The proteins kinases SNF1/AMPK/SnRK1 are a subfamily of serine/threonine kinases that act as metabolite sensors to constantly adapt metabolism to the supply of, and demand for, energy. In the yeast Saccharomyces cerevisiae, the SNF1 complex is a central component of the regulatory response to glucose starvation. AMP activated protein kinase (AMPK) the mammalian homologue of SNF1, plays a central role in the regulation of energy homeostasis at the cellular as well as the whole-body levels. In Arabidopsis thaliana, SnRK1.1 and SnRK1.2 have recently been described as central integrators of a transcription network for stress and energy signalling. In this study, biochemical analysis established SnRK1.1 as the major SnRK1 isoform both in isolated cells and leaves. In order to elucidate the function of SnRK1.1 in Arabidopsis thaliana, transgenic plants over-expressing SnRK1.1 were produced. Genetic, biochemical, physiological and molecular analyses of these plants revealed that SnRK1.1 is implicated in sugar and ABA signalling pathways. Modifications of the starch and soluble sugar content were observed in the 35S:SnRK1.1 transgenic lines. Our studies also revealed modifications of the activity of essential enzymes such as nitrate reductase or ADP-glucose pyrophosphorylase, and of the expression of several sugar-regulated genes, confirming the central role of the protein kinase SnRK1 in the regulation of metabolism.
The proteins kinases SNF1/AMPK/SnRK1 represent a subfamily of serine/threonine kinases that are highly conserved throughout evolution. They act as metabolite sensors that allow the organism to adapt its metabolism to the supply of, and demand for, energy. In response to metabolic stress, these kinases switch off anabolic pathways and switch on catabolic pathways via regulation of gene expression or via direct phosphorylation of key enzymes (Hardie, 2004; Polge and Thomas, 2007). In the yeast Saccharomyces cerevisiae, the SNF1 complex is a central component of the response to glucose starvation, mediating the shift from fermentative to oxidative metabolism by regulating de-repression of glucose-repressed genes (Carlson, 1999; Young et al., 2003). In response to an increase in the AMP/ATP ratio in the cell, AMPK inhibits lipid, carbohydrate and protein syntheses via phosphorylation of key enzymes to prevent consumption of ATP (Rutter et al., 2003; Hardie, 2007), and concomitantly activates glucose uptake, glycolysis and glucose and fatty acid oxidation to generate ATP (Hardie, 2004, 2007). Due to its involvement in the regulation of numerous processes, several diseases are provoked by misregulation of AMPK (Kahn et al., 2005; Hardie et al., 2006; Kola et al., 2006; Long and Zierath, 2006). In plants, SNF1-related kinase (SnRK1) responds to the availability of carbohydrate, and has been implicated in the control of carbohydrate and starch metabolism (Polge and Thomas, 2007). Evidence indicates a role for SnRK1 in the control of starch accumulation in non-photosynthetic organs (potato tubers), by regulation of redox-dependent ADP-glucose pyrophosphorylase (AGPase) (Tiessen et al., 2003; McKibbin et al., 2006). The SnRK1 protein kinases can also phosphorylate and inactivate several enzymes in cell-free assays, such as nitrate reductase (NR), sucrose phosphate synthase (SPS) and fructose-2,6-bisphosphatase (F2KP) (Sugden et al., 1999b; Kulma et al., 2004). Metabolic regulation induced by the SnRK1 protein kinases also occurs by transcriptional regulation of a large set of genes, including those that encode sucrose synthase, α-amylase or DIN6 (Purcell et al., 1998; Laurie et al., 2003; Baena-Gonzalez et al., 2007).
While AMPK is clearly activated by AMP binding to the γ subunits, the metabolites responsible for the regulation of the plant SnRK1 have not yet been identified. These kinases do not appear to be directly activated by AMP, although AMP inhibits their dephosphorylation (Sugden et al., 1999a). It has also been shown that these kinases are activated in response to sucrose (Bhalerao et al., 1999) and 2-deoxyglucose, a non-metabolized analogue of glucose (Harthill et al., 2006), but whether this is due to direct interaction is not known. Finally, glucose-6-phosphate may inhibit some SnRK1 complexes (Toroser et al., 2000).
Sugar signalling pathways and their interactions with each other and with hormonal signalling pathways are now being intensively studied (Rolland et al., 2006). Genetic approaches have demonstrated the importance of abscisic acid (ABA) in sugar response pathways, with both pathways using common signalling components (Cheng et al., 2002). Several reports have indicated that SnRK1 could be implicated in these interactions (Nemeth et al., 1998; Bradford et al., 2003; Thelander et al., 2004). Moreover, it has been suggested recently that SnRK1 plays a key role during germination, and could mediate ABA functions during seed maturation (Radchuk et al., 2006; Lu et al., 2007).
