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Modification of Potato Pectin by in Vivo Expression of an Endo-1,4-ß-D

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Modification of Potato Pectin by in Vivo Expression of an Endo-1,4-ß-D Pectin engineering: Modification of potato pectin by in vivo expression of an endo-1,4-␤-D-galactanase Susanne Oxenbøll Sørensen*†, Markus Pauly‡, Max Bush§, Michael Skjøt*, Maureen C. McCann§, Bernhard Borkhardt*, and Peter Ulvskov* *Biotechnology Group, Danish Institute of Agricultural Sciences, DK-1871 Copenhagen, Denmark; ‡Department of Plant Biology, The Royal Veterinary and Agricultural University, DK-1871 Copenhagen, Denmark; and §Department of Cell Biology, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom Edited by Adrienne Clarke, University of Melbourne, Victoria, Australia, and approved April 28, 2000 (received for review December 27, 1999) Potato tuber pectin is rich in galactan (oligomer of ␤-1,4-linked characterization. In addition, during the process of starch isolation, galactosyl residues). We have expressed a fungal endo-galactanase the potato starch industry generates large volumes of waste pulp cDNA in potato under control of the granule bound starch synthase that is relatively rich in pectins (11). Potato pectin is less suitable for promoter to obtain expression of the enzyme in tubers during many food industrial uses (e.g., as a gelling agent) than pectins from growth. The transgenic plants displayed no altered phenotype com- other sources in part because of its high content of neutral galac- pared with the wild type. Fungal endo-galactanase activity was tans. A remodeling of pectin structure by transgenic approaches quantified in the transgenic tubers, and its expression was verified by may allow improvement of potato pectin quality. Western blot analysis. The effect of the endo-galactanase activity on In this paper, we report the generation of potato transformants potato tuber pectin was studied by Fourier transform infrared mi- expressing a 38-kDa endo-galactanase (EC 3.2.1.89) isolated from crospectroscopy, immuno-gold labeling, and sugar analysis. All anal- Aspergillus aculeatus (12). The endo-galactanase degrades unsub- yses revealed alterations in pectin composition. Monosaccharide stituted 1,4-␤-D-galactan to galactose and galactobiose at optimal composition of total cell walls and isolated rhamnogalacturonan I conditions of pH 4.0 and 50–55°C (13, 14). The coding region of the fragments showed a reduction in galactosyl content to 30% in the endo-galactanase contains a signal sequence for secretion. Because transformants compared with the wild type. Increased solubility of signal sequences among eukaryotes are very similar (15), we expect pectin from transgenic cell walls by endo-polygalacturonase͞pectin the fungal signal sequence to function in a plant cell, resulting in methylesterase digestion points to other changes in wall architecture. secretion of the enzyme to the apoplast. We have determined PLANT BIOLOGY endo-galactanase activity in tubers of transgenic potato plants and ectic polysaccharides comprise between 30 and 50% of the studied the effect of the introduced gene on tuber pectic compo- Pcell walls of dicotyledonous plants (1). The pectic matrix of sition as determined by monosaccharide composition, Fourier plant cell walls is a complex mixture of homogalacturonan transform infrared microspectroscopy, and immuno-gold labeling (HGA), rhamnogalacturonan I (RGI), and rhamnoglacturonan with a galactan-specific antibody. This is a direct modification of the II polymers (2). HGA is an unbranched chain of ␣-1,4-linked monosaccharide profile of a plant cell-wall polysaccharide by galacturonic acid (GalA) residues that can be differentially transgenic experiment. methyl-esterified and͞or acetylated. RGI is a branched hetero- polymer of alternating ␣-1,2-linked rhamnose and ␣-1,4-linked Materials and Methods GalA residues (3) that carries neutral side-chains of predomi- Reagents and Enzymes. Unless otherwise stated, chemicals and nantly 1,4-␤-D-galactose and͞or 1,5-␣-L-arabinose residues at- reagents were purchased from Sigma. The porcine pancreatic tached to the rhamnose residues of the RGI backbone. RGI ␣-amylase was purchased from Merck, and the pullulanase from side-chains may be decorated with arabinosyl residues (arabi- Bacillus acidopullulyticus and endo-polygalacturonase (EPG) from nogalactan I) or other sugars, including fucose, xylose, and Aspergillus niger were purchased from Megazyme International. mannose. The highly conserved rhamnogalacturonan II mole- The purified recombinant pectin methylesterase (PME) from A. cule has a HGA backbone with side-chains containing the richest aculeatus was a gift from K. Schnorr (Novo Nordisk A͞S, Bagsværd, diversity of sugars and linkages known and can dimerize through Denmark). a borate