Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography– and gas chromatography-mass spectrometry-based metabolomics-derived data.

Casein kinase 2 is a ubiquitous protein kinase that has puzzled researchers for several decades because of its pleiotropic activity. Here, we set out to identify the in vivo targets of plastid casein kinase 2 (pCK2) in Arabidopsis thaliana. Survey phosphoproteome analyses were combined with targeted analyses with wild-type and pck2 knockdown mutants to identify potential pCK2 targets by their decreased phosphorylation state in the mutant. To validate potential substrates, we complemented the pck2 knockdown line with tandem affinity tag (TAP)-tagged pCK2 and found it to restore growth parameters, as well as many, but not all, putative pCK2-dependent phosphorylation events. We further performed a targeted analysis at the end-of-night to increase the specificity of target protein identification. This analysis confirmed light-independent phosphorylation of several pCK2 target proteins. Based on the aforementioned data, we define a set of in vivo pCK2-targets that span different chloroplast functions, such as metabolism, transcription, translation and photosynthesis. The pleiotropy of pCK2 functions is also manifested by altered state transition kinetics during short-term acclimation and significant alterations in the mutant metabolism, supporting its function in photosynthetic regulation. Thus, our data expand our understanding on chloroplast phosphorylation networks and provide insights into kinase networks in the regulation of chloroplast functions.

Functional gene clusters, containing two or more genes encoding different enzymes for the same pathway, are sometimes observed in plant genomes, most often when the genes specify the synthesis of specialized defensive metabolites. Here, we show that a cluster of genes in tomato (Solanum lycopersicum; Solanaceae) contains genes for terpene synthases (TPSs) that specify the synthesis of monoterpenes and diterpenes from cis-prenyl diphosphates, substrates that are synthesized by enzymes encoded by cis-prenyl transferase (CPT) genes also located within the same cluster. The monoterpene synthase genes in the cluster likely evolved from a diterpene synthase gene in the cluster by duplication and divergence. In the orthologous cluster in Solanum habrochaites, a new sesquiterpene synthase gene was created by a duplication event of a monoterpene synthase followed by a localized gene conversion event directed by a diterpene synthase gene. The TPS genes in the Solanum cluster encoding cis-prenyl diphosphate–utilizing enzymes are closely related to a tobacco (Nicotiana tabacum; Solanaceae) diterpene synthase encoding Z-abienol synthase (Nt-ABS). Nt-ABS uses the substrate copal-8-ol diphosphate, which is made from the all-trans geranylgeranyl diphosphate by copal-8-ol diphosphate synthase (Nt-CPS2). The Solanum gene cluster also contains an ortholog of Nt-CPS2, but it appears to encode a nonfunctional protein. Thus, the Solanum functional gene cluster evolved by duplication and divergence of TPS genes, together with alterations in substrate specificity to utilize cis-prenyl diphosphates and through the acquisition of CPT genes.

The regulation of shoot branching is an essential determinant of plant architecture, integrating multiple external and internal signals. One of the signaling pathways regulating branching involves the MAX (more axillary branches) genes. Two of the genes within this pathway, MAX3/CCD7 and MAX4/CCD8, encode carotenoid cleavage enzymes involved in generating a branch‐inhibiting hormone, recently identified as strigolactone. Here, we report the cloning of SlCCD7 from tomato. As in other species, SlCCD7 encodes an enzyme capable of cleaving cyclic and acyclic carotenoids. However, the SlCCD7 protein has 30 additional amino acids of unknown function at its C terminus. Tomato plants expressing a SlCCD7 antisense construct display greatly increased branching. To reveal the underlying changes of this strong physiological phenotype, a metabolomic screen was conducted. With the exception of a reduction of stem amino acid content in the transgenic lines, no major changes were observed. In contrast, targeted analysis of the same plants revealed significantly decreased levels of strigolactone. There were no significant changes in root carotenoids, indicating that relatively little substrate is required to produce the bioactive strigolactones. The germination rate of Orobanche ramosa seeds was reduced by up to 90% on application of extract from the SlCCD7 antisense lines, compared with the wild type. Additionally, upon mycorrhizal colonization, C13 cyclohexenone and C14 mycorradicin apocarotenoid levels were greatly reduced in the roots of the antisense lines, implicating SlCCD7 in their biosynthesis. This work demonstrates the diverse roles of MAX3/CCD7 in strigolactone production, shoot branching, source–sink interactions and production of arbuscular mycorrhiza‐induced apocarotenoids.

We present a comprehensive analysis of ADP-glucose pyrophosphorylase (AGP)-repressed pea (Pisum sativum) seeds using transcript and metabolite profiling to monitor the effects that reduced carbon flow into starch has on carbon-nitrogen metabolism and related pathways. Changed patterns of transcripts and metabolites suggest that AGP repression causes sugar accumulation and stimulates carbohydrate oxidation via glycolysis, tricarboxylic acid cycle, and mitochondrial respiration. Enhanced provision of precursors such as acetyl-coenzyme A and organic acids apparently support other pathways and activate amino acid and storage protein biosynthesis as well as pathways fed by cytosolic acetyl-coenzyme A, such as cysteine biosynthesis and fatty acid elongation/metabolism. As a consequence, the resulting higher nitrogen (N) demand depletes transient N storage pools, specifically asparagine and arginine, and leads to N limitation. Moreover, increased sugar accumulation appears to stimulate cytokinin-mediated cell proliferation pathways. In addition, the deregulation of starch biosynthesis resulted in indirect changes, such as increased mitochondrial metabolism and osmotic stress. The combined effect of these changes is an enhanced generation of reactive oxygen species coupled with an up-regulation of energy-dissipating, reactive oxygen species protection, and defense genes. Transcriptional activation of mitogen-activated protein kinase pathways and oxylipin synthesis indicates an additional activation of stress signaling pathways. AGP-repressed embryos contain higher levels of jasmonate derivatives; however, this increase is preferentially in nonactive forms. The results suggest that, although metabolic/osmotic alterations in iAGP pea seeds result in multiple stress responses, pea seeds have effective mechanisms to circumvent stress signaling under conditions in which excessive stress responses and/or cellular damage could prematurely initiate senescence or apoptosis.

Under the auspices of the European Training and Networking Activity programme of the European Union, a ‘Metabolic Profiling and Data Analysis’ Plant Genomics and Bioinformatics Summer School was hosted in Potsdam, Germany between 20 and 29 September 2006. Sixteen early career researchers were invited from the European Union partner nations and the so‐called developing nations (Appendix). Lectures from invited leading European researchers provided an overview of the state of the art of these fields and seeded discussion regarding major challenges for their future advancement. Hands‐on experience was provided by an example experiment – that of defining the metabolic response of Arabidopsis to treatment of a commercial herbicide of defined mode of action. This experiment was performed throughout the duration of the course in order to teach the concepts underlying extraction and machine handling as well as to provide a rich data set with which the required computation and statistical skills could be illustrated. Here we review the state of the field by describing both key lectures given at and practical aspects taught at the summer school. In addition, we disclose results that were obtained using the four distinct technical platforms at the different participating institutes. While the effects of the chosen herbicide are well documented, this study looks at a broader number of metabolites than in previous investigations. This allowed, on the one hand, not only to characterise further effects of the herbicide than previously observed but also to detect molecules other than the herbicide that were obviously present in the commercial formulation. These data and the workshop in general are all discussed in the context of the teaching of metabolomics.