Disruption of the microtubule network alters cellulose deposition and causes major changes in pectin distribution in the cell wall of the green alga, Penium margaritaceum

David S.Domozych 2 IbenSrensen 1 CarlySacks 2 HannahBrechka 2 AmandaAndreas 2 Jonatan U.Fangel 0 Jocelyn K. C.Rose 1 William G. T.Willats 0 Zo A.Popper 3 0 Department of Plant and Environmental Sciences, University of Copenhagen, Faculty of Science , Thorvaldsensvej 40, 1871 Frederiksberg, Denmark 1 Department of Plant Biology, Cornell University , Ithaca, NY 14853, USA 2 Department of Biology and Skidmore Microscopy Imaging Center, Skidmore College , Saratoga Springs, NY 12866, USA 3 Botany and Plant Science, School of Natural Sciences and Ryan Institute for Environmental, Marine and Energy Research, National University of Ireland , Galway, Ireland Application of the dintroaniline compound, oryzalin, which inhibits microtubule formation, to the unicellular green alga Penium margaritaceum caused major perturbations to its cell morphology, such as swelling at the wall expansion zone in the central isthmus region. Cell wall structure was also notably altered, including a thinning of the inner cellulosic wall layer and a major disruption of the homogalacturonan (HG)-rich outer wall layer lattice. Polysaccharide microarray analysis indicated that the oryzalin treatment resulted in an increase in HG abundance in treated cells but a decrease in other cell wall components, specifically the pectin rhamnogalacturonan I(RG-I) and arabinogalactan proteins (AGPs). The ring of microtubules that characterizes the cortical area of the cell isthmus zone was significantly disrupted by oryzalin, as was the extensive peripheral network of actin microfilaments. It is proposed that the disruption of the microtubule network altered cellulose production, the main load-bearing component of the cell wall, which in turn affected the incorporation of HG in the two outer wall layers, suggesting coordinated mechanisms of wall polymer deposition. - Plant cell walls are composites of polymers that are assembled and organized into intricate structures that surround the protoplast, where they serve multiple roles including defence, turgor resistance and controlled cell growth, water and mineral uptake, and communication (Baskin etal., 2004; Cosgrove, 2005; Sarkar et al., 2009; Keegstra, 2010; Fry, 2011). Cell wall architecture is highly dynamic, and synthesis, assembly, and any subsequent remodelling require precisely coordinated interactions between the cell endomembrane system, cytoskeletal network, plasma membrane, and multiple cross-talking signal transduction pathways. Cell wall production and maintenance therefore involve not just a substantial amount of the total photosynthate, but also a major portion of the genetic repertoire (Popper etal., 2011). The structural and developmental characteristics and functional competency of the plant wall are also fundamentally Abbreviations: AGP arabinogalactan protein; CBM, carbohydrate-binding module; CLSM, confocal laser scanning microscopy; DCB, dichloronitrobenzile; DE, degree of esterification; DIC-LM, differential interference contrast light microscopy; GA, Golgi apparatus; HG, homogalacturonan; LM, light microscopy; mAb, monoclonal antibody; PL, pectate lyase; RG-I, rhamnogalacturonan I; VPSEM, variable pressure scanning electron microscopy; WHM, Woods Hole medium. The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. affected by complex multipolymeric associations. The nature of these interactions, especially during development and in response to environmental stresses, is poorly understood and only recently has this been the focal point of detailed study. For example, cellulose microfibrils are generally described as being tethered by xyloglucan and other hemicellulosic (crosslinking glycan) polymers, and these have been proposed to influence microfibril slippage during wall and cell expansion (Popper and Fry, 2005, 2008; Fry, 2011); the nature, extent, and significance of this cross-linking have recently been discussed (Cosgrove and Jarvis, 2012; Park and Cosgrove, 2012). There is also recent evidence that the neutral sugar side chains (e.g. arabinans and galactans) of the pectin class rhamnogalacturonan-I (RG-I) may be directly bound to cellulose (Zykwinska etal., 2005, 2007). Yoneda etal. (2010) further suggested that pectin cross-bridges support and maintain the direction of cellulose microfibril orientation and slippage during cell expansion. However, there are doubtless many other interpolymeric associations that are critical for wall architecture and function, but that have yet to be recognized and characterized. Evaluating such interactions within the context of multicellular plants is very challenging, and the extraction of cell wall polymeric complexes inevitably disrupts or abolishes a number of the molecular associations. Moreover, the physical restriction of specific polymer probes in dense tissues and the inability to use live material in many labelling and analytical protocols effectively further limit dissection of interpolymeric interactions. In contrast, the identification and use of a unicellular plant system, particularly one with clearly defined cell wall polymer domains, would significantly enhance such studies. A unicellular taxon of the Charophycean green algae (CGA or Streptophyta; i.e. the group of green algae most closely related to land plants; Lewis and McCourt, 2004; Wodniok et al., 2011), Penium margaritaceum, has a number of characteristics that suggest it would provide a potentially valuable model system for the study of cell wall development, including interpolymeric associations. First, Penium only produces a permanent primary cell wall, comprising two prominent polymeric domains that are easily identified by microscopy: a pectic domain primarily consisting of homogalacturonan (HG) organized into a lattice-like network in the outer layer of the wall; and an inner domain consisting mostly of cellulose, together with smaller amounts of other glycan classes (Srensen etal., 2010, 2011; Domozych etal., 2011). Secondly, the focal point of HG secretion, which in Penium appears to drive cell wall growth and cell development, is a clearly defined narrow band located at the cell centre or isthmus, or the isthmus band (Domozych etal., 2009b). This facilitates visualization of wall polymer secretion in a spatially well-defined area. Thirdly, Penium can be grown in large, fast-growing cultures, enabling extraction of substantial amounts of cell wall material for biochemical and immuno-based screening (Mller et al., 2007; Srensen and Willats, 2011). Fourthly, wall polymer dynamics can be conveniently monitored by live cell labelling utilizing probes such as monoclonal antibodies (mAbs) directed a (...truncated)