Redox state-dependent modulation of plant SnRK1 kinase activity differs from AMPK regulation in animals.

The evolutionarily highly conserved SNF1-related protein kinase (SnRK1) protein kinase is a metabolic master regulator in plants, balancing the critical energy consumption between growth- and stress response-related metabolic pathways. While the regulation of the mammalian [AMP-activated protein kinase (AMPK)] and yeast (SNF1) orthologues of SnRK1 is well-characterised, the regulation of SnRK1 kinase activity in plants is still an open question. Here we report that the activity and T-loop phosphorylation of AKIN10, the kinase subunit of the SnRK1 complex, is regulated by the redox status. Although this regulation is dependent on a conserved cysteine residue, the underlying mechanism is different to the redox regulation of animal AMPK and has functional implications for the regulation of the kinase complex in plants under stress conditions.

A toolset of aequorin expression vectors for in planta studies of subcellular calcium concentrations in Arabidopsis thaliana.

Calcium has long been acknowledged as one of the most important signalling components in plants. Many abiotic and biotic stimuli are transduced into a cellular response by temporal and spatial changes in cellular calcium concentration and the calcium-sensitive protein aequorin has been exploited as a genetically encoded calcium indicator for the measurement of calcium in planta. The objective of this work was to generate a compatible set of aequorin expression plasmids for the generation of transgenic plant lines to measure changes in calcium levels in different cellular subcompartments. Aequorin was fused to different targeting peptides or organellar proteins as a means to localize it to the cytosol, the nucleus, the plasma membrane, and the mitochondria. Furthermore, constructs were designed to localize aequorin in the stroma as well as the inner and outer surface of the chloroplast envelope membranes. The modular set-up of the plasmids also allows the easy replacement of targeting sequences to include other compartments. An additional YFP-fusion was included to verify the correct subcellular localization of all constructs by laser scanning confocal microscopy. For each construct, pBin19-based binary expression vectors driven by the 35S or UBI10 promoter were made for Agrobacterium-mediated transformation. Stable Arabidopsis lines were generated and initial tests of several lines confirmed their feasibility to measure calcium signals in vivo.

Arabidopsis calcium-binding mitochondrial carrier proteins as potential facilitators of mitochondrial ATP-import and plastid SAM-import.

Chloroplasts and mitochondria are central to crucial cellular processes in plants and contribute to a whole range of metabolic pathways. The use of calcium ions as a secondary messenger in and around organelles is increasingly appreciated as an important mediator of plant cell signaling, enabling plants to develop or to acclimatize to changing environmental conditions. Here, we have studied the four calcium-dependent mitochondrial carriers that are encoded in the Arabidopsis genome. An unknown substrate carrier, which was previously found to localize to chloroplasts, is proposed to present a calcium-dependent S-adenosyl methionine carrier. For three predicted ATP/phosphate carriers, we present experimental evidence that they can function as mitochondrial ATP-importers.

Mining the soluble chloroplast proteome by affinity chromatography.

Chloroplasts are fundamental organelles enabling plant photoautotrophy. Besides their outstanding physiological role in fixation of atmospheric CO(2), they harbor many important metabolic processes such as biosynthesis of amino acids, vitamins or hormones. Technical advances in MS allowed the recent identification of most chloroplast proteins. However, for a deeper understanding of chloroplast function it is important to obtain a complete list of constituents, which is so far limited by the detection of low-abundant proteins. Therefore, we developed a two-step strategy for the enrichment of low-abundant soluble chloroplast proteins from Pisum sativum and their subsequent identification by MS. First, chloroplast protein extracts were depleted from the most abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase by SEC or heating. Further purification was carried out by affinity chromatography, using ligands specific for ATP- or metal-binding proteins. By these means, we were able to identify a total of 448 proteins including 43 putative novel chloroplast proteins. Additionally, the chloroplast localization of 13 selected proteins was confirmed using yellow fluorescent protein fusion analyses. The selected proteins included a phosphoglycerate mutase, a cysteine protease, a putative protein kinase and an EF-hand containing substrate carrier protein, which are expected to exhibit important metabolic or regulatory functions.

Cross-talk of calcium-dependent protein kinase and MAP kinase signaling.

Plants use different signalling pathways to acclimate to changing environmental conditions. Fast changes in the concentration of free Ca(2+) ions - so called Ca(2+) signals - are among the first responses to many stress situations. These signals are decoded by different types of calcium-dependent protein kinases, which - together with mitogen-activated protein kinases (MAPK) - present two major pathways that are widely used to adapt the cellular metabolism to a changing environment. Ca(2+)-dependent protein kinase (CDPK) and MAPK pathways are known to be involved in signalling of abiotic and biotic stress in animal, yeast and plant cells. In many cases both pathways are activated in response to the same stimuli leading to the question of a potential cross-talk between those pathways. Cross-talk between Ca(2+)-dependent and MAPK signalling pathways has been elaborately studied in animal cells, but it has hardly been investigated in plants. Early studies of CDPKs involved in the biotic stress response in tobacco indicated a cross-talk of CDPK and MAPK activities, whereas a recent study in Arabidopsis revealed that CDPKs and MAPKs act differentially in innate immune signalling and showed no direct cross-talk between CDPK and MAPK activities. Similar results were also reported for CDPK and MAPK activities in the salt stress response in Arabidopsis. Different modes of action are furthermore supported by the different subcellular localization of the involved kinases. In this review, we discuss recent findings on CDPK and MAPK signalling with respect to potential cross-talk and the subcellular localization of the involved components.

