma membrane but also a reduced cellulose synthesis inhibitor-induced accumulation of CSCs in intracellular compartments. This suggests a role for KOR1 both in the synthesis of cellulose microfibrils and in the intracellular trafficking of CSCs. Next, we used a multidisciplinary approach, including live cell imaging, gel filtration chromatography analysis, split ubiquitin assays in yeast (Saccharomyces cerevisiae NMY51), and bimolecular fluorescence complementation, to show that, in contrast to previous observations, KOR1 is an integral part of the primary cell wall CSC in the plasma membrane.

Interactions between membrane-bound cellulose synthases involved in the synthesis of the secondary cell wall

MINT‐6951199: CESA8 (uniprotkb:Q8LPK5) physically interacts (MI:0218) with CESA8 (uniprotkb:Q8LPK5) by bimolecular fluorescence complementation (MI:0809)

Spatio-temporal analysis of cellulose synthesis during cell plate formation in Arabidopsis

During cytokinesis a new crosswall is rapidly laid down. This process involves the formation at the cell equator of a tubulo-vesicular membrane network (TVN). This TVN evolves into a tubular network (TN) and a planar fenestrated sheet, which extends at its periphery before fusing to the mother cell wall. The role of cell wall polymers in cell plate assembly is poorly understood. We used specific stains and GFP-labelled cellulose synthases (CESAs) to show that cellulose, as well as three distinct CESAs, accumulated in the cell plate already at the TVN stage. This early presence suggests that cellulose is extruded into the tubular membrane structures of the TVN. Co-localisation studies using GFP-CESAs suggest the delivery of cellulose synthase complexes (CSCs) to the cell plate via phragmoplast-associated vesicles. In the more mature TN part of the cell plate, we observed delivery of GFP-CESA from doughnut-shaped organelles, presumably Golgi bodies. During the conversion of the TN into a planar fenestrated sheet, the GFP-CESA density diminished, whereas GFP-CESA levels remained high in the TVN zone at the periphery of the expanding cell plate. We observed retrieval of GFP-CESA in clathrin-containing structures from the central zone of the cell plate and from the plasma membrane of the mother cell, which may contribute to the recycling of CESAs to the peripheral growth zone of the cell plate. These observations, together with mutant phenotypes of cellulose-deficient mutants and pharmacological experiments, suggest a key role for cellulose synthesis already at early stages of cell plate assembly.

Transformation of Saccharomyces cerevisiae with a cDNA encoding a sterol C-methyltransferase from Arabidopsis thaliana results in the synthesis of 24-ethyl sterols.

Using an EST‐cDNA probe, a full‐length cDNA (411) sequence of 1411 bp was isolated from A. thaliana. This sequence contained features typical of methyltransferases in general and in particular showed 38% identity with ERG6, a S. cerevisiae gene which encodes the zymosterol‐C‐24‐methyltransferase. A yeast vector containing this ORF (4118‐pYeDP60) was used to transform a wild type S. cerevisiae which accumulates predominantly ergosterol, a 24‐methyl sterol as well as a mutant erg6 null mutant accumulating principally zymosterol, a sterol non‐alkylated at C‐24. In both cases, several 24‐ethyl‐ and 24‐ethylidene sterols were synthetized indicating that the 4118 cDNA encodes a plant sterol C‐methyltransferase able to perform two sequential methylations of the sterol side chain.

A methacrylate embedding procedure developed for immunolocalization on plant tissues is also compatible with in situ hybridization.

A recently described method that uses methacrylate embedding of aldehyde fixed plant tissues allows the immunolabelling of a range of antigens (Baskin et al. 1992). We have tested whether the same embedding procedure is also compatible with in situ hybridization. For this purpose we have used 2‐ 5 μm sections of methacrylate embedded plantlets of Arabidopsis thaliana. After removal of the resin the sections were prepared for in situ hybridization following standard procedures. Three different digoxygenin (dig)‐labelled probes were used, recognizing RNAs coding for the chlorophyll a/b binding protein cab‐140, the β‐tubulin tub5 and meri a member of the meri‐5 family. Each of the probes shows the labelling pattern expected from the literature. Moreover, the method allows a good structural preservation of very fragile tissues, in contrast to paraffin embedding. We conclude that methacrylate embedding, allowing both immunolabelling and in situ hybridization with high resolution and structural preservation, offers a high potential for the functional analysis of genes and proteins in plant development. This is especially true for Arabidopsis thaliana, a widely used model species where it seems to be the method of choice.