Direct Interference with Rhamnogalacturonan I Biosynthesis in Golgi Vesicles1 Michael Skjøt, Markus Pauly2, Maxwell S. Bush, Bernhard Borkhardt, Maureen C. McCann, and Peter Ulvskov* Biotechnology Group, Danish Institute of Agricultural Sciences, Thorvaldsensvej 40, 1871 Copenhagen, Denmark (M.S., B.B., P.U.); Department of Plant Biology, Plant Biochemistry Laboratory, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Copenhagen, Denmark (M.P.); and Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Colney Lane, NR4 7UH Norwich, United Kingdom (M.S.B., M.C.M.) Pectin is a class of complex cell wall polysaccharides with multiple roles during cell development. Assigning specific functions to particular polysaccharides is in its infancy, in part, because of the limited number of mutants and transformants available with modified pectic polymers in their walls. Pectins are also important polymers with diverse applications in the food and pharmaceutical industries, which would benefit from technology for producing pectins with specific functional properties. In this report, we describe the generation of potato (Solanum tuberosum L. cv Posmo) tuber transformants producing pectic rhamnogalacturonan I (RGI) with a low level of arabinosylation. This was achieved by the expression of a Golgi membrane-anchored endo-␣-1,5-arabinanase. Sugar composition analysis of RGI isolated from transformed and wild-type tubers showed that the arabinose content was decreased by approximately 70% in transformed cell walls compared with wild type. The modification of the RGI was confirmed by immunolabeling with an antibody recognizing ␣-1,5-arabinan. This is the first time, to our knowledge, that the biosynthesis of a plant cell wall polysaccharide has been manipulated through the action of a glycosyl hydrolase targeted to the Golgi compartment. Current models of the plant cell wall present pec- suggests roles for pectic side-chains, for example, tins as complex matrix polysaccharides embedding arabinans, the polymer of interest to the present in- the load-bearing structures of the wall (cellulose mi- vestigation. Arabinans are very flexible molecules in crofibrils and hemicelluloses) and forming the mid- aqueous solution (Cros et al., 1994), whereas 13C- dle lamella, which cements neighboring cells to- NMR studies by Renard and Jarvis (1999) demon- gether (Carpita and Gibeaut, 1993). The pectic matrix strate that they are also very mobile molecules in has been described as coextensive with the microfi- muro. The authors concluded that arabinans are not brillar and hemicellulosic polymers of the wall (Rob- structural components; rather, they propose a role for erts, 1994), suggesting that some pectic polymers them as plasticizers and water binding agents in the may be structural components rather than mere fill- wall. Testing this working hypothesis requires plants ers of cell wall pores. Pectin constitutes a very com- in which the arabinan structure or content is modi- plex class of polysaccharides (Ridley et al., 2001) and fied, and a technology for producing such plants is presented in this report. their large-scale organization in the cell wall is far Because they are the most abundant bio-polymers from resolved. The prevailing view of pectin fine on Earth (Prade et al., 1999), cell wall polysaccharides structure (Schols and Voragen, 1996) and conforma- are of fundamental interest and are used by industry tion and architecture (Pe´rez et al., 2000) has recently for both food and non-food applications. Biotechno- been challenged and a new pectin model is being logical approaches for their modification and further drafted (J.-P. Vincken, A. Voragen, and H. Schols, exploitation have so far been limited because modi- personal communication). Neither model directly fication and production of carbohydrates has focused primarily on the generation of novel starches and 1 This work was supported by the Danish Research Council’s fructans (Heyer et al., 1999). The primary reason for Technology by Highly Oriented Research program, by The Danish this slow progress in bioengineering is the fact that National Research Foundation, by the European Commission the biosynthetic pathways of cell wall polysaccha- (grant no. BIOTECH CT97–2224), and by a Royal Society Univer- rides have not been fully characterized at the molec- sity Research Fellowship (to M.C.M.). ular level. Despite significant efforts to elucidate the * Corresponding author; e-mail [email protected]; fax 45–3528–2581. biogenesis of cell wall carbohydrates through mutant 2 Present address: Max-Planck-Institute for Molecular Plant screening programs (Zablackis et al., 1996; Reiter et Physiology, Am Muehlenberg 1, 14476 Golm, Germany. al., 1997) and through