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Summary Arabidopsis thaliana SNF1-Related-Kinase1 (SnRK1)-Activating Kinase 1 and 2 (AtSnAK1 and 2) have been shown to phosphorylate in vitro and activate the energy signalling integrator, SnRK1. To clarify this signalling cascade in planta, a genetic- and molecular-based approach was developed. Homozygous single AtSnAK1 and AtSnAK2 T-DNA insertional mutants did not display an apparent phenotype. Crossing of the single mutants did not allow the isolation of double mutant plants while self-pollinating the S1-/- S2+/- sesquimutant specifically gave approximatively 22% individuals in their offspring that, when rescued on sugar-supplemented media in vitro, were shown to be AtSnAK1-AtSnAK2 double mutants. Interestingly, this was not obtained in the case of the other sesquimutant, S1+/- S2-/-. Although reduced in size, the double mutant had the capacity to produce flowers, but not seeds. Immunological characterization established the T-loop of the SnRK1 catalytic subunit to be non-phosphorylated in the absence of both SnAKs. When the double mutant was complemented with a DNA construct containing an AtSnAK2 open reading frame driven by its own promoter, a normal phenotype was restored. Therefore, wild-type plant growth and development is dependent on the presence of SnAK in vivo and this is correlated to SnRK1 phosphorylation. These data show that both SnAKs are kinases phosphorylating SnRK1 and thereby they contribute to energy signalling in planta. This article is protected by copyright. All rights reserved.

The CDPK-SnRK superfamily consists of seven types of serine-threonine protein kinases: calcium-dependent protein kinase (CDPKs), CDPK-related kinases (CRKs), phosphoenolpyruvate carboxylase kinases (PPCKs), PEP carboxylase kinase-related kinases (PEPRKs), calmodulin-dependent protein kinases (CaMKs), calcium and calmodulin-dependent protein kinases (CCaMKs), and SnRKs. Within this superfamily, individual isoforms and subfamilies contain distinct regulatory domains, subcellular targeting information, and substrate specificities. Our analysis of the Arabidopsis genome identified 34 CDPKs, eight CRKs, two PPCKs, two PEPRKs, and 38 SnRKs. No definitive examples were found for a CCaMK similar to those previously identified in lily (Lilium longiflorum) and tobacco (Nicotiana tabacum) or for a CaMK similar to those in animals or yeast. CDPKs are present in plants and a specific subgroup of protists, but CRKs, PPCKs, PEPRKs, and two of the SnRK subgroups have been found only in plants. CDPKs and at least one SnRK have been implicated in decoding calcium signals in Arabidopsis. Analysis of intron placements supports the hypothesis that CDPKs, CRKs, PPCKs and PEPRKs have a common evolutionary origin; however there are no conserved intron positions between these kinases and the SnRK subgroup. CDPKs and SnRKs are found on all five Arabidopsis chromosomes. The presence of closely related kinases in regions of the genome known to have arisen by genome duplication indicates that these kinases probably arose by divergence from common ancestors. The PlantsP database provides a resource of continuously updated information on protein kinases from Arabidopsis and other plants.

The recent gain in knowledge concerning enzymes involved in signal transduction pathways is a direct consequence of the considerable advances made in molecular biology. Protein kinases and protein phosphatases, the two major enzymes implicated in post-translational modifications, have been studied in particular. The number of characterized plant genes and/or cDNAs encoding these enzymes is increasing everyday. Since 1991, 26 genes and cDNAs coding for plant protein phosphatases have been isolated and characterized. The huge number of protein kinases (estimated at several thousands) makes it impossible to give an exhaustive list of the genes already identified, but a classification of these enzymes, based on phylogenetic criteria, allows us to appreciate the range of functions this protein family may play in plants.