Studying SnRK1s remains difficult because of their implication in multiple functions and mechanisms. Recently, Baena-Gonzalez et al. implicated SnRK1.1 and SnRK1.2 (also known as AKINα1/AKIN10/At3g01090 and AKINα2/AKIN11/At3g29160) as central integrators of transcription networks in response to stress and energy signalling, using transient expression in protoplasts and transgenic plants (Baena-Gonzalez et al., 2007; Baena-Gonzalez and Sheen, 2008). In order to delineate more precisely the function of the two protein kinases SnRK1.1 and SnRK1.2 in sugar signalling in Arabidopsis thaliana, we first estimated the contribution of each kinase to total SnRK1 activity using a biochemical approach. Transgenic plants over-expressing SnRK1.1 cDNA were obtained, and their analysis indicated that SnRK1.1 is involved in various sugar and ABA signalling pathways, resulting in modifications of essential enzyme activities and gene expression. The biochemical approaches emphasize the regulation of SnRK1 complexes in Arabidopsis thaliana by carbohydrates and the potential role of the upstream kinases.
SnRK1.1 is responsible for the major part of SnRK1 activity in extracts of A. thaliana suspension cells
In order to assess the contribution of SnRK1.1 and SnRK1.2 to total SnRK1 activity, kinase activities were partially purified from extracts of A. thaliana suspension cells and assayed using the AMARA peptide to detect SnRK1 activity (Dale et al., 1995). After preliminary purification steps that did not remove any AMARA kinase activity, purification by chromatography on MonoQ column (GE Healthcare, http://www.gehealthcare.com) revealed three peaks of kinase activity (I–III), eluting at 75, 250 and 320 mm NaCl, respectively (Figure 1a). None of these activities was inhibited by EGTA or stimulated by Ca2+ (data not shown). Because of their high molecular mass and Ca2+-independent activity, these three peaks must contain SnRK1 complexes rather than SnRK2, SnRK3 or Ca2+-dependent kinases (data not shown). In order to identify these kinases more precisely, antibodies raised against deduced protein sequences of SnRK1.1 and SnRK1.2 were used in Western blot analyses of the fractions from MonoQ purification. This revealed the presence of a 58 kDa polypeptide recognized by the anti-SnRK1.1 antibody in all fractions containing AMARA peptide kinase activity, whereas only peak III contained SnRK1.2 (Figure 1b). Finally, it was shown that, after immunoprecipitation assays using anti-SnRK1.1/SnRK1.2 antibodies, complexes containing SnRK1.1 contribute approximately 90% of the total AMARA kinase activity, while complexes containing SnRK1.2 contribute only approximately 10% in crude extracts of A. thaliana cell cultures (Figure 1c). In the leaves of 15-day-old in vitro-grown seedlings, the contribution of SnRK1.1 to total activity was even higher, with SnRK1.2 being undetectable under these conditions (Figure 1c). For this reason, our work has focused on the SnRK1.1 isoform.
Over-expression of SnRK1.1 confers a glucose-hypersensitive phenotype
A functional analysis of SnRK1.1 kinase was performed by using transgenic plants over-expressing this isoform. Three lines over-expressing SnRK1.1 were isolated and named 35S:SnRK1.1-1, 35S:SnRK1.1-2 and 35S:SnRK1.1-3. Using northern blotting, these three lines were found to express SnRK1.1 mRNA at levels 15-, 22- and 10-fold higher than the wild-type (Col), respectively (Figure 2a). These data were supported by the results of Western blot experiments performed on leaves of 2-week-old seedlings, showing that, while the SnRK1.1 protein was barely detectable in the wild-type (Col), the signal was 4-, 3.5- and 2.5-fold higher in 35S:SnRK1.1-1, 35S:SnRK1.1-2 and 35S:SnRK1.1-3 plants, respectively (Figure 2b).
As SNF1-related proteins are likely to play a major role in sugar signalling, we determined whether the increase in SnRK1.1 protein in 35S:SnRK1.1 plants was correlated with an increase in SnRK1.1 kinase activity when cultivated on glucose. Plants directly grown on high concentrations of glucose (>4%) showed important developmental alterations. We thus cultivated the three 35S:SnRK1.1 lines for 15 days on grids laid on half-strength MS solid culture medium in low glucose (0.2%). The grids were then transferred or not onto half-strength MS liquid medium containing either 6% glucose, 6% sorbitol or no sugar (control), and the leaves were harvested after 25 h (16 h light/8 h dark/1 h light). No difference in activity was observed for the plants that were not transferred (Figure 2c) or transferred to the non-signalling osmotic control, sorbitol (Figure 2c, Sorb). The three transgenic lines over-expressing SnRK1.1 protein showed 30% higher SnRK1.1 activity than the wild-type when transferred to half-strength MS medium (Figure 2c, Ctrl). When transferred to 6% glucose, the activity of both wild-type and over-expressing plants increased by a further 30% (Figure 2c, Glc). The activity of SnRK1.1 increased significantly only when plants were transferred to glucose (both wild-type and transgenic lines). Further experiments were thus conducted on plants transferred to half-strength MS medium, glucose or sorbitol.
These results show that an exogenous supply of glucose led to increased SnRK1.1 activity in vivo in both wild-type and in 35S:SnRK1.1 transgenic plants. Interestingly, when growing plants directly on half-strength MS medium containing increasing amounts of glucose (2–6%), the over-activation of SnRK1.1 led to a clear glucose hypersensitivity phenotype (Figure 2d). Indeed, after 7 days of culture in the presence of 4% glucose, 35S:SnRK1.1 lines showed growth delay and an absence of root growth compared to wild-type seedlings. When grown on 6% glucose, the development of transgenic lines stopped just after germination (Figure 2d). The growth-delay phenotype was also observed in the presence of 4 or 6% of sucrose, while no difference was observed between wild-type and 35S:SnRK1.1 transgenic seedlings grown on palatinose (a non-metabolizable analogue of sucrose). Together, these results suggest a specific activation of SnRK1 by glucose.
