Establishing the developmental function of the rhamnogalacturonan II component of pectin

Abstract

The primary plant cell wall consists of a complex set of polysaccharides including pectin, cellulose and hemicelluloses that are critical for normal plant development. There are three major forms of pectin, rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II) and homogalacturonan (HGA). Of these, the pectic polysaccharide RG-II, is the least abundant but the most complex. Despite this, RG-II is highly conserved among vascular plants, suggesting animportant function which is dependent upon structure. RG-II consists of four structurally welldefined side chains attached to a backbone of 1,4-linked galacturonic acid (GalA) residues and exists predominately as a dimer in plant cell walls. RG-II function has yet to be identified; however, mutations affecting RG-II structure have severe growth and development defects. 3-deoxy-D-manno-2-octulosonic acid (Kdo) is a rarely found sugar and is a component of the RGII side chain C. Kdo biosynthesis has been well characterised and a number of Kdo synthesis genes identified in Arabidopsis. Traditional gene knockout approaches to study the effect of disrupting Kdo biosynthesis have been limited by the apparent lethality of these mutants. Alternative approaches using partial knockout, inducible gene silencing and chemical approaches have being employed with the primary aim of specifically altering the structure of RG-II to determine the developmental function of RG-II. By combination of a GAL4/VP16 expression system and ALCR/alcA ethanol-switch to achieve temporal and spatial control of transgene expression, it has been possible to generate a genetic tool kit consisting of a series of Arabidopsis lines in which it should be possible to disrupt Kdo biosynthesis in specific tissues at strictly defined developmental stages. As a proof of concept the J0951/iKdsB line, in which expression of an AtKdsB antisense sequence is restricted to the epidermis and root cap in the presence of ethanol, is shown to be almost completely devoid of root hairs when grown under induced conditions. This result is suggestive of a role for RG-II in tip growth processes and is consistent with the phenotypes of null mutants in which a failure in pollen tube elongation results in gametophyte lethality. In silico and in vitro approaches are used to investigate the potential application of an inhibitor of AtKdsB, 2?-deoxy Kdo, as a tool for the disruption of CMP-Kdo synthesis in plants. Using homology modelling the Arabidopsis and E. coli enzymes are shown to have a near identical active site conformation. Using recombinantly expressed AtKdsB in enzyme kinetic and inhibition studies the substrate analogue 2?-deoxy-Kdo was shown to be a potent in vitro inhibitor of AtKdsB with a Ki of 1.26 ± 0.15 ??, consistent with measures of the Kd made by isothermal titration calorimetry (ITC) analysis. The 2?-deoxy-Kdo was subsequently applied in vivo and results in a severe inhibition of cell elongation of Arabidopsis root cells that can be partially rescued by either Kdo or boron. It is likely that 2?-deoxy-Kdo application disrupts CMP-Kdo biosynthesis with consequences for RG-II structure and dimer formation.

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