International audience; SNF1-related protein kinase-1 (SnRK1), the plant kinase homolog of mammalian AMP-activated protein kinase (AMPK), is a sensor that maintains cellular energy homeostasis via control of anabolism/catabolism balance. AMPK-dependent phosphorylation of p27(KIP1) affects cell-cycle progression, autophagy and apoptosis. Here, we show that SnRK1 phosphorylates the Arabidopsis thaliana cyclin-dependent kinase inhibitor p27(KIP1) homologs AtKRP6 and AtKRP7, thus extending the role of this kinase to regulation of cell-cycle progression. AtKRP6 and 7 were phosphorylated in vitro by a recombinant activated catalytic subunit of SnRK1 (AtSnRK11). Tandem mass spectrometry and site-specific mutagenesis identified Thr152 and Thr151 as the phosphorylated residues on AtKRP6- and AtKRP7, respectively. AtSnRK1 physically interacts with AtKRP6 in the nucleus of transformed BY-2 tobacco protoplasts, but, in contrast to mammals, the AtKRP6 Thr152 phosphorylation state alone did not modify its nuclear localization. Using a heterologous yeast system, consisting of a cdc28 yeast mutant complemented by A.thaliana CDKA;1, cell proliferation was shown to be abolished by AtKRP6(WT) and by the non-phosphorylatable form AtKRP6(T152A), but not by the phosphorylation-mimetic form AtKRP6(T152D). Moreover, A.thaliana SnRK11/KRP6 double over-expressor plants showed an attenuated AtKRP6-associated phenotype (strongly serrated leaves and inability to undergo callogenesis). Furthermore, this severe phenotype was not observed in AtKRP6(T152D) over-expressor plants. Overall, these results establish that the energy sensor AtSnRK1 plays a cardinal role in the control of cell proliferation in A.thaliana plants through inhibition of AtKRP6 biological function by phosphorylation.

The perception of nutrient and energy levels inside and outside the cell is crucial to adjust growth and metabolism to available resources. The signaling pathways centered on the conserved TOR and SnRK1/Snf1/AMPK kinases have crucial and numerous roles in nutrient and energy sensing and in translating this information into metabolic and developmental adaptations. In plants evidence is mounting that, like in other eukaryotes, these signaling pathways have pivotal and antagonistic roles in connecting external or intracellular cues to many biological processes, including ribosome biogenesis, regulation of translation, cell division, accumulation of reserves and autophagy. Data on the plant TOR pathway have been hitherto rather scarce but recent findings have shed new light on its roles in plants. Moreover, the distinctive energy metabolism of photosynthetic organisms may reveal new features of these ancestral eukaryotic signaling elements.

Arabidopsis thaliana sucrose nonfermenting 1-related protein kinase 1 complexes belong to the SNF1/AMPK/SnRK1 protein kinase family that shares an ancestral function as central regulators of metabolism. In A. thaliana, the products of AtSnAK1 and AtSnAK2, orthologous to yeast genes, have been shown to autophosphorylate and to phosphorylate/activate the AtSnRK1.1 catalytic subunit on Thr(175). The phosphorylation of these kinases has been investigated by site-directed mutagenesis and tandem mass spectrometry. The autophosphorylation site of AtSnAK2 was identified as Thr(154), and it was shown to be required for AtSnAK catalytic activity. Interestingly, activated AtSnRK1 exerted a negative feedback phosphorylation on AtSnAK2 at Ser(261) (Ser(260) of AtSnAK1) that was dependent on AtSnAK autophosphorylation. The dynamics of these reciprocal phosphorylation events on the different kinases was established, and structural modeling allowed clarification of the topography of the AtSnAK phosphorylation sites. A mechanism is proposed to explain the observed changes in the enzymatic properties of each kinase triggered by these phosphorylation events.

The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized. The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region. The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1). SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase. Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A. thaliana. Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves. AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.

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.

International audience; The SNF1/AMPK/SnRK1 kinases are evolutionary conserved kinases involved in yeast, mammals, and plants in the control of energy balance. These heterotrimeric enzymes are composed of one alpha-type catalytic subunit and two gamma- and beta-type regulatory subunits. In yeast it has been proposed that the beta-type subunits regulate both the localization of the kinase complexes within the cell and the interaction of the kinases with their targets. In this work, we demonstrate that the three beta-type subunits of Arabidopsis (Arabidopsis thaliana; AKINbeta1, AKINbeta2, and AKINbeta3) restore the growth phenotype of the yeast sip1Deltasip2Deltagal83Delta triple mutant, thus suggesting the conservation of an ancestral function. Expression analyses, using AKINbeta promoterbeta-glucuronidase transgenic lines, reveal different and specific patterns of expression for each subunit according to organs, developmental stages, and environmental conditions. Finally, our results show that the beta-type subunits are involved in the specificity of interaction of the kinase with the cytosolic nitrate reductase. Together with previous cell-free phosphorylation data, they strongly support the proposal that nitrate reductase is a real target of SnRK1 in the physiological context. Altogether our data suggest the conservation of ancestral basic function(s) together with specialized functions for each beta-type subunit in plants.

