Isoxaben is a pre-emergence herbicide that inhibits cellulose biosynthesis in higher plants. Two loci identified by isoxabenresistant 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 function procuste1 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.Cellulose synthesis remains a poorly understood process (for review, see Delmer, 1999). This linear 1,4--linked glucan is synthesized in terrestrial plants by a hexameric protein complex referred to as the terminal complex or rosette embedded in the plasma membrane (Kimura et al., 1999). The composition of this complex is unknown. Each of the six particles in the complex is thought to contain several catalytic subunits with an unknown stoichiometry. The relationship between the rosette and the crystalline microfibril structure remains controversial. It is commonly held that one rosette produces one microfibril. This idea was based on calculations based upon the spacing of 1,4--glucan chains in algal celluloses and it assumed the presence of 36 glucan chains in a single microfibril (Herth, 1983). Each rosette particle would then contribute six glucan chains to the microfibril. More recent studies using solidstate nuclear magnetic resonance lead to the conclusion that microfibrils in the primary wall consist of 2-nm crystalline units or elementary fibrils, each of which would not contain more than 10 to 15 glucan chains (Ha et al., 1998). The authors propose a scenario in which each of the six particles in the rosette produce the glucan chains of an elementary fibril, which in turn associate into a hexagonal 8-to 10-nm microfibril observed in most primary cell walls.Genes encoding the cellulose synthase catalytic subunits have been identified in bacteria and plants (Pear et al., 1996). The Arabidopsis genome encodes 10 isoforms of the cellulose synthase catalytic subunit, CESA (http://cellwall.stanford.edu). The ...