RESEARCH ARTICLE Arabidopsis sucrose non-fermenting-1-related protein kinase-1 and calcium-dependent protein kinase phosphorylate conserved target sites in ABA response element binding proteins Y. Zhang 1 , P.J. Andralojc 1 , S.J. Hey 1 , L.F. Primavesi 1 , M. Specht 2 , J. Koehler 2 , M.A.J. Parry 1 & N.G. Halford 1 1 Plant Science Department, Rothamsted Research, Harpenden, Hertfordshire, UK 2 Biomathematics and Bioinformatics Department, Rothamsted Research, Harpenden, Hertfordshire, UK Keywords AREBP; CDPK; metabolic signalling; SnRK1; SnRK2; SnRK3; stress. Correspondence N.G. Halford, Plant Science Department, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK. Email: nigel.halford@bbsrc.ac.uk Received: 23 October 2008; revised version accepted: 27 October 2008. doi:10.1111/j.1744-7348.2008.00302.x Abstract Evidence is provided that plant transcription factors of the ABA response element binding protein (AREBP) class are phosphorylated by sucrose non-fermenting-1- related protein kinase-1 (SnRK1) and a calcium-dependent protein kinase at highly conserved target sites. Two target sites for SnRK1 were identified in AREBPs using a specially developed motif search pipeline. Peptides containing the AREBP sites were phosphorylated by purified SnRK1 in vitro and by cal- cium-dependent and calcium-independent activities present in soluble protein extracted from Arabidopsis seedlings grown in liquid culture. Most (69–77%) of the calcium-independent phosphorylation of these peptides was removed by immunoprecipitation using anti-SnRK1 antisera, linking this activity unambig- uously to SnRK1. It is possible that the protein kinase responsible for the cal- cium-dependent activity was SnRK3, a protein kinase that is related to SnRK1 and which has similar requirements for target site recognition. However, the involvement of other calcium-dependent protein kinases cannot be ruled out. Introduction The last few years has seen a burgeoning of interest in links between stress and metabolite signalling in plants. This began with the results of screens carried out by several independent groups to identify Arabidopsis (Arabidopsis thaliana) mutants that are impaired in their response to sugar (sugar response mutants). Several of the mutants identified in these screens turned out to be ABA related, such as ABI5 mutants (Brocard et al., 2002). Interest has been stimulated further by evidence placing sucrose non-fermenting-1 (SNF1)-related protein kinases (SnRKs) in stress signalling pathways. Sucrose non-fermenting-1-related protein kinases can be divided into three classes, SnRK1, SnRK2 and SnRK3 (Halford et al., 2003; Halford, 2006). SnRK1 is a meta- bolic regulator that is activated in response to high cellu- lar sucrose/low cellular glucose and regulates carbon metabolism through the modulation of enzyme activity and gene expression (Halford, 2006). It is structurally and functionally related to the yeast protein kinase, SNF1, which gives the family its name, and to mamma- lian AMP-activated protein kinase. SnRK1 phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-coenzyme A reductase, sucrose phosphate synthase, nitrate reductase, trehalose-phosphate synthase and 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase (Kulma et al., 2004; Halford, 2006; Harthill et al., 2006). In addition, SnRK1 is required for redox modulation of ADP-glucose pyro- phosphorylase activity in response to sucrose (Tiessen et al., 2003) and, like its fungal and animal counterparts, is involved in regulating the expression of genes in- volved in carbon metabolism. Antisense inhibition of SnRK1 gene expression has been shown to reduce su- crose synthase gene expression in potato tubers (Purcell et al., 1998) and a-amylase gene expression in cultured Annals of Applied Biology ISSN 0003-4746 Ann Appl Biol 153 (2008) 401–409 ª 2008 The Authors Journal compilation ª 2008 Association of Applied Biologists 401