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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.

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Abbreviations

APW:

Artificial pond water

FB 28:

Fluorescent brightener 28

GFP:

Green fluorescent protein

PBS:

Phosphate-buffered saline

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Acknowledgments

This work was supported by the Natural Sciences and Engineering Research Council of Canada through Discovery Grants awarded to M.G. and G.W., the Collaborative Research and Training Experience program, which supports R.C.E., and a Research Capacity Development in Small Universities Grant awarded to St. Francis Xavier University, as well as by funding from the US National Science Foundation (J.S.), and the Australian Research Council (G.W.). At St. Francis Xavier University, Norma Mitchell, Daniel Morrison and Haixin Xu provided skilled technical assistance; graduate Elham A. Albahi and undergraduates Kristen Brown, Corinne Dewar and Tracy Smith (recipient of a Natural Sciences and Engineering Research Council of Canada Undergraduate Summer Research Award) performed hair rupture analysis, hair length measurements and immunofluorescence experiments. We also thank Roger Heady (Australian National University) for cryo-scanning electron microscopy, Thomas Magill (formerly at the Australian National University) for video clip creation, and Angela Whittington (formerly at the Australian National University) for expert assistance with DNA extraction and PCR amplification. Development and distribution of the CCRC-M1 antibody by the Complex Carbohydrate Research Center was supported in part by a grant from the NSF Plant Genome Program (DBI-0421683).

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Correspondence to Moira E. Galway.

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Movie S1. Four-hour time-lapse recording of hair from Fig. 5 (f-h) shows that ruptured root hair outgrowths can resume growth and survive subsequent multiple ruptures of differing severity. Numbers in lower left show elapsed time in hours:minutes:seconds; images were recorded at 2.5 min intervals. Tip growth initially resumed through a ring of debris produced by outgrowth rupture; cytoplasm was released in a mild rupture at 1:02 and a vacuole appeared at the hair tip before tip growth resumed. All of the tip cytoplasm was released in a rupture at 1:39 and the tip became vacuolated; tip growth resumed at 2:52, followed by a final rupture at 4:37 (MOV file, 614 KB)

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Supplementary material 2 (PDF 512 kb)

Movie S1. Four-hour time-lapse recording of hair from Fig. 5 (f-h) shows that ruptured root hair outgrowths can resume growth and survive subsequent multiple ruptures of differing severity. Numbers in lower left show elapsed time in hours:minutes:seconds; images were recorded at 2.5 min intervals. Tip growth initially resumed through a ring of debris produced by outgrowth rupture; cytoplasm was released in a mild rupture at 1:02 and a vacuole appeared at the hair tip before tip growth resumed. All of the tip cytoplasm was released in a rupture at 1:39 and the tip became vacuolated; tip growth resumed at 2:52, followed by a final rupture at 4:37 (MOV file, 614 KB)

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Galway, M.E., Eng, R.C., Schiefelbein, J.W. et al. Root hair-specific disruption of cellulose and xyloglucan in AtCSLD3 mutants, and factors affecting the post-rupture resumption of mutant root hair growth. Planta 233, 985–999 (2011). https://doi.org/10.1007/s00425-011-1355-6

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Keywords

  • Arabidopsis
  • Root hair
  • Cellulose
  • Xyloglucan
  • CCRC-M1
  • Cellulose synthase-like gene