Martine Thomas

The Arabidopsis CDPK-SnRK Superfamily of Protein Kinases

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.

Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase

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.

Arabidopsis homologs of the shaggy and GSK-3 protein kinases: molecular cloning and functional expression in Escherichia coli.

The conservation in evolution of fundamental signal transduction modules offers a means of isolating genes likely to be involved in plant development. We have amplified by PCR Arabidopsis cDNA and genomic sequences related to the product of the shaggy/zeste-white 3 (sgg) segment polarity gene of Drosophila. This regulatory protein is functionally homologous to glycogen synthase kinase-3 in mammals (GSK-3), which regulates, among others, the DNA-binding activity of the c-jun/AP1 transcription factor. Analysis of PCR products led to the identification of five genes; for two of which, corresponding full-length cDNAs, ASK-α and γ (for Arabidopsis shaggy-related protein kinase), were characterized. The encoded proteins were 70% identical to GSK-3 and sgg over the protein kinase catalytic domain and, after production in Escherichia coli, autophosphorylated mainly on threonine and serine residues, but phosphotyrosine was also detected. ASK-α and ASK-γ also phosphorylated phosphatase inhibitor-2 and myelin basic protein, on threonine and serine, respectively. The high conservation of the protein kinases of GSK-3 family, and their action at the transcriptional level, suggest that the ASK proteins have important functions in higher plants.

β-Subunits of the SnRK1 Complexes Share a Common Ancestral Function Together with Expression and Function Specificities; Physical Interaction with Nitrate Reductase Specifically Occurs via AKINβ1-Subunit

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 α-type catalytic subunit and two γ- and β-type regulatory subunits. In yeast it has been proposed that the β-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 β-type subunits of Arabidopsis (Arabidopsis thaliana; AKINβ1, AKINβ2, and AKINβ3) restore the growth phenotype of the yeast sip1Δsip2Δgal83Δ triple mutant, thus suggesting the conservation of an ancestral function. Expression analyses, using AKINβ promoter∷β-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 β-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 β-type subunit in plants.

Cross-phosphorylation between Arabidopsis thaliana Sucrose Nonfermenting 1-related Protein Kinase 1 (AtSnRK1) and Its Activating Kinase (AtSnAK) Determines Their Catalytic Activities

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 Thr175. The phosphorylation of these kinases has been investigated by site-directed mutagenesis and tandem mass spectrometry. The autophosphorylation site of AtSnAK2 was identified as Thr154, and it was shown to be required for AtSnAK catalytic activity. Interestingly, activated AtSnRK1 exerted a negative feedback phosphorylation on AtSnAK2 at Ser261 (Ser260 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.

Light-regulation of phosphoenolpyruvate carboxylase mRNA in leaves of C4 plants: Evidence for phytochrome control on transcription during greening and for rhythmicity

Abstract 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.

SNF1-related protein kinases (SnRKs) — Regulators at the heart of the control of carbon metabolism and partitioning

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.

Gene density and organization in a small region of the Arabidopsis thaliana genome

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, Atcysl and Athypl (for Arabidopsis thaliana cystathionine γ-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. Atcysl, Athypl, 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 Atcysl shows a high degree of similarity with the cystathionine γ-synthase from Escherichia coli. The putative product of the Athypl 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 Atcysl, Athypl and AKin10 per haploid genome, and Northern blot analysis demonstrates that the three genes are differentially expressed in roots, shoots and leaves.

Opportunities for manipulating the seed protein composition of wheat and barley in order to improve quality

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.

Identification of cdc2cAt: a new cyclin-dependent kinase expressed in Arabidopsis thaliana flowers.

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.