The mechanism underlying the accumulation of specific RNA encoding the G form (photosynthetic) of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) has been studied during the greening process of Sorghum and maize leaves. From a Γgt11 cDNA library, a cDNA clone for Sorghum leaf PEPC was identified, sequenced and found to correspond to the photosynthetic type of the protein by means of monoclonal antibodies. ‘Run off transcription experiments showed that Sorghum or maize leaf nuclei extracted from light-grown plants produced considerably more PEPC mRNAs than those obtained from dark-grown plants. Ribosomal RNA and LHCP mRNAs increased concomitantly. ‘Run off’ analyses also indicated that transcription of these mRNAs was phytochrome-mediated. In plants grown in normal photoperiodic conditions, the PEPC-RNA pool followed a rhythmical behaviour, being low at night and recovering its maximum level the following day. However, after 24 h of dark treatment, both the RNA pool and the transcription capacity were found to be strongly decreased. In contrast, the PEPC protein remained constant during the same developmental period.

The SNF1-related kinases are considered to be crucial elements of transcriptional, metabolic and developmental regulation in response to stress. In yeast, SNF1 is one of the main regulators in the shift from fermentation to aerobic metabolism; AMPK, its mammalian counterpart, is a master metabolic regulator involved in a variety of metabolic disorders such as diabetes and obesity. The aim of this review is to examine the literature concerning SnRK1 proteins, the SNF1 homologues in plants. The remarkable structural similarities between the plant complexes and those of yeast and mammalian suggest the existence of a common ancestral function in the regulation of energy and carbon metabolism. We will also highlight some distinctive features acquired by the plant proteins during evolution.

International audience; The sucrose nonfermenting-1 protein kinase (SNF1)/AMP-activated protein kinase subfamily plays a central role in metabolic responses to nutritional and environmental stresses. In yeast (Saccharomyces cerevisiae) and mammals, the beta- and gamma-noncatalytic subunits are implicated in substrate specificity and subcellular localization, respectively, and regulation of the kinase activity. The atypical betagamma-subunit has been previously described in maize (Zea mays), presenting at its N-terminal end a sequence related to the KIS (kinase interacting sequence) domain specific to the beta-subunits (Lumbreras et al., 2001). The existence of two components, SNF1-related protein kinase (SnRK1) complexes containing the betagamma-subunit and one SnRK1 kinase, had been proposed. In this work, we show that, despite its unusual features, the Arabidopsis (Arabidopsis thaliana) homolog AKINbetagamma clearly interacts with AKINbeta-subunits in vitro and in vivo, suggesting its involvement in heterotrimeric complexes located in both cytoplasm and nucleus. Unexpectedly, a transcriptional analysis of AKINbetagamma gene expression highlighted the implication of alternative splicing mechanisms in the regulation of AKINbetagamma expression. A two-hybrid screen performed with AKINbetagamma as bait, together with in planta bimolecular fluorescence complementation experiments, suggests the existence of interactions in the cytosol between AKINbetagamma and two leucine-rich repeats related to pathogen resistance proteins. Interestingly, this interaction occurs through the truncated KIS domain that corresponds exactly to a GBD (glycogen-binding domain) recently described in mammals and yeast. A phylogenetic study suggests that AKINbetagamma-related proteins are restricted to the plant kingdom. Altogether, these data suggest the existence of plant-specific SnRK1 trimeric complexes putatively involved in a plant-specific function such as plant-pathogen interactions.