Over-activation of SnRK1.1 alters regulation of carbon and nitrogen metabolism
SnRK1 protein kinases are implicated in the regulation of metabolism and of several key enzymes such as nitrate reductase (NR), which interacts with AKINβ1 and is inhibited in cell-free assays by phosphorylation by SnRK1 (Sugden et al., 1999b; Polge et al., 2008). We investigated the consequences of over-activation of SnRK1.1 on metabolism. To confirm the invlovement of SnRK1.1 in in vivo phosphorylation of NR, experiments were performed on leaves of 2-week-old plants harvested after 7 h of light and transferred to the dark for 2 h. The total NR activity was measured in the presence of EDTA, and the activity of non-phosphorylated NR was measured in the presence of Mg2+, in wild-type (Col) and the three lines over-expressing SnRK1.1 (Figure 3a). In response to glucose, the plants over-expressing SnRK1.1 showed a decrease in non-phosphorylated NR of approximately 25–30% compared to the wild-type. Thus, the proportion of phosphorylated NR (inactive NR) increased in the 35S:SnRK1.1 lines, confirming the role of this kinase in the phosphorylation and regulation of NR.
Moreover, after transfer onto glucose, leaves of 2-week-old 35S:SnRK1.1 lines showed an over-accumulation of fructose and glucose of approximately 30%, and a concomitant 50% decrease in starch (Figure 3b). Under our conditions, sucrose was barely detectable in leaves of plants transferred to half-strength MS medium (Col, 2.9 ± 0.3 nmol mg−1 dry weight; 35S:SnRK1.1-1, 2.5 ± 0.4 nmol mg−1 dry weight; 35S:SnRK1.1-2, 3.6 ± 0.2 nmol mg−1 dry weight; 35S:SnRK1.1-3, 2.8 ± 0.1 nmol mg−1 dry weight), and was not detectable in leaves of plants transferred to either glucose or sorbitol. These modifications in sugar content could be the result of the modification of AGPase activity, as SnRK1 has already been implicated in the regulation of this enzyme (Tiessen et al., 2003; McKibbin et al., 2006). The redox activation of AGPase has been quantified through the amount of reduced small subunit AGPase (50 kDa) monomer by Western blotting of leaves of 2-week-old plants (Prioul et al., 1994). The amount of monomeric AGPase in the wild-type was very low (approximately 5%) after transfer onto half-strength MS medium or 6% sorbitol, but rose to 25% when plants were transferred to 6% glucose. By contrast, no significant AGPase activation in response to glucose was observed in plants over-expressing SnRK1.1 (Figure 3c), suggesting that de-regulation of AGPase activity contributes to the modifications of sugar and starch contents observed in these plants.
Metabolism signalling genes are misregulated in 35S:SnRK1.1 transgenic lines
In order to study the potential involvement of SnRK1.1 in the three glucose signalling pathways (hexokinase-dependent, hexokinase-independent and glycolysis-dependent), expression of some of the genes regulated by these pathways was followed in the leaves of 2-week-old plants transferred to various media as described above. cwINV1 and CHS genes expression show that they can be activated only by a supply of 6% glucose and not by the osmotic control sorbitol (Figure 4) as previously shown (Jang et al., 1997). These results allowed us to validate the approach used in this experiment. When comparing wild-type and 35S:SnRK1.1 transgenic plants, no significant differences in gene expression were observed for cwINV1 and CHS or for PLDα1 and RBCS, which are regulated by the hexokinase-independent and -dependent pathways, respectively (Figure 4). In wild-type plants, the pathogenesis-related genes PR1, PR2 and PR5, which are regulated by hexokinase catalytic activity through downstream metabolites (Xiao et al., 2000), were highly induced by glucose. Surprisingly, PR2 and PR5 were also activated by osmotic stress, thus these two PR genes can be activated either by glucose or osmotic stress under these conditions. To our knowledge, this response of PR2 and PR5 to sorbitol has never been studied. Interestingly, in the over-expressing lines, these three PR genes (PR1, PR2 and PR5) were no longer induced by glucose (PR1) or to a much lower extent (PR2 and PR5) than in the wild-type, but the response to sorbitol remained mostly unchanged. These results suggest that SnRK1.1 could be involved in metabolism signalling pathways.