cloning and characterization of Article, publication date, and citation information can be found enzymes involved in cellulose (Arioli et al., 1998), at www.plantphysiol.org/cgi/doi/10.1104/pp.010948. xyloglucan (Perrin et al., 1999), and galactomannan Plant Physiology, May 2002, Vol. 129, pp. 95–102, www.plantphysiol.org © 2002 American Society of Plant Biologists 95 Skjøtetal. (Edwards et al., 1999) biosynthesis, the cell wall poly- saccharide biosynthetic apparatus will remain elu- sive for quite a while given the large number of genes predicted to be involved (Mohnen, 1999). Simpler approaches are called for. We have previously dem- onstrated that ␤-1,4-galactan side-chains of the pectic polymer rhamnogalacturonan I (RGI) can be enzy- matically cleaved post deposition in the cell wall Figure 1. Expression cassettes. 1, The pGED/ARA expression cassette without compromising plant viability (Sørensen et for apoplastic targeting. 2, The pGED/ST::ARA cassette conferring ␣ al., 2000). This was achieved through the targeting of Golgi targeting. Dotted bar, Region encoding the A. aculeatus -1,5- a fungal endo-1,4-␤-d-galactanase to the apoplast in endo-arabinanase signal sequence. Black bar, Mature A. aculeatus ␣-1,5-endo-arabinanase. Gray bar, Region encoding the potato (Solanum tuberosum L. cv Posmo) tubers. In ␤-galactoside ␣-2,6-sialyltransferase cytoplasmic domain. Hatched this paper, we present technology for direct interfer- bar, Region encoding the ␤-galactoside ␣-2,6-sialyltransferase mem- ence with pectin biosynthesis in Golgi vesicles. By brane spanning/signal sequence domain. White bar, Region encod- targeting a rat ␣-2,6 sialyl transferase-endo-␣-1,5- ing a truncated ␤-galactoside ␣-2,6-sialyltransferase catalytic do- arabinanase fusion protein to the Golgi compartment main. Pro, Tuber-specific granule-bound starch synthase promoter. of potato tuber cells, arabinan side-chains on RGI can Term, Nopaline synthase terminator and polyadenylation site. be hydrolyzed at the site of pectin biosynthesis. We demonstrate that this approach reduces the biosyn- thesis of RGI-arabinans in transgenic potato tubers starch synthase promoter was induced using the without compromising the viability of plants. method described previously (Visser et al., 1991). In addition, polyclonal antibodies raised against the endo-arabinanase recognized a protein in the extracts RESULTS from induced plantlets. The molecular mass of this The Endo-Arabinanase Displays Activity toward Potato protein was in range with the 34 kD (Skjøt et al., 2001) Rhamnogalacturonan I in Vitro determined for the arabinanase when expressed in Aspergillus oryzae (Fig. 3). The absence of tubers from A purified recombinant endo-arabinanase from As- the pGED/ARA plants made it particularly relevant pergillus aculeatus shows endo-activity in vitro to develop a Golgi-anchored version of the arabinan- against debranched sugar beet arabinan releasing ase as an alternative approach to modifying pectins primarily arabinobiose and arabinotriose (Skjøt et al., in planta. 2001). We verified that it is also active toward RGI isolated from wild-type (WT) potato tubers. Monosaccharide analysis of isolated RGI from potato Generation of Potato Plants Expressing a treated with the arabinanase, showed that enzyme Golgi-Localized Arabinanase treatment resulted in a 75% reduction in the Ara To generate a Golgi-localized enzyme, we fused the content compared with the untreated sample (not 52 N-terminal amino acids of a rat ␣-2,6 sialyl trans- shown). ferase (ST; Munro, 1991) to the mature part of the arabinanase creating pGED/ST::ARA (Fig. 1). The Tubers Are Not Recovered if Arabinanase Is fusion protein has a calculated molecular mass of 39 Targeted to the Apoplast kD. Previous analyses of the primary structure of ST suggest that the protein comprises a nine-residue The cDNA encoding an A. aculeatus endo-␣-1,5- N-terminal cytoplasmic domain, a 17-residue hydro- arabinanase including the fungal secretion signal phobic sequence that serves as a membrane anchor (Skjøt et al., 2001) was transcriptionally fused to the and signal sequence, and a large Golgi-lumenal, cat- tuber-specific granule-bound starch synthase pro- alytic domain (Weinstein et al., 1987). The region moter (Visser et al., 1991), giving the vector pGED/ including the N-terminal cytoplasmic domain, the ARA (Fig. 1). Transformation with pGED/ARA re- hydrophobic sequence, and 26 residues of the cata- duced the transformation frequency: 27% compared lytic domain has previously been shown to direct with approximately 80% for the empty pGED vector. Golgi targeting of green fluorescent protein (Boevink Regenerated in vitro plantlets were tested for the et al., 1998) and lysozyme (Munro, 1991)