LETTERS A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells Sungjin Park 1 , Amy L. Szumlanski 1 , Fangwei Gu 1 , Feng Guo 1 and Erik Nielsen 1,2 In plants, cell shape is defined by the cell wall, and changes in cell shape and size are dictated by modification of existing cell walls and deposition of newly synthesized cell-wall material 1 . In root hairs, expansion occurs by a process called tip growth, which is shared by root hairs, pollen tubes and fungal hyphae 1 . We show that cellulose-like polysaccharides are present in root-hair tips, and de novo synthesis of these polysaccharides is required for tip growth. We also find that eYFP–CSLD3 proteins, but not CESA cellulose synthases, localize to a polarized plasma-membrane domain in root hairs. Using biochemical methods and genetic complementation of a csld3 mutant with a chimaeric CSLD3 protein containing a CESA6 catalytic domain, we provide evidence that CSLD3 represents a distinct (1 → 4)-β-glucan synthase activity in apical plasma membranes during tip growth in root-hair cells. Cellulose is the most abundant polysaccharide in plant cell walls, and provides the majority of the cell wall’s tensile strength 1,2 . Cellulose microfibrils contain multiple (1 → 4)-β-glucan polysaccharides, and are synthesized by plasma-membrane-localized cellulose synthase (CESA) complexes 1,2 . The Arabidopsis thaliana genome contains ten CESA genes, and at least three, CESA1, CESA3 and CESA6, are involved in primary cell-wall formation 3–5 . By comparison, secondary cell-wall formation requires CESA4, CESA7 and CESA8 (refs 6–8). These CESA cellulose synthase complexes have been investigated during diffuse growth 9–13 , but their roles in cell-wall synthesis in tip-growing cells remain uncharacterized. The root-hair cell apex contains only a thin primary cell wall with no obvious directionality to the orientation of cell- wall polysaccharides 14–16 . Although mutations in cellulose synthase components affect root-hair development 3,4,17,18 , tip-growing root-hair cells still form. To clarify whether cellulose, or cellulose-like (1 → 4)-β- glucan polysaccharides, were present in growing root-hair tips, we labelled cells with cellulose-specific reagents. Both Pontamine fast scarlet 4B (S4B; Fig. 1a; ref. 19) and a carbohydrate-binding module (CBM3a; Fig. 1b; ref. 20) strongly labelled cellulose, or cellulose-like polysaccharides, in actively growing root-hair tips. Whereas strong S4B 1 Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA. 2 Correspondence should be addressed to E.N. (e-mail: nielsene@umich.edu) Received 18 October 2010; accepted 7 June 2011; published online 17 July 2011; DOI: 10.1038/ncb2294 labelling was seen primarily in growing root-hair tips, mature root hairs showed further S4B labelling along root-hair shafts (Supplementary Fig. S1a), consistent with earlier reports that cellulose microfibrils selectively accumulate during secondary reinforcement in mature root-hair cell walls 21,22 . The cellulose synthase inhibitor 2,6-dichlorobenzene (DCB) inhibits root-hair formation 23,24 , but it was unclear if this occurred during or before initiation of tip growth. Addition of 20 μM DCB to growing root hairs resulted in rapid cessation of elongation and rupture at the cell apex (Fig. 1c and Supplementary Movie S1). DCB effects on root-hair growth were concentration dependent, and both cell rupture and formation of bulges occurred under a range of DCB concentrations (Fig. 1d). DCB has also been implicated in microtubule dynamics 25 , but when considered together with enrichment of cellulose-specific markers at root-hair tips these results indicate that cellulose synthesis or synthesis of cellulose-like (1 → 4)-β-glucan polysaccharides is required in the tips of growing root hairs. Growing root hairs were also incubated with purified, recombinant cellulose-specific endo-(1 → 4)-β-glucanase (cellulase), xyloglucan- specific endo-(1 → 4)-β-glucanase (xyloglucanase), or pectate lyase (Supplementary Fig. S1b–d). Cellulase treatment rapidly induced cellular rupture (Fig. 1e and Supplementary Movie S2A), and this bursting could be inhibited by co-incubation with excess carboxymethylated cellulose (CM-cellulose). Treatment of root hairs with either xyloglucanase (Fig. 1e and Supplementary Movie S2B) or pectate lyase (Fig. 1e and Supplementary Movie S2C) also inhibited root-hair elongation, but surprisingly neither induced cell rupture. Inhibition of root-hair elongation by either xyloglucanase or pectate lyase was unaffected by co-incubation with CM-cellulose (Fig. 1e). Because xyloglucan biosynthesis mutants show root-hair defects 26 and xyloglucan deposition was altered in a csld3 (cellulose synthase-like D3, also known as At3g03050.1) mutant 27 , we analysed xyloglucanase effects on cell-wall polysaccharides. Root hairs were probed before and after xyloglucanase treatment with S4B, or the xyloglucan- specific monoclonal antibody CCRC-M1. Whereas S4B (Fig. 1a and Supplementary Fig. S1a,e) was strongly enriched in tips of untreated root hairs, CCRC-M1 immunolabelling was more uniformly NATURE CELL BIOLOGY VOLUME 13 | NUMBER 8 | AUGUST 2011 973 © 20 11 M acmillan Publishers Limited. 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