Over-expression of SnRK1.1 confers an ABA-hypersensitive phenotype
The existence of cross-talk between glucose and ABA signalling pathways led us to investigate the effect of SnRK1.1 over-expression on ABA responses. No difference in germination was observed between wild-type and transgenic plants when seeds were plated for 5 days on increasing concentrations of ABA (Figure 5a). However, seedlings over-expressing SnRK1.1 clearly exhibited a marked ABA-hypersensitive response on half-strength MS medium supplemented with 2.5 μm ABA (Figure 5b). Moreover, when glucose was added to the medium, the ABA-hypersensitive phenotype was enhanced (Figure 5c). However, no significant difference in kinase activity was observed in leaves of both wild-type and transgenic lines transferred for 25 h onto half-strength MS medium without ABA or with 2.5 or 10 μm ABA (Figure 5d) or in 7-day-old seedlings directly grown on half-strength MS medium containing or not 0.2% glucose and 1 μm ABA (Figure 5e). Finally, the hypersensitivity to glucose was not a consequence of a change in ABA content, as shown in Figure 5f, and the ABA hypersensitivity phenotype of 35S:SnRK1.1 lines was not due to a modification in ABA content in seedlings (Figure 5g).
To identify the interaction between SnRK1.1 and ABA signalling, a double mutant was generated between 35S:SnRK1.1-3 and gin1-1/aba2. GIN1-1/ABA2 encodes an enzyme that is essential for ABA synthesis and necessary for sugar signalling (Cheng et al., 2002). The gin1-1 mutant exhibits a glucose-insensitive phenotype when grown on a high concentration of glucose. 35S:SnRK1.1-3 × gin1-1 seedlings grown for 7 days on half-strength MS medium supplemented with 6% glucose exhibited the same glucose-insensitive phenotype as gin1-1 (Figure 5h). Thus, the hypersensitive phenotype of 35S:SnRK1.1 requires the presence of ABA, suggesting that GIN1-1/ABA2 is epistatic to SnRK1.1.
SnRK1.1 is activated by phosphorylation in response to sugars
SNF1 protein kinases are activated by phosphorylation on a threonine residue in the ‘activation loop’. In plants, it has been reported that activation of spinach SnRK1 is associated with the phosphorylation of Thr175 (Sugden et al., 1999a). We have investigated whether a similar mechanism might explain the regulation of SnRK1 in response to sugar in plant cells. SnRK1 purified from peak II (Figure 1a), which contains only the SnRK1.1 kinase from cells grown in the presence of sugar, was inactivated by incubation with the homogeneous catalytic subunit of mammalian protein phosphatase PP2A. Inactivation by PP2A was then blocked by 100 nm okadaic acid. SnRK1.1 inactivated by PP2A treatment was not re-activated after the addition of Mg-ATP, Ca2+or AMP alone (Figure 6a). However, reactivation was observed when Mg-ATP plus the partially purified mammalian LKB1 complex (Hawley et al., 2003) or pig CaMKK (Lee and Edelman, 1994) were added (Figure 6a). The phosphorylation state of Thr175 was assessed in the proteins from the purified peak II from cells that were grown without (−) or with (+) 3% of sucrose using an antibody raised against the sequence around the phosphorylated Thr172 of mammalian AMPKα1. Figure 6(b) shows that phosphorylation was reduced in the absence of the sugar (surprisingly, a polypeptide with a slightly higher mobility was detectable under these conditions). In order to confirm that the lower level of SnRK1.1 signal was due to an absence of phosphorylation rather than a lower amount of protein, same experiment was performed using the anti-SnRK1.1 antibody, and no significant difference in signal was observed (Figure 6b).
To confirm that SnRK1.1 is activated by phosphorylation in response to glucose treatment in plants, we performed a purification of phosphorylated proteins (Meimoun et al., 2007) extracted from plants grown on grids and transferred to half-strength MS medium (Ctrl) or 6% glucose (Glc). After blotting with SnRK1.1 antibodies, the results revealed that the amount of SnRK1.1 proteins phosphorylated in plants transferred to half-strength MS medium was not significantly different between the wild-type (Col) and 35S:SnRK1.1-3. On 6% glucose, the amount of SnRK1.1 phosphorylated in the transgenic line was twofold higher than in wild-type (Figure 6c), which is in accordance with the difference in SnRK1.1 activity measured between the 35S:SnRK1.1 and wild-type plants.
SnRK1 protein kinases are considered to be central regulators of metabolism (Baena-Gonzalez et al., 2007; Polge and Thomas, 2007). Their functions may depend on the availability of carbohydrates due to changes in light quality (in source tissues) or the supply of sucrose (in sink tissues). This study aimed to obtain a better understanding of the regulation of these key signalling pathways.
In cauliflower, only two Ca2+-independent kinases (HRKA and HRKB) have been identified, although there are four in spinach leaves (HRKA, HRKB, HRKC and HRKD), two of which, HRKA and HRKC, are forms of SnRK1 (Ball et al., 1994). Biochemical studies on suspension cells suggest that, in A. thaliana cells, SnRK1 complexes are present only in HRKA, HRKC and HRKD. Moreover, size exclusion chromatography revealed complexes of 150 and 200 kDa, which may correspond to the forms HRKA and HRKC previously described in spinach leaves. Peak III was the only peak containing SnRK1.2 as a catalytic subunit, while peaks I and II contained only SnRK1.1. As the sizes of both catalytic subunits are identical, the difference observed between the two first peaks and the third is probably due to a difference in the composition of the non-catalytic subunits, or different post-translational modifications. Immunoprecipitation experiments in crude cell extracts revealed that SnRK1.1 complexes are responsible for 90% of the total SnRK1 activity, with the remaining 10% corresponding to SnRK1.2 complexes. In leaves of 15-day-old plants, SnRK1.1 complexes appear to be responsible for all SnRK1 activity, as SnRK1.2 activity is undetectable under these conditions. For this reason, the main part of the study focused on SnRK1.1. Analysis of the SnRK1.1 over-expressing transgenic lines 35S:SnRK1.1-1, 2 and 3, indicated that this isoform is involved in sugar and ABA signalling, suggesting that this kinase could link these two signalling pathways. Transgenic plants over-expressed SnRK1.1 mRNA to a great extent (with mRNA levels 10–22 times those in the wild-type). Nevertheless, the amount of kinase protein was only approximately 2.5–4-fold higher in 35S:SnRK1.1 lines than in wild-type. These data suggest translational or post-translational regulation of expression of the kinase protein.
