Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis

Abstract

The irregular xylem 1 (irx1) mutant of Arabidopsis has a severe deficiency in the deposition of cellulose in secondary cell walls, which results in collapsed xylem cells. This mutation has been mapped to a 140-kb region of chromosome 4. A cellulose synthase catalytic subunit was found to be located in this region, and genomic clones containing this gene complemented the irx1 mutation. IRX1 shows homology to a previously described cellulose synthase (IRX3). Analysis of the irx1 and irx3 mutant phenotypes demonstrates that both IRX1 and IRX3 are essential for the production of cellulose in the same cell. Thus, IRX1 and IRX3 define distinct classes of catalytic subunits that are both essential for cellulose synthesis in plants. This finding is supported by coprecipitation of IRX1 with IRX3, suggesting that IRX1 and IRX3 are part of the same complex.

Figure 1.

Map Position of irx1 on Chromosome 4 Relative to Several Molecular Markers. Black bar represents a portion of chromosome 4. Markers are shown above, with the number of recombinants with irx1 given in parentheses. Bars underneath represent the positions of BACs spanning this region. Hatched box denotes region in which irx1 falls.

Figure 2.

Alignment of the Amino Acid Sequences of Several Plant Cellulose Synthase Genes. Solid boxes indicate regions in which more than half of the residues are identical; gray boxes indicate conserved residues. The positions of three aspartic acid (D) residues and QxxRW motifs are indicated by vertical arrowheads and asterisks, respectively. Variable regions VR1 and VR2 are also indicated. Dashes were introduced to optimize alignment. Antibodies were raised against the VR1 from IRX1 (amino acids 75 to 149) and against the VR1 from IRX3 (amino acids 114 to 203).

Figure 3.

Toluidine Blue–Stained Sections of Arabidopsis Vascular Bundles Showing Complementation of irx1-1. (A) Wild type transformed with pP8GUS. (B) irx1-1 transformed with pP8GUS. (C) irx1-1 transformed with pCS52. xe, xylem elements. .

Figure 4.

Cellulose Measurements Showing Complementation of irx1-1. Error bars represent standard error. Values are the mean of measurements from five independent transformants. WT Ler, wild-type Landsberg erecta.

Figure 5.

RNA Gel Blots Showing Expression of the IRX1 Gene. Blots containing RNA from developing stems and leaves from wild-type (WT) and irx1-1 plants were probed with IRX1, IRX3, COMT, and rRNA.

Figure 6.

Ultrastructure of Xylem Element Cell Walls. Transmission electron microscopy was used to study cell wall structure. (A) Wild type. (B) irx3. (C) irx1-1. Arrows indicate middle lamellae. .

Figure 7.

Specificity of IRX1 and IRX3 Antibodies. Protein gel blots of wild-type and irx3 extracts probed with anti-IRX1 antibody and anti-IRX3 antibody. Molecular mass markers are given at right in kilodaltons. WT, wild type.

Figure 8.

Solubilization of IRX1 in Triton X-100. Protein gel blot probed with the anti-IRX1 antibody. Lane 1 contains total protein after clarification; lane 2, supernatant after centrifugation at 100,000g; lane 3, pellet after 100,000g centrifugation; lane 4, supernatant of solubilized extract after 100,000g centrifugation; lane 5, pellet from solubilized extract after 100,000g centrifugation. Molecular mass markers are given at right in kilodaltons.

Figure 9.

Copurification of IRX3 and IRX1 as Shown by Protein Gel Blots. (A) NHisIRX3 probed with anti-RGSHHHH antibody (top) and anti-IRX1 antibody (bottom). (B) Wild type probed with anti-IRX1 antibody. (C) NHisIRX3 probed with anti-RGSHHHH antibody (top) and anti-aquaporin antibody (bottom). Molecular mass markers are shown at right in kilodaltons.