Protein N-acylation overrides differing targeting signals.

In a bioinformatics based screen for chloroplast-localized protein kinases we noticed that available protein targeting predictors falsely predicted chloroplast localization. This seems to be due to interference with N-terminal protein acylation, which is of particular importance for protein kinases. Their N-myristoylation was found to be highly overrepresented in the proteome, whereas myristoylation motifs are almost absent in known chloroplast proteins. However, only abolishing their myristoylation was not sufficient to target those kinases to chloroplasts and resulted in nuclear accumulation instead. In contrast, inhibition of N-myristoylation of a calcium-dependent protein kinase was sufficient to alter its localization from the plasma membrane to chloroplasts and chloroplast localization of ferredoxin-NADP+ reductase and Rubisco activase could be efficiently suppressed by artificial introduction of myristoylation and palmitoylation sites.

VIP1 response elements mediate mitogen-activated protein kinase 3-induced stress gene expression.

The plant pathogen Agrobacterium tumefaciens transforms plant cells by delivering its T-DNA into the plant cell nucleus where it integrates into the plant genome and causes tumor formation. A key role of VirE2-interacting protein 1 (VIP1) in the nuclear import of T-DNA during Agrobacterium-mediated plant transformation has been unravelled and VIP1 was shown to undergo nuclear localization upon phosphorylation by the mitogen-activated protein kinase MPK3. Here, we provide evidence that VIP1 encodes a functional bZIP transcription factor that stimulates stress-dependent gene expression by binding to VIP1 response elements (VREs), a DNA hexamer motif. VREs are overrepresented in promoters responding to activation of the MPK3 pathway such as Trxh8 and MYB44. Accordingly, plants overexpressing VIP1 accumulate high levels of Trxh8 and MYB44 transcripts, whereas stress-induced expression of these genes is impaired in mpk3 mutants. Trxh8 and MYB44 promoters are activated by VIP1 in a VRE-dependent manner. VIP1 strongly enhances expression from a synthetic promoter harboring multiple VRE copies and directly interacts with VREs in vitro and in vivo. Chromatin immunoprecipitation assays of the MYB44 promoter confirm that VIP1 binding to VREs is enhanced under conditions of MPK3 pathway stimulation. These results provide molecular insight into the cellular mechanism of target gene regulation by the MPK3 pathway.

Experimental testing of predicted myristoylation targets involved in asymmetric cell division and calcium-dependent signalling.

Evolutionary conservation of N-terminal N-myristoylation within protein families indicates significant functional impact of this lipid posttranslational modification for function. In the MYRbase study (Maurer-Stroh et al., (2004) Genome Biology 5, R21), protein families with relevance to asymmetric cell division in animals and the group of plant calcium-dependent protein kinases (CPKs) have surfaced with many predicted myristoylated members. Here, we describe experimental in vitro verification of predicted myristoylation and explore its impact on subcellular localization for these targets in vivo. Our results confirm that, indeed, Numb isoform A, Neuralized isoforms C and D from Drosophila melanogaster and two Neuralized-like homologues from Mus musculus have the capability for N-terminal myristoylation in vitro and in vivo (in fly tissue and in mouse 3T3 cells respectively) whereas other isoforms such as Neuralized A and B have not. The latter two cases are an examples of different potential of various isoforms for posttranslational modifications. Additionally, the Arabidopsis thaliana CDPKs CPK6, CPK9 and CPK13 are shown to be substrates for myristoylation in vitro, which also affects their subcellular localization (in Arabidopsis protoplasts and tobacco leaves). At the same time, CPK6 and CPK13 do not appear to be substrates of a NMT1-like enzyme; the reasons for differing substrate specificities of NMT homologues in plants are derived from the evolutionary divergence of their N-myristoyl transferase sequences. As a methodical advance, we describe a fast and very sensitive technique (compared to traditional autoradiography) for in vitro testing of myristoylation based on thin layer chromatography read-out of the incorporated radioactive myristoyl anchor with subsequent Western blotting detection for protein yield determination using the same membrane.

The PP2C-type phosphatase AP2C1, which negatively regulates MPK4 and MPK6, modulates innate immunity, jasmonic acid, and ethylene levels in Arabidopsis.

Wound signaling pathways in plants are mediated by mitogen-activated protein kinases (MAPKs) and stress hormones, such as ethylene and jasmonates. In Arabidopsis thaliana, the transmission of wound signals by MAPKs has been the subject of detailed investigations; however, the involvement of specific phosphatases in wound signaling is not known. Here, we show that AP2C1, an Arabidopsis Ser/Thr phosphatase of type 2C, is a novel stress signal regulator that inactivates the stress-responsive MAPKs MPK4 and MPK6. Mutant ap2c1 plants produce significantly higher amounts of jasmonate upon wounding and are more resistant to phytophagous mites (Tetranychus urticae). Plants with increased AP2C1 levels display lower wound activation of MAPKs, reduced ethylene production, and compromised innate immunity against the necrotrophic pathogen Botrytis cinerea. Our results demonstrate a key role for the AP2C1 phosphatase in regulating stress hormone levels, defense responses, and MAPK activities in Arabidopsis and provide evidence that the activity of AP2C1 might control the plant's response to B. cinerea.