The accumulation of SnRK1.1 kinase in 35S:SnRK1.1 plants leads to a clear phenotype of glucose sensitivity, which is consistent with recent results obtained by Baena-Gonzalez et al. (2007). We show here that this hypersensitivity is dependent on glucose availability, as no difference in growth was observed between wild-type and 35S:SnRK1.1 lines on palatinose or sorbitol. When transferred to half-strength MS medium, over-expressing plants exhibited a 30% increase in activity compared to wild-type. For both wild-type and transgenic lines, a 30–50% increase of activity was observed after transfer to medium containing glucose compared to half-strength MS conditions. Thus, the increase in SnRK1.1 activity in wild-type as well as in the 35S:SnRK1.1 lines depends on glucose availability. Moreover, it has been recently shown that SnRK1.1 activation in response to a high concentration of glucose (6%) requires the presence of myo-inositol polyphosphate 5-phosphatase, which is essential to maintain levels of SnRK1 activity under low-nutrient as well as high-sugar conditions (Ananieva et al., 2008). As no direct relationship between the kinase amount and its activity and the consequence of this activity (modification of NR and AGPase activity and gene expression) was observed in response to glucose treatment, our data suggest strong post-translational regulation. The capacity of SnRK1.1 to phosphorylate its targets could be regulated by the availability of the non-catalytic subunits AKINβ1, 2 or 3, which are differentially regulated in response to sugar (Polge et al., 2008). No difference in the phosphorylation state of SnRK1.1 at Thr175 has been observed in response to either darkness, DCMU, hypoxia or sucrose treatment (Baena-Gonzalez et al., 2007), or in the present study between wild-type and 35S:SnRK1.1 lines under control conditions. Using various biochemical approaches, we show that activation of SnRK1.1 in response to glucose occurs by phosphorylation of Thr175 in the activation loop. It has been previously shown that the mammalian upstream kinase (LKB1) is able to activate SnRK1 activity in Arabidopsis extracts (Harthill et al., 2006). Here, we show that the kinase SnRK1.1, can also be activated in cell-free assays by CaMKK, like the mammalian AMPK. It will be interesting to determine whether the phosphorylation of SnRK1.1 in response to glucose is due to activation of the potential upstream kinases SnAK1/GRIK2 and SnAK2/GRIK1 (Shen and Hanley-Bowdoin, 2006; Hey et al., 2007). Very recently, it was shown in A. thaliana protoplasts that regulation of the DIN1 and DIN6 genes by SnRK1 was inhibited by sugars, suggesting that this SnRK1-mediated response is inhibited by carbohydrates. We show an increase of SnRK1.1 activity in plants in response to glucose. As the SnRK1 protein kinases are implicated in the global regulation of metabolism, SnRK1.1 could respond to changes in carbohydrate level – both deprivation and excess. Indeed, SnRK1.1 is able to regulate DIN1 and DIN6 expression in response to glucose or sucrose, but a supply of glucose can also activate SnRK1.1 and lead to the modification of expression of other genes (PR1, PR2 and PR5) or of enzyme activity (NR, AGPase). As SnRK1.1 is included in a heterotrimeric complex, various non-catalytic subunits may be available depending on the conditions, the tissue or the cellular compartment, allowing SnRK1.1 to respond to various stimuli. For example, AKINβ1 is expressed in cotyledons and leaves, its expression responds to sugars, and it can be differentially localized in the cell depending on its myristoylation by NMT1 (N-myristoyltransferase) (Pierre et al., 2007; Polge et al., 2008).
In previous genetic studies, the SnRK1 protein kinases were mainly implicated in regulation of the expression or activity of metabolic enzymes. Nevertheless, until now, SnRK1 phosphorylation of these enzymes have only been demonstrated in cell-free assays (Sugden et al., 1999b; Kulma et al., 2004). We have shown recently that NR regulation by SnRK1 occurs via binding to AKINβ1 (Polge et al., 2008). Here we confirm by a genetic approach that SnRK1.1 can inhibit NR in response to glucose. Over-expression of SnRK1.1 led to modifications in the content of soluble sugars, starch and AGPase activity in response to glucose. Such modifications have been observed in potato tubers over-expressing SnRK1 (Tiessen et al., 2003; McKibbin et al., 2006), but with an opposite effect to what we observed in 35S:SnRK1.1 transgenic lines. However, the data of McKibbin et al. (2006) were obtained on sink tissues, i.e. tubers, while our experiments were performed on source tissues, i.e. leaves. It can thus be hypothesized that SnRK1 could have different functions in sink or source organs, to coordinate metabolism in various organs as shown in mammals. This hypothesis is reinforced by the capacity of the tobacco SnRK1 complex to reallocate the carbon resources from leaves to roots in response to wounding (Schwachtje et al., 2006).
