Inhibition of SNF1-related protein kinase1 activity and regulation of metabolic pathways by trehalose-6-phosphate

Trehalose-6-phosphate (T6P) is a proposed signaling molecule in plants, yet how it signals was not clear. Here, we provide evidence that T6P functions as an inhibitor of SNF1-related protein kinase1 (SnRK1; AKIN10/AKIN11) of the SNF1-related group of protein kinases. T6P, but not other sugars and sugar phosphates, inhibited SnRK1 in Arabidopsis (Arabidopsis thaliana) seedling extracts strongly (50%) at low concentrations (1–20 μm). Inhibition was noncompetitive with respect to ATP. In immunoprecipitation studies using antibodies to AKIN10 and AKIN11, SnRK1 catalytic activity and T6P inhibition were physically separable, with T6P inhibition of SnRK1 dependent on an intermediary factor. In subsequent analysis, T6P inhibited SnRK1 in extracts of all tissues analyzed except those of mature leaves, which did not contain the intermediary factor. To assess the impact of T6P inhibition of SnRK1 in vivo, gene expression was determined in seedlings expressing Escherichia coli otsA encoding T6P synthase to elevate T6P or otsB encoding T6P phosphatase to decrease T6P. SnRK1 target genes showed opposite regulation, consistent with the regulation of SnRK1 by T6P in vivo. Analysis of microarray data showed up-regulation by T6P of genes involved in biosynthetic reactions, such as genes for amino acid, protein, and nucleotide synthesis, the tricarboxylic acid cycle, and mitochondrial electron transport, which are normally down-regulated by SnRK1. In contrast, genes involved in photosynthesis and degradation processes, which are normally up-regulated by SnRK1, were down-regulated by T6P. These experiments provide strong evidence that T6P inhibits SnRK1 to activate biosynthetic processes in growing tissues.Suc and trehalose are widespread nonreducing disaccharides that function as translocated carbon sources and stress protection compounds. Plants and cyanobacteria are the only organisms in which the pathways of trehalose and Suc synthesis coexist. In the majority of plants, trehalose occurs in trace amounts only, prohibiting a function as a carbon source. This raises the question of the role of the trehalose pathway in plants, given the large number and ubiquity of putative genes encoding enzymes for trehalose synthesis (Avonce et al., 2006; Lunn, 2007). Many of these genes are subject to a high level of regulation at the gene (Paul et al., 2008) and posttranslational (Harthill et al., 2006) levels, suggesting an important function. This is confirmed in transgenic and mutant plants with modified trehalose pathway gene expression, which show a range of phenotypes. For example, a trehalose-6-P synthase (TPS1) has been shown to be essential for embryo development (Eastmond et al., 2002) and for normal vegetative growth and the transition to flowering (Van Dijken et al., 2004). Overexpression of TPS from different species, for example, otsA encoding Escherichia coli TPS, produces effects on sugar utilization in seedlings (Schluepmann et al., 2003) and on vegetative and photosynthetic phenotypes (Pellny et al., 2004; Almeida et al., 2007; Stiller et al., 2008), opposite in nature to seedling and photosynthetic phenotypes of plants expressing E. coli otsB encoding trehalose-6-P phosphatase (TPP; Schluepmann et al., 2003; Pellny et al., 2004). The phenotype of the maize (Zea mays) ramosa3 mutant has been attributed to a knockout of a TPP gene normally expressed in discrete domains subtending axillary meristems (Satoh-Nagasawa et al., 2006). This gives rise to an inflorescence architecture that aids the efficient packing and harvesting of seeds. Trehalose-6-P (T6P) has been found to stimulate starch synthesis via redox activation of ADP-Glc pyrophosphorylase (Kolbe et al., 2005), and T6P responds to light and sugar in relation to carbon status (Lunn et al., 2006). This supports a role for T6P in signaling the sugar status of the cytosol to the chloroplast and thereby activating starch synthesis. However, most of the phenotypes produced where the pathway has been genetically modified in mutant and transgenic lines, which also extend to effects on abiotic stress resistance (Garg et al., 2002; Avonce et al., 2004), cell division, cell walls (Gómez et al., 2006), and cell shape (Chary et al., 2008), cannot be explained simply in terms of the effect on starch metabolism but rather support a more central function. In some fungi, T6P inhibits hexokinase, an enzyme implicated in sugar signaling in plants and other organisms (Moore et al., 2003), but there is no evidence that T6P affects plant hexokinases (Eastmond et al., 2002). Another important signaling route in plants is through SNF1-related protein kinase1 (SnRK1) of the family of calcium-independent Ser/Thr protein kinases that includes AMPK of mammals and SNF1 of yeast (Hardie, 2007; Polge and Thomas, 2007). These conserved kinases perform a fundamental role in transcriptional, metabolic, and developmental regulation in response to energy limitation and starvation of the carbon source (Hardie, 2007). SnRK1 in plants is thought to consist of a heterotrimeric complex, as in AMPK and SNF1, composed of an AKIN10 or AKIN11 catalytic α-subunit and β- and γ-subunits together with a number of additional interacting and regulatory factors (Pierre et al., 2007; Polge and Thomas, 2007; Ananieva et al., 2008). Recent work established that AKIN10 catalytic activity regulates 1,000 or so target genes involved in the response of metabolism and growth to starvation (Baena-González et al., 2007). It was shown that SnRK1 activates genes involved in degradation processes and photosynthesis and inhibits those involved in biosynthetic processes and, by so doing, regulates metabolism and growth in response to available carbon (Baena-González et al., 2007). SnRK1 can phosphorylate class II TPSs (Glinski and Weckwerth, 2005; Harthill et al., 2006) and regulate their transcription (Baena-González et al., 2007), although the importance of this is not clear. SnRK1 transcript increased in response to 100 mm trehalose feeding (Schluepmann et al., 2004) and was decreased 2-fold in tps1 mutants (Gómez et al., 2006). Given a possible interaction between the two pathways, we went on to determine whether T6P affects the catalytic activity of SnRK1. Here, we provide evidence for a function of T6P as an inhibitor of SnRK1 activity. First, we show that low micromolar concentrations of T6P inhibit SnRK1 activity in Arabidopsis (Arabidopsis thaliana) seedling extracts and other young plant material, but not in mature leaves. Second, we show that T6P inhibits SnRK1 at a site distinct and separable from the SnRK1 catalytic site via an intermediary factor. This as yet unknown intermediary factor was not found in mature leaves. Third, we establish effects on gene expression in seedlings with elevated T6P consistent with inhibition of SnRK1 in vivo. Overall, the data provide strong evidence for a function of T6P as an inhibitor of SnRK1 to promote biosynthetic reactions in growing tissues.