SummaryHigher plants assimilate inorganic sulfate into cysteine, which is subsequently converted to methionine, and into a variety of other sulfur-containing organic compounds. To resist sulfur deficiency, plants must demonstrate physiological flexibility: the expression of an extensive set of genes and gene regulators that act in the affected pathways or signalling cascades must be delicately tuned in response to environmental challenges. To elucidate this network of interactions, we have applied an array hybridisation/transcript profiling method to Arabidopsis plants subjected to 6, 10 and 13 days of constitutive and induced sulfur starvation. The temporal expression behaviour of approximately 7200 non-redundant genes was analysed simultaneously. The experiment was designed in a way to identify statistically significant changes of gene expression based on sufficient numbers of repeated hybridisations performed with five uniform pools of plant material. The expression profiles were processed to select differentially expressed genes. Among the 1507 sulfur-responsive clones implicated in this way, 632 genes responded specifically to sulfur deficiency by significant over-expression. The sulfur-responsive genes were grouped according to functional categories or biosynthetic pathways. As expected, genes of the sulfur assimilation pathway were altered in expression. Furthermore, genes involved in flavonoid, auxin, and jasmonate biosynthesis pathways were upregulated in conditions of sulfur deficiency. Based on the correlative analysis of gene expression patterns, we suggest that a complex co-ordination of systematic responses to sulfur depletion is provided via integration of flavonoid, auxin and jasmonate pathway elements. Plait concept for transduction of specificity via the main non-specific signalling stream is proposed.

SUMMARYThe unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled 13 C, 15 N and 34 S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a highresolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.

Sulfur is an essential macroelement in plant and animal nutrition. Plants assimilate inorganic sulfate into two sulfur-containing amino acids, cysteine and methionine. Low supply of sulfate leads to decreased sulfur pools within plant tissues. As sulfurrelated metabolites represent an integral part of plant metabolism with multiple interactions, sulfur deficiency stress induces a number of adaptive responses, which must be coordinated. To reveal the coordinating network of adaptations to sulfur deficiency, metabolite profiling of Arabidopsis has been undertaken. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry techniques revealed the response patterns of 6,023 peaks of nonredundant ion traces and relative concentration levels of 134 nonredundant compounds of known chemical structure. Here, we provide a catalogue of the detected metabolic changes and reconstruct the coordinating network of their mutual influences. The observed decrease in biomass, as well as in levels of proteins, chlorophylls, and total RNA, gives evidence for a general reduction of metabolic activity under conditions of depleted sulfur supply. This is achieved by a systemic adjustment of metabolism involving the major metabolic pathways. Sulfur/carbon/nitrogen are partitioned by accumulation of metabolites along the pathway O-acetylserine to serine to glycine, and are further channeled together with the nitrogen-rich compound glutamine into allantoin. Mutual influences between sulfur assimilation, nitrogen imbalance, lipid breakdown, purine metabolism, and enhanced photorespiration associated with sulfur-deficiency stress are revealed in this study. These responses may be assembled into a global scheme of metabolic regulation induced by sulfur nutritional stress, which optimizes resources for seed production.For living organisms with a complex hierarchical organization, such as plants, the necessity for close coordination of various elements requires systemic organization at the level of the whole organism. Additional complexity is imposed on the system through environmental variability. Due to the inability to escape unfavorable environmental conditions, plants have evolved complex mechanisms to sense and transmit external signals to the internal decision points to trigger the adaptive response program for homeostatic maintenance. Such adaptive programs are accomplished at multiple organizational levels, e.g. gene and enzymatic activities, being finally manifested in altered metabolite concentrations. This study describes the metabolic component of the whole-system response to sulfur deficiency, as an environmental perturbation, with special emphasis on the mechanisms of the response coordination.Despite the difficulties in measuring metabolites due to their dynamic behavior and complex chemistry, new methods allow profiling of low molecular weight compounds, with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) being the most robust (Stitt and Fernie, 2003). In th...