The hypersensitivity of 35S:SnRK1.1 plants to glucose, and the modification of expression of the PR1, 2 and 5 genes in response to glucose, suggest involvement of SnRK1.1 in the metabolism-dependent pathway (Xiao et al., 2000). Moreover, the hypersensitivity to ABA of 35S:SnRK1.1 lines without modification of ABA content, and the hyposensitivity to glucose of the 35S:SnRK1.1 × aba2 cross, suggest an interaction between SnRK1.1 and ABA signalling. Repression of SnRK1 in pea revealed pleiotropic defects of maturation similar to an ABA-insensitive phenotype (Radchuk et al., 2006). The kinase could respond to this hormone by a modification in the composition of the complex, as the LeSNF4 γ subunit in tomato is induced by ABA treatment (Bradford et al., 2003). Nevertheless, in 35S:SnRK1.1 lines, ABA sensitivity does not occur at the time of germination, but during seedling development. Indeed, in response to ABA or glucose, the development of 35S:SnRK1.1 lines stops after germination at the second developmental checkpoint defined by Lopez-Molina et al. (2001). Thus, SnRK1.1 could be one of the elements that allow interactions between sugar and ABA signalling during transition from the heterotrophic to the autotrophic stage as found for SNF1 in yeast during the diauxic shift.
The work of Baena-Gonzalez et al. has demonstrated the role of the A. thaliana SnRK1 protein kinases in regulation of gene expression in stress and energy signalling responses (Baena-Gonzalez et al., 2007). The present study highlights the regulation of SnRK1.1 in response to glucose and its involvement in glucose and ABA signalling, confirming, as for AMPK and SNF1 in mammals and yeast, its involvement at the heart of metabolism regulation.
Plant material and cell culture
Wild-type Arabidopsis thaliana ecotype Columbia (Col) was used to produce transgenic plants over-expressing AKINα1. The gin1-1/aba2 is in the Wassilewskija (WS) ecotype (Cheng et al., 2002). Plants were grown in a culture room and were maintained in soil with a 16 h light/8 h dark regime (long day). The relative humidity was 65% and the temperature was 20/17°C during the light/dark cycle. For in vitro culture, seeds were germinated on Murashige and Skoog medium diluted twice (half-strength MS) containing 2–6% w/v glucose, 2–6% w/v sorbitol, 2–6% w/v sucrose, 2–6% w/v palatinose) or 1–10 μm ABA, at 20°C under a 16 h light/8 h dark regime for 7 or 15 days. For plant transfer experiments, seeds were germinated on solid or liquid half-strength MS medium containing 0.2% w/v glucose and grown for 2 weeks at 20°C under a 16 h light/8 h dark regime before being transferred for 25 h (16 h light/8 h dark/1 h light) to liquid half-strength MS medium containing 6% w/v glucose, 6% w/v sorbitol or 2.5 or 10 μm ABA. Leaves were collected after 1 h of light, immediately frozen in liquid N2, and stored at −80°C prior to analysis. Arabidopsis suspension cells (cv. Landsberg erecta; May and Leaver, 1993) were cultured in standard medium containing Murashige and Skoog basal salts with minimal organics (MSMO; Sigma, http://www.sigmaaldrich.com/), sucrose (3%), 1-naphthaleneacetic acid (0.5 mg l−1) and kinetin (0.05 mg l−1), pH 5.7, under continuous light at 20°C.
Preparation of antibodies and Western blot
Production of anti-phosphoThr172 antibodies has been described previously (Sugden et al., 1999a). Arabidopsis thaliana SnRK1.1 antibodies were raised against the regulatory domain of SnRK1.1. Arabidopsis thaliana SnRK1.2 antibodies were raised against a specific peptide derived from the deduced amino acid sequence of SnRK1.2 (TDSGSNPMRTPEAG). The antibodies were used in Western blot experiments as described by Meimoun et al. (2007).
Gene constructs and plant transformation
For over-expression of SnRK1.1 in transgenic plants, SnRK1.1 cDNA in pBluescript (Le Guen et al., 1992) was excised using EcoRI. The resulting 1.5 kb fragment was ligated into the pDH51 vector (Stratagene, http://www.stratagene.com). This vector was digested using EcoRI to release the fragment p35S-SnRK1.1-ter35S, which was then sub-cloned into the pBIN19 vector (Bevan, 1984). Transgenic plants were selected on half-strength MS medium containing kanamycin (50 mg l−1). Seeds were obtained by self-fertilization, and homozygous lines from the T3 generation were used in this study.
