Cellulose, an abundant, crystalline polysaccharide, is central to plant morphogenesis and to many industries. Chemical and ultrastructural analyses together with map-based cloning indicate that the RSW1 locus of Arabidopsis encodes the catalytic subunit of cellulose synthase. The cloned gene complements the rsw1 mutant whose temperature-sensitive allele is changed in one amino acid. The mutant allele causes a specific reduction in cellulose synthesis, accumulation of noncrystalline beta-1,4-glucan, disassembly of cellulose synthase, and widespread morphological abnormalities. Microfibril crystallization may require proper assembly of the RSW1 gene product into synthase complexes whereas glucan biosynthesis per se does not.

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-␤-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. C ellulose microfibrils are synthesized from a multiprotein complex inserted into the plasma membrane. These ''rosette'' complexes consist of six globules, each of which contains multiple cellulose synthase catalytic subunits (CESAs). These complexes migrate in the plasma membrane along microtubules, propelled by the polymerization of the ␤-1,4-glucan chains (1).Plant CESA genes are members of multigene families. Arabidopsis has 10 CESA isoforms that, based on sequence comparison with other plant species, can be classified into six orthologous groups (2). Mutational analysis shows that these six groups of isoforms have nonredundant functions in cellulose synthesis. Mutants for three isoforms (CESA4, CESA7, and CESA8) show defects in cellulose synthesis specifically in secondary walls (3)(4)(5). Microarray data show that the mRNAs for the three genes are coregulated (6, 7). The three proteins are expressed in the same cell types during secondary cell wall deposition, and co-immunoprecipitation (IP) experiments show that all three proteins interact (3). Although the interactions remain to be validated in vivo, these data strongly suggest that at least in these cells the complexes contain three isoforms. Mutants for isoforms CESA1, CESA3, and CESA6 have cellulose defects in primary cell walls (8-11). The three genes are also coregulated at the mRNA level (12). It is not known, however, whether the corresponding proteins are ...

Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by plasma membrane-bound complexes containing cellulose synthase proteins (CESAs). Here, we establish a role for the cytoskeleton in intracellular trafficking of cellulose synthase complexes (CSCs) through the in vivo study of the green fluorescent protein (GFP)-CESA3 fusion protein in Arabidopsis thaliana hypocotyls. GFP-CESA3 localizes to the plasma membrane, Golgi apparatus, a compartment identified by the VHA-a1 marker, and, surprisingly, a novel microtubule-associated cellulose synthase compartment (MASC) whose formation and movement depend on the dynamic cortical microtubule array. Osmotic stress or treatment with the cellulose synthesis inhibitor CGA 325'615 induces internalization of CSCs in MASCs, mimicking the intracellular distribution of CSCs in nongrowing cells. Our results indicate that cellulose synthesis is coordinated with growth status and regulated in part through CSC internalization. We find that CSC insertion in the plasma membrane is regulated by pauses of the Golgi apparatus along cortical microtubules. Our data support a model in which cortical microtubules not only guide the trajectories of CSCs in the plasma membrane, but also regulate the insertion and internalization of CSCs, thus allowing dynamic remodeling of CSC secretion during cell expansion and differentiation.

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Tissue mechanics have been shown to play a key role in the regulation of morphogenesis in animals [1-4] and may have an equally important role in plants [5-9]. The aerial organs of plants are formed at the shoot apical meristem following a specific phyllotactic pattern [10]. The initiation of an organ from the meristem requires a highly localized irreversible surface deformation, which depends on the demethylesterification of cell wall pectins [11]. Here, we used atomic force microscopy (AFM) to investigate whether these chemical changes lead to changes in tissue mechanics. By mapping the viscoelasticity and elasticity in living meristems, we observed increases in tissue elasticity, correlated with pectin demethylesterification, in primordia and at the site of incipient organs. Measurements of tissue elasticity at various depths showed that, at the site of incipient primordia, the first increases occurred in subepidermal tissues. The results support the following causal sequence of events: (1) demethylesterification of pectin is triggered in subepidermal tissue layers, (2) this contributes to an increase in elasticity of these layers-the first observable mechanical event in organ initiation, and (3) the process propagates to the epidermis during the outgrowth of the organ.

The Arabidopsis thaliana hypocotyl is widely used to study the effects of light and plant growth factors on cell elongation. To provide a framework for the molecular-genetic analysis of cell elongation in this organ, here we describe, at the cellular level, its morphology and growth and identify a number of characteristic, developmental differences between light-grown and dark-grown hypocotyls. First, in the light epidermal cells show a characteristic differentiation that is not observed in the dark. Second, elongation growth of this organ does not involve significant cortical or epidermal cell divisions. However, endoreduplication occurs, as revealed by the presence of 4C and 8C nuclei. In addition, 16C nuclei were found specifically in dark-grown seedlings. Third, in the dark epidermal cells elongate along a steep, acropetal spatial and temporal gradient along the hypocotyl. In contrast, in the light all epidermal cells elongated continuously during the entire growth period. These morphological and physiological differences, in combination with previously reported genetic data (1. Desnos, V. Orbovic, C. Bellini, j. Kronenberger, M. Caboche, j. Traas, H. Hofte [19961 Development 122: 683-693), illustrate that light does not simply inhibit hypocotyl growth in a cell-autonomous fashion, but that the observed growth response to light is a part of an integrated developmental change throughout the elongating organ.

High-rate electron transfer toward an anode in microbial fuel cells (MFCs) has thus far not been described for bacteria-producing soluble redox mediators. To studythe mechanism of electron transfer, we used a MFC isolate, Pseudomonas aeruginosa strain KRP1. Bacterial electron transfer toward the MFC anode was enabled through pyocyanin and phenazine-1-carboxamide. The presence of the anode stimulated pyocyanin production. Mutant strains, deficient in the synthesis of pyocyanin and phenazine-1-carboxamide, were unable to achieve substantial electron transfer and reached only 5% of the wild type's power output. Upon pyocyanin addition, the power output was restored to 50%. Pyocyanin was not only used by P. aeruginosa to improve electron transfer but as well enhanced electron transfer by other bacterial species. The finding that one bacterium can produce electron shuttles, which can be used also by other bacteria, to enhance electron-transfer rate and growth, has not been shown before. These findings have considerable implications with respect to the power output attainable in MFCs.

The results show that THE1 mediates the response of growing plant cells to the perturbation of cellulose synthesis and may act as a cell-wall-integrity sensor.