Plant Biotechnology Journal (2006) 4, pp. 409–418 doi: 10.1111/j.1467-7652.2006.00190.x © 2006 Blackwell Publishing Ltd 409 Blackwell Publishing Ltd Oxford, UK PBI Plant Biotechnology Journal 1467-7644 © 2006 Blackwell Publishing Ltd ? 2006 2 ? Original Article Production of high-starch, low-glucose potatoes Rowan S. McKibbinet al. Production of high-starch, low-glucose potatoes through over-expression of the metabolic regulator SnRK1 Rowan S. McKibbin 1, †, Nira Muttucumaru 2 , Matthew J. Paul 2 , Stephen J. Powers 3 , Michael M. Burrell 4, ‡, Steve Coates 4 , Patrick C. Purcell 1, §, Axel Tiessen 5, ¶, Peter Geigenberger 5 and Nigel G. Halford 2, * 1 Long Ashton Research Station, Bristol BS41 9AF, UK 2 Crop Performance and Improvement, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK 3 Biomathematics and Bioinformatics, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK 4 Advanced Technologies (Cambridge) Ltd, 210 Cambridge Science Park, Milton Road, Cambridge CB4 0WA, UK 5 Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Golm, Germany Summary Transgenic potato (Solanum tuberosum cv. Prairie) lines were produced over-expressing a sucrose non-fermenting-1-related protein kinase-1 gene (SnRK1) under the control of a patatin (tuber-specific) promoter. SnRK1 activity in the tubers of three independent transgenic lines was increased by 55%-167% compared with that in the wild-type. Glucose levels were decreased, at 17%-56% of the levels of the wild-type, and the starch content showed an increase of 23%-30%. Sucrose and fructose levels in the tubers of the transgenic plants did not show a significant change. Northern analyses of genes encoding sucrose synthase and ADP-glucose pyrophosphorylase, two key enzymes involved in the biosynthetic pathway from sucrose to starch, showed that the expression of both was increased in tubers of the transgenic lines compared with the wild-type. In contrast, the expression of genes encoding two other enzymes of carbohydrate metabolism, α-amylase and sucrose phosphate synthase, showed no change. The activity of sucrose synthase and ADP-glucose pyrophosphorylase was also increased, by approximately 20%– 60% and three- to five-fold, respectively, whereas the activity of hexokinase was unchanged. The results are consistent with a role for SnRK1 in regulating carbon flux through the storage pathway to starch biosynthesis. They emphasize the importance of SnRK1 in the regulation of carbohydrate metabolism and resource partitioning, and indicate a specific role for SnRK1 in the control of starch accumulation in potato tubers. Received 28 October 2005; revised 10 February 2006; accepted 13 February 2006. *Correspondence (fax (44) (0) 1582 763 010; e-mail nigel.halford@bbsrc.ac.uk) †Present address: Biotechnology and Biological Sciences Research Council (BBSRC), Polaris House, North Star Avenue, Swindon SN2 1UH, UK ‡ Present address : Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK §Present address: Patent Office, PO Box 49, Cardiff Road, Newport NP10 8YU, UK ¶ Present address : CIMMYT (International Maize and Wheat Improvement Center), Apdo. Postal 6-641, Col. Juárez. C.P. 06600, México, D.F. Keywords: ADP-glucose pyrophosphorylase, carbohydrate metabolism, glucose, metabolite signalling, protein kinase, SNF1, Solanum tuberosum , sucrose synthase, sugar sensing, starch. Introduction High starch and low glucose levels are desirable traits in commer- cial potato tubers. More than two million tonnes of starch are pro- duced annually from potato for industrial uses, and an increase in starch content also reduces the processing costs of potato food products such as crisps and French fries. Low glucose improves processing properties because glucose causes blackening during frying. The aim of this study was to modify the carbohydrate content of potato tubers by manipulating the metabolic regulator sucrose non-fermenting-1-related protein kinase-1 (SnRK1). SnRK1 is a serine/threonine protein kinase that takes its name from sucrose non-fermenting-1 (SNF1), its homologue in yeast ( Saccharomyces cerevisiae ) (Celenza and Carlson, 1986). There is also a member of the family in the animal kingdom, AMP-activated protein kinase (AMPK). SnRK1, SNF1 and AMPK are heterotrimeric complexes. In animals, these comprise an α subunit containing the protein kinase catalytic domain, a β subunit and a γ subunit that interacts with a regulatory domain in the α subunit (Woods et al ., 1996). The yeast complex comprises a catalytic subunit encoded by the SNF1 gene itself, a second subunit that can be any one of a class of proteins that includes SIP1, SIP2 and GAL83, which are homologues of the animal β subunit, and a regulatory subunit called SNF4 (Celenza et al ., 1989) that is homologous to AMPK γ .