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

Abstract

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-beta-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.

Fig. 1.

Visualization of similar distribution and dynamics of GFP-CESA3 and GFP-CESA6. (A–D) Functional GFP-CESA3 and GFP-CESA6 fusion proteins expressed, respectively, in mutant backgrounds cesa3^je5 and cesa6^prc1–1 label similar intracellular compartments and complexes that migrate with comparable velocities at the cell surface. Spinning disk microscope images (A and B) and projection of a time series (10 min, 30 s, C; 10 min, D) of epidermal cells at the top of a dark-grown hypocotyl expressing GFP-CESA3 (A and C) or GFP-CESA6 (B and D). (Scale bars: 4 μm.) (E) Distribution of the velocities of the surface particles.

Fig. 2.

CESA3 and CESA6 are in the same cellulose synthase complex. (A and B) CESA3 and CESA6 protein levels, measured in dark-grown seedlings by quantitative immunoblotting, are reduced in mutants cesa6^prc1–1, cesa3^eli1–1 (A) and cesa1^rsw1–10 (B). Protein levels are expressed as percentage of wild-type levels, and error bars are SDs of three biological repeats. (C) CESA3 coimmunoprecipitates with CESA6 and vice versa, but not with membrane-bound cellulase KOR1. IP was carried out in nondenaturing or denaturing conditions. Shown are total Triton X-100-solubilized extracts (lane 1), IP with anti-NKOR, unbound (lane 2) and bound (lane 3) fractions; with anti-NCESA3, unbound (lane 4) and bound (lane 5) fractions or with anti-NCESA6, unbound (lane 6) and bound (lane 7) fractions. Immunoblotting was carried out with anti-NCESA3 anti-NCESA6 or anti-NKOR antibodies. (D) BiFC in N. benthamiana leaf epidermal cells shows in vivo CESA6 homodimer and CESA3/CESA6 heterodimer formation. (Upper Left) YN-PIP/YC-PIP-positive control. (Upper Right) YN-CESA6/YC-PIP. (Lower Left) YN-CESA6/YC-CESA6. (Lower Right) YN-CESA6/YC-CESA3. Other CESA combinations are shown in SI Fig. 8.

Fig. 3.

Partial redundancy between CESA6-related CESAs. Five-day-old light-grown (A), dark-grown seedlings (B), and 32-day-old greenhouse-grown plants (C) of (from left to right) Col0, cesa2, cesa5, cesa2/cesa5, cesa6^prc1–1, cesa2/cesa6^prc1–1, and cesa5/cesa6^prc1–1 are shown.

Fig. 4.

CESA6, CESA2, and CESA5 are targets for isoxaben. (Upper) The effect of isoxaben on hypocotyl length: dose–response curve. Hypocotyl length is expressed in percentage of the untreated control. Average values of ≈35 individuals are shown. (Lower) For clarity, SDs of untreated and 100 nM isoxaben-treated seedlings are shown.