The SNF1/AMPK/SnRK1 heterotrimeric kinase complex is involved in the adaptation of cellular metabolism in response to diverse stresses in yeast, mammals and plants. Following a model proposed in yeast, the kinase targets are likely to bind the complex via the non-catalytic beta-subunits. These proteins currently identified in yeast, mammals and plants present a common structure with two conserved interacting domains named Kinase Interacting Sequence (KIS) and Association with SNF1 Complex (ASC), and a highly variable N-terminal domain. In this paper we describe the characterisation of AKINbeta3, a novel protein related to AKINbeta subunits of Arabidopsis thaliana, containing a truncated KIS domain and no N-terminal extension. Interestingly the missing region of the KIS domain corresponds to the glycogen-binding domain (beta-GBD) identified in the mammalian AMPKbeta1. In spite of its unusual features, AKINbeta3 complements the yeast sip1Deltasip2Deltagal83Delta mutant. Moreover, interactions between AKINbeta3 and other AKIN complex subunits from A. thaliana were detected by two-hybrid experiments and in vitro binding assays. Taken together these data demonstrate that AKINbeta3 is a beta-type subunit. A search for beta-type subunits revealed the existence of beta3-type proteins in other plant species. Furthermore, we suggest that the AKINbeta3-type subunits could be plant specific since no related sequences have been found in any of the other completely sequenced genomes. These data suggest the existence of novel SnRK1 complexes including AKINbeta3-type subunits, involved in several functions among which some could be plant specific.

This paper reviews the contribution of P. Gadal’s group to the study of phosphoenolpyruvate carboxylase (PEPC) in plants. It highlights how molecular biology and genetics have helped to advance our understanding of the PEPC multigene family, including evolutionary aspects, and the regulatory mechanisms controlling the expression of the gene encoding the photosynthetic isoform of C4 plants. Preliminary experiments using plant transformation with the aim of determining the role of PEPC isoforms in different physiological contexts and to improve crop yield are also reported.

Publisher Summary This chapter discusses sucrose nonfermenting-1 (SNF1) and adenosine monophosphate-activated protein kinase (AMPK)—global regulators of carbon metabolism in their respective systems. SNF1-related protein kinases (SnRKs) are a family of plant protein kinases with catalytic domains similar to that of SNF1) of yeast and AMPK of animals. The plant SnRK family comprises at least three subfamilies (SnRKs1, 2, and 3). Relatively little is known about the functions of SnRK2s and SnRK3s, but it is already clear that SnRK1s play an important role in the regulation of carbon metabolism and in the cross-talk between metabolic and other signaling pathways in plants. This role not only has some similarities with the roles of SNF1 and AMPK but also has some intriguing differences. The genetic modification of SnRK1 activity and function may be one way of improving this important trait in crop plants.

AKINalpha1, a Ser/Thr kinase from Arabidopsis thaliana belongs to the highly conserved SNF1 family of protein kinases in eukaryotes. Recent data suggest that the plant SNF1-related kinases (SnRK1 family) are key enzymes implicated in the regulation of carbohydrate and lipid metabolism. In Saccharomyces cerevisiae and mammals, the SNF1 and AMPKalpha protein kinases interact with two other families of proteins, namely SNF4/AMPKgamma and SIP1/SIP2/GAL83/AMPKbeta, to form active heterotrimeric complexes. In this paper, we describe the characterisation of three novel cDNAs. AKINbeta1 and AKINbeta2 encode proteins similar to SIP1, SIP2 and GAL83 and AKINgamma codes for a protein showing similarity with SNF4. Using the two-hybrid system, specific interactions have been shown between A. thaliana AKINbeta1/beta2, AKINgamma and AKINgamma as well as between the A. thaliana and S. cerevisiae subunits. Interestingly, AKINbeta1, AKINbeta2 and AKINgamma mRNAs accumulate differentially in A. thaliana tissues and are modulated during development and under different growth conditions. These data suggest the presence in higher plants of a conserved heterotrimeric complex. Moreover, the differential transcription of different non-catalytic subunits can constitute a first level of regulation of the SNF1-like complex in plants.

