Plant Growth Regulation 22: 181–188, 1997. 181 c 1997 Kluwer Academic Publishers. Printed in the Netherlands. Identification of a sucrose nonfermenting-1-related protein kinase in sugar beet (Beta vulgaris L.) Wendy A. Monger , Tudor H. Thomas, Patrick C. Purcell & Nigel G. Halford IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS18 9AF, UK ( To whom correspondence should be addressed; current address: School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK) Received 21 July 1997; accepted 21 July 1997 Key words: cellular signalling, metabolism, phosphorylation, SNF1, sugar-sensing Abstract A 154 bp polymerase chain reaction product, SBKIN154, showing 76–83% sequence identity with sucrose nonfermenting-1 (SNF1)-related protein kinase nucleotide sequences from other plant species was amplified from sugar beet storage root RNA. Southern blot analysis using SBKIN154 as a hybridisation probe suggested that sugar beet contains either a single-copy SNF1-related gene or a small gene family of highly conserved genes. An antibody raised to a heterologously-expressed fusion of the rye SNF1-related protein kinase, RKIN1, and maltose binding protein, recognised a protein of the expected size (M r approx. 60,000) on western blots of storage root, stalk, leaf and root extracts. Measurements of SNF1-related activity were made using a specific peptide (SAMS) phosphorylation assay. Activity was highest (0.38 nmol min 1 mg 1 protein) in developing storage roots and lowest (0.035 nmol min 1 mg 1 ) in fibrous roots. Abbreviations: SNF1 = sucrose nonfermenting-1; MBP = maltose binding protein; AMPK = AMP-activated protein kinase; HMG-CoA = 3-hydroxy-3-methylglutaryl-Coenzyme A; PCR = polymerase chain reaction; RT = reverse transcriptase; TBE = Tris, borate, EDTA gel running buffer; SSPE = standard saline phosphate EDTA; SSC = standard saline citrate; SDS = sodium dodecyl sulphate 1. Introduction Members of the sucrose nonfermenting-1 (SNF1) subfamily of protein kinases have been identified in a wide range of eukaryotes [16]. The first to be characterised genetically was SNF1 itself from the budding yeast (Saccharomyces cerevisiae) [6]. SNF1 is required for the derepression of many genes which are repressed when sufficient levels of glucose are available to satisfy the carbon requirement of the culture [30], including several which encode enzymes of carbohydrate catabolism, such as SUC2 (invertase). SNF1 also directly modulates the phosphorylation state of a number of metabolic enzymes, including acetyl- CoA carboxylase [32] and glycogen synthase [17], and is required for the arrest of growth and the cell cycle when yeast runs out of a carbon source [31]. The vertebrate homologue of SNF1 is the AMP-activated protein kinase (AMPK) [5]. AMPK has not yet been shown to be involved in transcriptional regulation, but does control the activity of acetyl-CoA carboxylase and HMG-CoA reductase [28, 7]. SNF1-related sequences have been isolated from several plant species, including rye (RKIN1) [1], Arabidopsis (AKIN10) [22], barley (BKIN2, BKIN12) [14, 15], potato (PKIN1) [24] and tobacco (NPK5) [26]. RKIN1, which was the first to be isolated, was shown to complement a yeast snf1 mutant [1]. It has also been demonstrated that plant SNF1-related protein kinases will phosphorylate HMG-CoA reductase and a synthetic peptide (SAMS peptide) [2, 4], both of which are substrates for AMPK, and, intriguingly, spinach nitrate reductase will also act as a substrate in vitro [10]. The plant, animal and yeast SNF1-related protein