SnRK1.1 immunoprecipitation and peptide kinase assays
Total proteins were extracted from 50 mg fresh leaves of 2-week-old seedlings or whole 7-day-old seedlings in 1 ml of extraction buffer containing 50 mm Tris/HCl pH 7.6, 0.25 m mannitol, 50 mm NaF, 1 mm EDTA, 1 mm EGTA, 1% v/v Triton X-100, 1 mm DTT and 1% of protease inhibitor mixture for plant extracts diluted 1:2000 (P9599; Sigma). Protein concentrations in extracts were determined using the Bradford method (Bradford, 1976). Saturating amounts of the anti-SnRK1.1 antibody were bound for 1 h at 4°C on 5 mg of protein A–Sepharose (P3391; Sigma) with 300 μl of ‘150 mm NaCl’ buffer (50 mm Tris/HCl pH 6.8, 150 mm NaCl, 50 mm NaF, 1 mm EDTA, 1 mm EGTA, 1% v/v Triton X-100, 1 mm DTT, 1% of protease inhibitor mixture for plant extracts diluted 1:2000), and then incubated with 25 μg of total proteins for 3 h at 4°C under gentle shaking. For determination of the relative proportions of SnRK1.1/SnRK1.2 activities, the supernatant was removed after immunoprecipitation and centrifugation (1 min, 12 000 g, 4°C), and subjected to further immunoprecipitation. SnRK1 immunocomplexes were washed once with 1 ml of ‘1 m NaCl’ buffer and three times with 50 mm HEPES pH 7.
For the peptide kinase assays, the SnRK1 immunocomplexes were resuspended in buffer containing 50 mm HEPES pH 7, 1 mm DTT, 5 mm MgCl2, 50 μm ATP and 450 μm AMARA peptide. The reaction is initiated by addition of 2 μCi of γ32P-ATP. Following a 20 min incubation at 30°C, a 10 μl aliquot of the reaction mixture was spotted onto a square of phosphocellulose paper (P81, Whatman, http://www.whatman.com). The paper was then immediately washed three times in 1% w/v H3PO4 (5 min per wash) in order to remove unincorporated γ32P-ATP. 32P incorporation into the synthetic peptides was determined by scintillation counting using a liquid scintillation spectrometer.
CaMKK was purified from pig brain (Lee and Edelman, 1994), and kinase activity was assayed using ATP (200 mm), calmodulin (1 μm), CaCl2 (1 mm) and MgCl2 (5 mm). LKB1 (LKB1, STRADα and MO25α) was partially purified from rat liver, and kinase activity assayed as described by Hawley et al. (2003).
Nitrate reductase assay and analysis of AGPase redox activation
Total proteins were extracted from 50 mg of leaves of 2-week-old seedlings, and the soluble protein content was determined in crude leaf extract. The maximal extractable activity and activation state of nitrate reductase (NR) were measured as described by Ferrario-Mery et al. (1998). The activity of non-phosphorylated NR was measured in the presence of 10 mm MgCl2, and the total NR activity was measured in the presence of 5 mm EDTA.
The extraction and analysis of the redox activation of AGPase were performed by analysing the proportion of ADP-glucose pyrophosphorylase B subunit (AGPB) monomer as described previously (Tiessen et al., 2002).
Measurement of carbohydrate content
Carbohydrate contents were determined by enzyme assays (Bergmeyer and Bernt, 1974). Sugars were extracted from 200 mg of leaves of 2-week-old seedlings using 1 ml of perchloric acid. The supernatant was used for the measurement of hexose and sucrose contents, and the pellet for measurement of the starch content. The hexose content was determined in a 200 μl reaction mixture containing 235 mm Tris/NaOH pH 7.6, 3 mm MgSO4, 3.2 mm NADP+, 11.5 mm ATP and an enzyme mixture (0.7 units phosphoglucose isomerase, 0.15 units glucose-6-phosphate dehydrogenase, and 0.3 units hexokinase) at 25°C for 15 min. The NAD+ reduction was measured at 340 nm. The sucrose content was determined in a 200 μl reaction mixture containing β-fructosidase (0.36 units), 0.32 m sodium citrate pH 4.6 at 30°C for 20 min. After adding 235 mm Tris/NaOH pH 7.6, 3 mm MgSO4, 3.2 mm NADP+ and 11.5 mm ATP, the absorbance was measured at 340 nm (OD1). The enzyme mixture (0.15 units glucose-6-phosphate dehydrogenase, 0.3 units hexokinase) was added, and the NAD+ reduction was measured at 340 nm (OD2) after 10 min at 25°C. The sucrose content was determined by the difference between OD2 and OD1. Starch in the pellet was heated for 2 h at 100°C with 1 ml of water. After addition of amyloglucosidase (4 units), α-amylase (0.045 units) and β-amylase (0.07 units) dissolved in 200 mm sodium acetate (pH 4.8), starch hydrolysis was performed overnight at 50°C. The extract was cleared by centrifugation (15 min, 12 000 g, 4°C), and any glucose derived from starch was determined spectrophotometrically as previously described for hexose measurements, but without addition of phosphoglucose isomerase.