In the present paper, the isolation of a third cyclin-dependent kinase gene and its cognate cDNA from Arabidopsis thaliana is described. Whereas other characterised cdc2 genes are ubiquitously expressed in Arabidopsis, expression of cdc2cAt is restricted to flowers. This gene, named cdc2cAt, differs from the two previously reported cdc2 genes in its organisation. Comparison with other cdc2 genes suggests that the deduced protein belongs to a new family of CDC2-like proteins related to the human CHED protein kinase.

We have characterized a 6.4 kb genomic fragment from Arabidopsis thaliana ecotype Columbia overlapping the 5' end of the AKin10 gene which encodes a protein Ser/Thr kinase. Using, as probes, various restriction fragments located upstream of AKin10, two cDNA clones have been isolated from a cDNA library prepared from young shoot tissue. A comparison between the cDNA and the above genomic sequences allowed us to locate two novel genes, Atcys1 and Athyp1 (for Arabidopsis thaliana cystathionine gamma-synthase 1 and hypothetical protein 1). The coding sequences of both genes are interrupted by introns and the exons match the sequences of the corresponding cDNAs. Further analysis of the genomic fragment revealed the presence of an open reading frame (ORF) of 609 nucleotides situated between the two genes. Atcys1, Athyp1, AKin10 and the ORF are very close to each other and organized in the same polarity; hence, the intergenic regions probably contain, within less than 0.5 kb, all the regulatory elements necessary to govern initiation and termination of transcription. The deduced protein sequence of Atcys1 shows a high degree of similarity with the cystathionine gamma-synthase from Escherichia coli. The putative product of the Athyp1 gene contains seven hydrophobic regions flanked by hydrophilic domains, reminiscent of membrane-spanning proteins. Southern blot hybridization experiments suggest the presence of one copy of Atcys1, Athyp1 and AKin10 per haploid genome, and Northern blot analysis demonstrates that the three genes are differentially expressed in roots, shoots and leaves.

Wheat and barley are the major temperate cereals, being used for food, feed and industrial raw material. However, in all cases the quality may be limited by the amount, composition and properties of the grain storage proteins. We describe how a combination of biochemical and molecular studies has led to an understanding of the molecular basis for breadmaking quality in wheat and feed quality in barley, and also provided genes encoding key proteins that determine quality. The control of expression of these genes has been studied in transgenic tobacco plants and by transient expression in cereal protoplasts, providing the basis for the production of transgenic cereals with improved quality characteristics.

The mechanism underlying the light effect on phosphoenolpyruvate carboxylase (PEPC) from the C(4) plant sorghum (Sorghum vulgare Pers., var Tamaran) leaves was investigated. Following exposure to light a new isozyme of PEPC, specific for the green leaf and responsible for primary CO(2) fixation in photosynthesis, was established. Northern blot experiments revealed the presence of PEPC mRNA showing a molecular weight of 3.4 kilobases. During the greening process, concomitant to enzyme activity, PEPC protein and PEPC messenger RNA amounts increased considerably. This photoresponse was shown to be under phytochrome control.

Monoclonal antibodies were produced against the G isozyme subunit of PEP carboxylase (PEPC) from Sorghum leaves by the hybridoma technique. More than 400 antibodies-producing hybridomas to PEPC were produced from the fusion of spleen cells from immunized mice with NS1 myeloma cells. By using an ELISA, three hybridomas (91-G, 83-G, 49-EG) were selected. Monoclonal antibodies were subsequently characterized in a Western experiment; Mabs 83-G and 91-G were found to be highly specific to the G isozyme whereas Mab 49-EG recognized both forms (E and G isozymes) of the enzyme. Addition of Mabs to the enzyme preparation did not modify its catalytic activity nor its activation by glycine. Use of these probes provided direct and definite evidence of the specific enhancing effect of light on the G form and on its corresponding mRNA.

The use of immunological techniques allowed us to purify close to homogeneity phosphoenolpyruvate carboxylase (PEPc, EC 4.1.1.31) from sorghum leaf. It was thus established that: 1) this protein is phosphorylated in vivo on seryl residues; 2) in C4-type photosynthesis, the phosphorylation process mainly concerns the PEPC isozyme form G; 3) enzyme phosphorylation displays significant variations through a day-night alternation which therefore suggests light control of the process.