di-ester cross-link (4). Although the composition of these molecules is well characterized, their assembly into higher- Transformation and Regeneration of Potato Plants. The granule order structures and their function in wall architecture is unclear. bound starch synthase promoter was amplified by PCR from Diverse roles have been proposed for pectic polymers (5–9), the plasmid pPGB121s (16) by using oligonucleotide primers including the regulation of cell-cell adhesion, cell expansion, wall 5Ј-GATTACGCCAAGCTTTAACG (HindIII site italicized) and mechanical properties, mediation of wall porosity, a source of 5Ј-GGTTTTCTAGAGTCGACGAAATCAGAAATAATT- signaling molecules (oligosaccharins), and involvement in cell GGAGG (XbaI site italicized) and was inserted as a HindIII-XbaI differentiation and organogenesis. To address the function of fragment into pBI121 (17), generating pPGB121-new. The coding pectins in these processes, we need to manipulate the quantities region of ␤-glucuronidase was removed by digestion with SmaI and of different types of wall polysaccharides and examine the SacI followed by Klenow DNA polymerase and religation, resulting downstream consequences. One strategy is the random gener- in pPGB121s-new. The HindIII site upstream of the promoter ation of cell wall mutants with altered monosaccharide compo- sition (10). Only one of these, the Arabidopsis mur8, is deficient in rhamnose and is likely to be a mutant in which pectin This paper was submitted directly (Track II) to the PNAS office. composition is specifically altered (10). An alternative strategy Abbreviations: EPG, endo-polygalacturonase; FTIR, Fourier transform infrared microspec- troscopy; GalA, galacturonic acid; HGA, homogalacturonic acid; PCA, principal component is to directly manipulate pectins in transgenic plants that over- analysis; PME, pectin methylesterase; RGI, rhamnogalacturonan I; UA, uronic acid. express cell-wall degrading enzymes. As many fungal enzymes †To whom reprint requests should be addressed. E-mail: [email protected]. have evolved to degrade specific parts of the pectic network, this The publication costs of this article were defrayed in part by page charge payment. This strategy offers a wide range of potential cell-wall phenotypes. article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. We have chosen potato (Solanum tuberosum L.) as a model §1734 solely to indicate this fact. system for these studies because it is easy to transform and provides Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.130568297. large quantities of transformed tissue and hence cell walls for Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.130568297 PNAS ͉ June 20, 2000 ͉ vol. 97 ͉ no. 13 ͉ 7639–7644 Downloaded by guest on September 24, 2021 region was deleted by digestion with HindIII, fill in of the 5Ј- through a nylon mesh (pore size 30 ␮m), and the resulting residue overhangs with Taq DNA polymerase and religation to generate on the filter was added back to the pellet. The pellet was resus- pPGB121s-B. A polylinker with restriction sites for HindIII, XbaI, pended in 100 ml of ice-cold buffer C (0.5% sodium deoxy- ͞ ͞ and XhoI was produced by annealing of the two synthetic oligonu- cholate 20 mM Hepes, pH 7.5 3mMNa2S2O5,) and was stirred at cleotides 5Ј-TCGACAAGCTTTCTAGAGCCTCGAGG (SalI 4°C for 30 min. The centrifugations and subsequent washes in buffer overhang italicized) 5Ј- GATCCCTCGAGGCTCTAGAAAGC- C were repeated three times. The final wash was done overnight. TTG (BamHI overhang italicized) and was cloned into SalI͞ The pellet was resuspended and centrifuged three times in 150 ml BamHI-cleaved pPGB121s-B, generating the plasmid pGED. The of ice-cold Milli-Q water before extraction with 150 ml PAW 1.3-kb cDNA encoding an A. aculeatus endo-galactanase was (phenol:acetic acid:water, 2:1:1 by volume) for3hatroomtem- ϫ excised from the vector pC1G1 (a gift from S. Kauppinen, Novo- perature followed by centrifugation (15,000 gmax) for 20 min at Nordisk A͞S, Bagsværd, Denmark) by digestion with HindIII and 24°C. The pellet was washed three times with 150 ml of water before XbaI and was cloned into pGED, giving rise to the plasmid resuspension in 150 ml of 25 mM NaOAc (pH 4.0); it was then pGED-GAL. This plasmid was introduced into Agrobacterium heated for 20 min at 80°C to gelatinize the starch and was tumefaciens strain LBA 4404 by electroporation. Transformation of subsequently cooled on ice. The pH was adjusted to 5.0 with 1 M potato leaf discs followed a two-media regeneration procedure NaOH before addition of 6 mg of ␣-amylase and 60 ␮l of pullu- essentially as described by Edwards et al. (18) by using kanamycin lanase (1,000 units͞ml). The mixture was incubated for3hat37°C, selection. Both transformed and wild-type plants were regenerated the enzymes were inactivated by stirring in
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