RG_II

RG-II is highly complex and unique to vascular plants. We hypothesized that enzymes involved in its biosynthesis probably were also unique to plants and hence unlikely to be included databases like CAZy. We thus undertook bioinformatics GT-discovery effort to identify plant specific GTs that might be involved in the biosynthesis of unique polymers like RG-II. Functional roles are assigned to the GTs based on cellwall composition of T-DNA insertional mutants and on biochemical characterization of heterologously expressed enzymes. One method is called “the free sugar assay”. The idea is to use a nucleotide diphosphate sugar as donor and a monosaccharide as acceptor in high concentration under formation of a new glycosidic bond. The product saccharides are purified by chromatography and analysed by NMR to determine their precise structure. The mass spectrometric techniques Dionex-MS and MALDI-TOFF are being used as well. The drawback of the free sugar assay is the limited number of available acceptors and more (complex) acceptors are needed. We prepare well-defined acceptors by chemical synthesis and obtain larger oligosaccharides and polysaccharides by controlled hydrolysis of pectin fragments.

Expression of plant CW GTs have turned out to be non-trivial. We use the Baculo virus system and Pichia pastoris and, for ectopic expression in pea or tobacco, a Pea Early Browning Virus (PEBV) expression vector. We have expressed two xylosyltransferases, designated rhamnogalacturonan xylosyltransferase 1 & 2 (RGXT1, At4g01770 and RGXT2, At4g01750), which originally were identified by the bioinformatics screen.

 

Protein-protein interactions
We hypothesize that synthesis of a complex CW polymer like RG II proceeds in a highly orderly manner, involving dedicated metabolons. This is a joint effort with Henrik V. Scheller and colleagues regarding the possible interactions between RGXT1/2 and a Glucuronosyltransferase (GlcAT, At5g61840), both of which use the RG II-A-chain fucosyl residue as acceptor.

 

Pod shatter
Dehiscence refers to the opening of dry fruits for seed dispersal. The siliques of Arabidopsis and oilseed rape and the pods of soybean are typical examples. Loss of seed due to unsynchronized seed shattering is economically significant in oilseed rape in particular and also in soybean under some circumstances. We have contributed to our understanding of the hormonal regulation of silique opening, to the identification of the endo-polygalacturonase that is responsible for the middle lamella depolymerization that precedes silique opening, its regulated secretion as well as identification of a soybean ortholog of the endo-polygalacturonase.

 

The involvement of glucanases in biosynthesis
In the course of the pod shatter work, we serendipitously cloned the oilseed rape orthologue of the KOR membrane anchored beta-glucanase, This eventually led to the characterization of the remaining members of this little gene family in Arabidopsis as well as the proposition that these glucanases act as proof reading enzymes during cellulose synthesis at the plasma membrane.

 

Publications
Østergaard,L., Borkhardt, B., Ulvskov, P. (2006) Dehiscence. In: Cell Separation and Adhesion in Plants (Jerry Roberts & Zinnia Gonzalez-Carranza eds.) Blackwell. In press

Dal Degan, F., Child, R., Svendsen, I., Ulvskov, P. (2001) The Cleavable N-terminal Domain of Plant Endopolygalacturonases from Clade B May Be Involved in a Regulated Secretion Mechanism. JBC: 176(38): 35297-35304

Sander, L., Child, R., Ulvskov, P., Albrechtsen, M., Borkhardt, B. (2001) Analysis of a dehiscence zone endo-polygalacturonase in oilseed rape (Brassica napus) and Arabidopsis thaliana: evidence for roles in cell separation in dehiscence and abscission zones, and in stylar tissues during pollen tube growth.PMB: 46: 469-479

Christiansen,L. C., Dal Degan, F.,. Ulvskov, P., Borkhardt, B. (2002) Examination of the dehiscence zone in soybean pods and isolation of a dehiscence-related endopolygalacturonase gene. Plant Cell Environ. 25: 479-490

Mølhøj M, Ulvskov P & Dal Degan F (2001) Characterization of a functional soluble form of a Brassica napus membrane-anchored endo-1,4-b-D-glucanase heterologously expressed in Pichia pastoris. Plant Physiology127(2): 674-684

Mølhøj, M. Jørgensen, B., Ulvskov, P., Borkhardt, B. (2001) Two Arabidopsis thaliana genes, KOR2 and KOR3, which encode membrane-anchored endo-1,4-ß-D-glucanases, are differentially expressed in developing leaf trichomes and their support cells. PMB 46: 263-275

Mølhøj, M., Johansen, B., Ulvskov, P., Borkhardt, B. (2001) Expression of a membrane-anchored endo-1,4-ß-glucanase from Brassica napus, orthologous to KOR from Arabidopsis thaliana, is inversely correlated to elongation in light-grown plants. PMB 45: 93-105

Chauvaux, N., Child, R., John, K., Ulvskov, P., Borkhardt, B., Prinsen, E., van Onckelen, H.A. (1997) The role of auxin in cell separation in the dehiscence zone of oilseed rape pods. J. Exp. Bot. 48(312): 1423-1429

Child, R.D., Chauvaux, N., John, K., Ulvskov, P., van Onckelen, H.A. (1998) Ethylene biosynthesis in oilseed rape pods in relation to pod shatter. J. Exp. Bot. 49(322): 829-838

 

Petersen, M., Sander,L., Child, R., Van Onckelen., H., Ulvskov,P, Borkhardt, B. (1996). Isolation and characterization of a pod dehiscence zone specific polygalacturonase from Brassica napus. Plant Molecular Biology 31(3): 517-527