Root hair-specific disruption of cellulose and xyloglucan in AtCSLD3 mutants, and factors affecting the post-rupture resumption of mutant root hair growth

Abstract

The glycosyl transferase encoded by the cellulose synthase-like gene CSLD3/KJK/RHD7 (At3g03050) is required for cell wall integrity during root hair formation in Arabidopsis thaliana but it remains unclear whether it contributes to the synthesis of cellulose or hemicellulose. We identified two new alleles, root hair-defective (rhd) 7-1 and rhd7-4, which affect the C-terminal end of the encoded protein. Like root hairs in the previously characterized kjk-2 putative null mutant, rhd7-1 and rhd7-4 hairs rupture before tip growth but, depending on the growth medium and temperature, hairs are able to survive rupture and initiate tip growth, indicating that these alleles retain some function. At 21°C, the rhd7 tip-growing root hairs continued to rupture but at 5ºC, rupture was inhibited, resulting in long, wild type-like root hairs. At both temperatures, the expression of another root hair-specific CSLD gene, CSLD2, was increased in the rhd7-4 mutant but reduced in the kjk-2 mutant, suggesting that CSLD2 expression is CSLD3-dependent, and that CSLD2 could partially compensate for CSLD3 defects to prevent rupture at 5°C. Using a fluorescent brightener (FB 28) to detect cell wall (1 → 4)-β-glucans (primarily cellulose) and CCRC-M1 antibody to detect fucosylated xyloglucans revealed a patchy distribution of both in the mutant root hair cell walls. Cell wall thickness varied, and immunogold electron microscopy indicated that xyloglucan distribution was altered throughout the root hair cell walls. These cell wall defects indicate that CSLD3 is required for the normal organization of both cellulose and xyloglucan in root hair cell walls.

Root Hair Development in the Grasses: What We Already Know and What We Still Need to Know

“…2). The formation of the RH bulge has been well researched in Arabidopsis (Galway et al, 2011;Pei et al, 2012), but among the grasses, only a handful of mutants in which RH development has been arrested at this stage have been described. Even in these, the RH bulge is still present, as the mutant phenotype results from the inhibition of RH elongation (Szarejko et al, 2005).…”

Root Hairs

“…Additional inner cell wall layers containing parallel arrays of cellulose microfibrils appear to be deposited later, and these are often organized in a helical orientation along the length of the root hair. Cellulose synthesis is required for root hair tip growth (Park et al, 2011;Galway et al, 2011), and while known components of the cellulose synthase complexes CESA3 and CESA6 are not observed in apical plasma membranes in actively growing root hair cells, a functional, fluorescently-tagged CELLULOSE SYNTHASE-LIKE PROTEIN D3 (CSLD3/KJK/RHD7: At3g03050) was enriched at the tips of growing root hairs (Park et al, 2011). Both CSLD2 (At5g16910) and CSLD3/KJK/RHD7, members of the Cellulose-Synthase-Like (CSL) super family, are required for root hair growth (Wang et al, 2001;Bernal et al, 2008;Galway et al, 2011;Yin et al, 2011;Yoo et al, 2012).…”

“…Cellulose synthesis is required for root hair tip growth (Park et al, 2011;Galway et al, 2011), and while known components of the cellulose synthase complexes CESA3 and CESA6 are not observed in apical plasma membranes in actively growing root hair cells, a functional, fluorescently-tagged CELLULOSE SYNTHASE-LIKE PROTEIN D3 (CSLD3/KJK/RHD7: At3g03050) was enriched at the tips of growing root hairs (Park et al, 2011). Both CSLD2 (At5g16910) and CSLD3/KJK/RHD7, members of the Cellulose-Synthase-Like (CSL) super family, are required for root hair growth (Wang et al, 2001;Bernal et al, 2008;Galway et al, 2011;Yin et al, 2011;Yoo et al, 2012). While mannan synthase activity has been detected for some CSLD proteins (Yin et al, 2011), a CSLD3 chimera containing a CESA6 catalytic domain restored root hair growth in a csld3 null mutant (Park et al, 2011), raising the intriguing possibility that CSLD proteins may synthesize cellulose-like polysaccharides in tipgrowing root hair cells.…”

The Microtubule Plus-End Tracking Protein ARMADILLO-REPEAT KINESIN1 Promotes Microtubule Catastrophe in Arabidopsis

“…A second PCR with the attB-adapter primers was performed according to the manufacturer's protocol (Invitrogen). The mRNA extraction and cDNA synthesis protocol were performed as described (Galway et al, 2011). Following the BP reaction with the various attB-PCR products and the pDONR221 vector (Invitrogen), an LR reaction was performed with the pMDC107 vector (Curtis and Grossniklaus, 2003) for the ARK1 genomic sequence and the pUBC-RFP DEST vector (Grefen et al, 2010) for the various ARK1 coding sequences.…”