Quantification of ABA
Determination of ABA content was performed on leaves of 2-week-old seedlings or on whole 7-day-old seedlings frozen in liquid nitrogen and lyophilized. Extractions were performed in non-oxidative methanol:water (80:20, v/v), with pre-purification through SepPak C18 cartridges (Waters, http://www.waters.com), and HPLC fractionation was performed using a Luna C18 4.6 × 250 mm column (Phenomenex, http://www.phenomenex.com) with a 0.1% v/v trifluoroacetic acid:acetonitrile gradient. The efficiency of the purification was measured by means of 3H-ABA added to the extracts and scintillation counting of aliquots of the purified fractions. The ELISA procedure was based upon competition for a limited amount of monoclonal anti-ABA antibody (LPDP 229; Jussieu) between standard ABA–BSA conjugate adsorbed on the wells of a microtitration plate and free ABA extracted from the samples. Then, the plates were washed, and bound antibodies were labelled using a peroxidase-conjugated goat antibody to mouse immunoglobulins (Sigma). Peroxidase activity was measured using ABTS [2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid, Sigma) added as substrate (1 mm in 3.25 mm perborate buffer, pH 4.6]. A standard curve was established for each microtitration plate. ABA content was determined five times for each sample.
Northern blot hybridization
Total RNA (20 μg) was extracted from the leaves of 2-week-old seedlings, subjected to electrophoresis, transferred to membranes and hybridized as previously described (Bouly et al., 1999). The probes were obtained by PCR amplification using specific primers, and correspond to full-length EF1α1 (At1g07920), SnRK1.1 (At3g01090), CHS (At5g13930), PR1 (At2g14610), PR2 (At3g57260), PR5 (At1g75040), PLDα1 (At3g15730), RBCS (At1g67090) and cwINV1 (At3g13790) cDNAs.
Purification of SnRK1 activity from Arabidopsis thaliana cell culture
The purification procedure used previously by Ball et al. (1994) was used with modifications. All procedures were carried out at 4°C. Cells (500 g) were ground in a mortar in the presence of liquid nitrogen, and homogenized with 2 g of polyvinylpolypyrrolidone, 500 ml of homogenization buffer (50 mm Tris/HCl pH 8.2, 0.25 m mannitol, 1 mm NaPPi, 50 mm NaF, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 1 mm benzamidine, 0.1 mm phenylmethane sulfonyl fluoride) and 1% v/v Triton X-100. The homogenate was centrifuged at 18 000 g for 20 min. The supernatant was removed and passed through two layers of miracloth (http://www.miracloth.com). Ammonium sulfate was added to give a 40% satured solution. After stirring for 15 min, the precipitate was collected by centrifugation at 18 000 g for 20 min, dissolved in 200 ml of buffer A (50 mm Tris/HCl pH 8, 50 mm NaF, 1 mm NaPPi, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 1 mm benzamidine, 0.1 mm phenylmethane sulfonyl fluoride, 0.02% v/v brij-35 (Sigma, http://www.sigmaaldrich.com) and 10% v/v glycerol and dialysed against 2 × 2 litres of buffer A. The preparation was applied to a 100 ml DEAE Sepharose Fast Flow anion-exchange column (Amersham, http://www.gehealthcare.com) equilibrated in buffer A. After washing with 1 l of buffer A, the preparation was eluted with a linear gradient of 0–0.5 m NaCl in buffer A. Fractions containing the kinase were pooled, and 40% polyethylene glycol (PEG6000 in buffer A) (Sigma) was added to give a 20% PEG final solution. After stirring for 15 min, the precipitate was collected by centrifugation at 18 000 g for 20 min. The precipitate was dissolved in 30 ml of buffer B (buffer A except at pH 7.0). The sample was applied to a 50 ml Blue-Sepharose column (Amersham) equilibrated in buffer B. Once the column had been washed with 1 l of buffer B, the preparation was eluted with buffer B containing 0.5 m NaCl. Active fractions were pooled, and 40% PEG in buffer B was added to a 20% final concentration. The pellet was dissolved in buffer C (50 mm Na-HEPES pH 8, 50 mm NaF, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 1 mm benzamidine, 0.1 mm phenylmethane sulfonyl fluoride, 0.02% v/v Brij-35 and 10% v/v glycerol). The sample was applied at 0.3 ml min−1 to an FPLC MonoQ HR 5/5 column equilibrated in buffer C, and the kinase activity was eluted using a gradient from 0 to 1 m NaCl in buffer C.
Phosphoprotein enrichment was performed using Qiagen columns according to the manufacturer’s instructions with minor modifications. Denatured proteins (2 mg) extracted from the leaves of 2-week-old seedlings were resuspended in loading buffer containing 0.25% CHAPS at a concentration of 0.1 mg ml−1 and loaded onto the Qiagen mini-column (200 μl) at a slow flow rate of 125 μl min−1 and room temperature (Meimoun et al., 2007). After washing with the loading buffer (1 ml), the bound phosphoproteins were eluted with 2.5 ml of elution buffer. Eluted proteins were precipitated with 10% trichloroacetic acid, and the pellet (after centrifugation at 20 000 g for 10 min at 4°C) was solubilized in 30 μl of dissociation buffer for SDS–PAGE prior to Western blot analysis.
We are grateful to W. Bourbon (Université Paris-Sud XI, Orsay, France) for technical support and J. Vidal (Université Paris-Sud XI) for critical reading of the manuscript. We thank B. Sotta (Université Paris VI, Paris, France) for ABA quantification, and J.-L. Prioul (Université Paris-Sud XI, Orsay, France) for the AGPB antibodies. M.J. and P.M. are supported by the Ministère de l’Education National et de la Recherche, France.