Rhamnogalacturonan-I

Potato tuber tissue is generally rich in RG-I and potato RG-I is rich in galactan side chains, the socalled type-I galactans. We have studied the biosynthesis of pectic beta-1,4-galactans extensively in potato suspension cells. Galactosyl transferase activities have been detected in isolated Golgi vesicles, and we have provided evidence that the first galactosyl residue added to the RG-I back-bone and the subsequent galactosyl residues of the side chain are not transferred by the same enzyme. We have further shown that the transferases face the Golgi lumen and proposed that they are type-II membrane anchored glycosyl transferases.

While potato is well suited for the biochemical studies of RG-I biosynthesis and also for the in vivo remodeling of RG-I in transgenic plants, it is not the model system of choice for identification and cloning of the gene that encodes the galactosyl transferases. For this purpose we have selected Lupinus angustifolius.

It has been known since the late 19th century that the cotyledons of several lupin species have thickened cell walls, and that the content of these walls are largely mobilized after germination. The main polysaccharide component of the thickened cell walls in the storage parenchyma of Lupinus angustifolius cotyledons is a linear (1-4)-ß-linked D-galactan that is probably attached to RG-I. This galactan is deposited into the cell wall late in seed development and occurs within a relatively short time period and results in an increase in wall thickness from 0.2 to 20 µm. The increase in wall thickness probably requires de novo synthesis of all the enzymes involved in galactan biosynthesis

We are now in the progress of sequencing a cDNA library derived from RNA isolated from tissue with thickened cells walls with the purpose to identify transcript that encodes glycosyl transferases and/or synthases.

Publications
Geshi N, Jørgensen B, Ulvskov P (2004) Subcellular localization and topology of beta-1-4-galactosyltransferase that elongates beta-4-galactan side chains in rhamnogalacturonan I in potato. Planta 218: 862-868

Geshi, N., Pauly, M., Ulvskov, P. (2002) Solubilization of galactosyltransferase that synthesizes 1,4-ß-galactan side chains in pectic rhamnogalacturonan I Physiol. Plant. 114:540-548

Geshi, N., Jørgensen, B., Scheller, H.V., Ulvskov, P.(2000) In vitro biosynthesis of 1,4-ß-galactan attached to rhamnogalacturonan. Planta 210: 622-629

Orfila, C., Oxenbøll Sørensen, S., Harholt, J., Geshi, N., Crombie, H., Truong H-N., Grant Reid, J.S. Knox, J.P. Vibe Scheller, H. (2005) QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana inflorescence stems, and affects homogalacturonan and xylan biosynthesis. Planta 222: 613-622

 

 

In vivo remodeling of RG-I
The roles of the different RG-I features in the wall are largely unknown. In order to address these questions, we have generated transgenic potato plants with modified pectin composition by expressing different fungal (A. aculeatus) cell-wall degrading enzymes under the control of the tuber-specific granule bound starch synthase and patatin promoters. Modified pectic polysaccharides have been obtained by the apoplastic expression of an endo-galactanase and a RG-I hydrolase. The apoplastic expression approach, however, did not work for an endo-arabinanase as this led to tuber-less potato plants, but redirecting the expression of the arabinanse to the Golgi apparatus resulted in plants with reduced pectic arabinan content.

The transformants that are reduced in pectic galactan or arabinan are similar to WT, bur a biophysical characterization of the transformed tissues has demonstrated that the precise RG-I side chain structures are functionally significant and contribute to wall mechanical properties.

 

Publications
Borkhardt, B., Skjøt, M., Mikkelsen, R. Jørgensen, B. and Ulvskov, P. 2005. Expression of a fungal endo-a-1,5-L-arabinanase during stolon differentiation in potato inhibits tuber formation and results in accumulation of starch and tuber-specific transcripts in the stem. Plant Science, 169, 872-881.

 

Ulvskov, P., Wium, H., Bruce, D., Jørgensen, B., Qvist K.B., Skjøt, M., Hepworth, D., Borkhardt, B. and Sørensen, S.O. 2005. Biophysical consequences of remodeling the neutral side chains of rhamnogalacturonan I in tubers of transgenic potatoes. Planta 220: 609-620.

 

Oomen, R.J.F.J., Doeswijk-Voragen, C.H.L., Bush, M.S., Vincken, J.-P., Borkhardt, B., Broek, L.A.M., Corsar, J., Ulvskov, P., Voragen, A.G.J., McCann, M.C. and Visser, R.G.F. 2002. In muro fragmentation of the rhamnogalacturonan I backbone in potato ( Solanum tuberosum L.) results in a reduction and altered localization of the galactan and arabinan side-chains and abnormal periderm development. Plant Journal, 30(4), 403-413.

 

Skjøt, M., Pauly, M., Bush, M.S., Borkhardt, B., McCann, M.C. and Ulvskov, P. 2002. Direct interference with rhamnogalacturonan I biosynthesis in Golgi vesicles. Plant Physiology, 129 (1), 95-102.

 

Ulvskov, P., Shols, H., Visser, R., Borkhardt, B., Sørensen, S.O., Oomen, R., Vincken, J.-P., McCann, M., Skjøt, M., Bush, M., Doeswijk Voragen, C. and Beldman, G. 2000. Method for remodelling polysaccharide structure of pectins. Patent application 00610020.0

 

Sørensen, S.O., Pauly, M., Bush, M., Skjøt, M., McCann, M., Borkhardt, B. and Ulvskov, P. 2000. Pectin engineering: Modification of potato pectin by in vivo expression of an endo-1,4-ß-D-galactanase. PNAS, 97 (13), 7639-7644.

 

Vincken, J.-P., Borkhardt, B., Bush, M., Doeswijk-Voragen, C. H. L., Dopico, B., Labrador, E., Lange, L., McCann, M., Morvan, C., Schols, H. A., Oomen, R., Peugnet, I., Rudolph, B., Schols, H., Sørensen, S., Ulvskov, P., Voragen, A., and Visser, R. (2000) Remodelling pectin structure in potato. In Conference Proceedings of Phytosfere'99 European Plant Biotechnology Network (Vries de, G. E. and Metzlaff, K., eds). Amsterdam: Elsevier Science B.V., pp. 245-256.

 

Coating of medical devices
Higher plants synthesize that widest variety of polysaccharide structures of any group of eukaryotes. Although plants do not produce all the carbohydrate structures found in mammals, we propose that cell wall polysaccharides may be tailored to mimic mammalian glycans in vivo using transgenic approaches or in vitro using fungal enzymes. We wish to use tailored rhamnogalacturonan-I for the coating of medical devices. The rationale is that we can impart the surfaces with biological properties so as to stimulate the cell colonization of the implant surfaces while suppressing inflammatory and rejection responses. The first step towards proof of concept is presented in the publicantion by Morra et al listed below, and more information is available at www.pecticoat.org.

 

Publications
Morra, M., Cassinelli, C., Cascardo, G., Nagel,M-D., Della Volpe,C., Siboni, S., Maniglio, D., Brugnara, M., Ceccone, G., Schols, H.S., Ulvskov, P. (2004) Effects on interfacial properties and cell adhesion of surface modification by pectic hairy regions. Biomacromolecules 5: 2094-2114