Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants.

Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO2-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment.

The evolutionarily conserved SNF1-related protein kinase 1 (SnRK1) kinase complex is a key regulator in adjusting cellular metabolism during starvation, stress conditions, and growth-promoting conditions. Over the last two decades, extensive genetic evidence for a widespread SnRK1 signaling network has accumulated. It is now well established that SnRK1 is a central integrator of energy signaling. However, little is known about the connections between the cytoplasmic and nuclear-localized SnRK1 and plastids and mitochondria as the main energy-producing compartments in the cell. Here, we review recent findings indicating how SnRK1 affects metabolic adaptation, including plastidial and mitochondrial functions. Special emphasis is put on identified direct targets of SnRK1, which would eventually enable cross talk with organelles. In this context, a number of transcription factors (TFs) are emerging as mediators of SnRK1 signaling, potentially linking SnRK1 activity to organellar functions. Furthermore, many SnRK1 targets act in various hormonal signaling pathways, which are at least partly localized in plastids. With this review, we summarize the current knowledge on SnRK1 organelle interaction and provide ideas on the potential molecular mechanisms governing these interactions. METABOLIC REPROGRAMMING BY SNRK1 KINASE ACTIVITY UNDER DIFFERENT GROWTH AND STRESS CONDITIONSAlready under optimal growth conditions, plants need to continuously adjust their metabolic balance between autotrophic growth based on photosynthesis during the day and respiration during the night. At daytime, sugars and other metabolites are produced by photosynthesis in chloroplasts and further distributed to be used in other metabolic pathways at different cellular compartments or transported to nonphotosynthetic sink tissues. During the night, starch and sugars supply carbon equivalents for respiration, providing the necessary energy for further growth and metabolic activities. Additionally, metabolic reprogramming is required for plants to adjust their metabolism to diverse biotic and abiotic environmental stimuli. This often leads to a stop of plant growth involving a reduction in ribosomal protein synthesis, and in parallel, accumulation of protective metabolites or defense compounds. This switching of cellular energy metabolism is mediated by the activity of the evolutionarily conserved AMPK/ SNF1/SnRK1 kinase complex (Box 1; Crozet et al.,

SUMMARYHere, we describe a method for the combined metabolomic, proteomic, transcriptomic and genomic analysis from one single sample as a major step for multilevel data integration strategies in systems biology. While extracting proteins and DNA, this protocol also allows the separation of metabolites into polar and lipid fractions, as well as RNA fractionation into long and small RNAs, thus allowing a broad range of transcriptional studies. The isolated biomolecules are suitable for analysis with different methods that range from electrophoresis and blotting to state-of-the-art procedures based on mass spectrometry (accurate metabolite profiling, shot-gun proteomics) or massive sequencing technologies (transcript analysis). The low amount of starting tissue, its cost-efficiency compared with the utilization of commercial kits, and its performance over a wide range of plant, microbial, and algal species such as Chlamydomonas, Arabidopsis, Populus, or Pinus, makes this method a universal alternative for multiple molecular isolation from plant tissues.

SUMMARYTheobroma cacao and its popular product, chocolate, are attracting attention due to potential health benefits including antioxidative effects by polyphenols, anti-depressant effects by high serotonin levels, inhibition of platelet aggregation and prevention of obesity-dependent insulin resistance. The development of cacao seeds during fruit ripening is the most crucial process for the accumulation of these compounds. In this study, we analyzed the primary and the secondary metabolome as well as the proteome during Theobroma cacao cv. Forastero seed development by applying an integrative extraction protocol. The combination of multivariate statistics and mathematical modelling revealed a complex consecutive coordination of primary and secondary metabolism and corresponding pathways. Tricarboxylic acid (TCA) cycle and aromatic amino acid metabolism dominated during the early developmental stages (stages 1 and 2; cell division and expansion phase). This was accompanied with a significant shift of proteins from phenylpropanoid metabolism to flavonoid biosynthesis. At stage 3 (reserve accumulation phase), metabolism of sucrose switched from hydrolysis into raffinose synthesis. Lipids as well as proteins involved in lipid metabolism increased whereas amino acids and N-phenylpropenoyl amino acids decreased. Purine alkaloids, polyphenols, and raffinose as well as proteins involved in abiotic and biotic stress accumulated at stage 4 (maturation phase) endowing cacao seeds the characteristic astringent taste and resistance to stress. In summary, metabolic key points of cacao seed development comprise the sequential coordination of primary metabolites, phenylpropanoid, N-phenylpropenoyl amino acid, serotonin, lipid and polyphenol metabolism thereby covering the major compound classes involved in cacao aroma and health benefits.

Stress impacts negatively on plant growth and crop productivity, caicultural production worldwide. Throughout their life, plants are often confronted with multiple types of stress that affect overall cellular energy status and activate energy-saving responses. The resulting low energy syndrome (LES) includes transcriptional, translational, and metabolic reprogramming and is essential for stress adaptation. The conserved kinases sucrose-non-fermenting-1-related protein kinase-1 (SnRK1) and target of rapamycin (TOR) play central roles in the regulation of LES in response to stress conditions, affecting cellular processes and leading to growth arrest and metabolic reprogramming. We review the current understanding of how TOR and SnRK1 are involved in regulating the response of plants to low energy conditions. The central role in the regulation of cellular processes, the reprogramming of metabolism, and the phenotypic consequences of these two kinases will be discussed in light of current knowledge and potential future developments.

BackgroundThe variation of growth and cold tolerance of two natural Arabidopsis accessions, Cvi (cold sensitive) and Rschew (cold tolerant), was analysed on a proteomic, phosphoproteomic and metabolomic level to derive characteristic information about genotypically distinct strategies of metabolic reprogramming and growth maintenance during cold acclimation.ResultsGrowth regulation before and after a cold acclimation period was monitored by recording fresh weight of leaf rosettes. Significant differences in the shoot fresh weight of Cvi and Rschew were detected both before and after acclimation to low temperature. During cold acclimation, starch levels were found to accumulate to a significantly higher level in Cvi compared to Rschew. Concomitantly, statistical analysis revealed a cold-induced decrease of beta-amylase 3 (BAM3; AT4G17090) in Cvi but not in Rschew. Further, only in Rschew we observed an increase of the protein level of the debranching enzyme isoamylase 3 (ISA3; AT4G09020). Additionally, the cold response of both accessions was observed to severely affect ribosomal complexes, but only Rschew showed a pronounced accumulation of carbon and nitrogen compounds. The abundance of the Cold Regulated (COR) protein COR78 (AT5G52310) as well as its phosphorylation was observed to be positively correlated with the acclimation state of both accessions. In addition, transcription factors being involved in growth and developmental regulation were found to characteristically separate the cold sensitive from the cold tolerant accession. Predicted protein-protein interaction networks (PPIN) of significantly changed proteins during cold acclimation allowed for a differentiation between both accessions. The PPIN revealed the central role of carbon/nitrogen allocation and ribosomal complex formation to establish a new cold-induced metabolic homeostasis as also observed on the level of the metabolome and proteome.ConclusionOur results provide evidence for a comprehensive multi-functional molecular interaction network orchestrating growth regulation and cold acclimation in two natural accessions of Arabidopsis thaliana. The differential abundance of beta-amylase 3 and isoamylase 3 indicates a central role of transitory starch degradation in the coordination of growth regulation and the development of stress tolerance. Finally, our study indicates naturally occurring differential patterns of C/N balance and protein synthesis during cold acclimation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0668-1) contains supplementary material, which is available to authorized users.

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.