Systems rebalancing of metabolism in response to sulfur deprivation, as revealed by metabolome analysis of Arabidopsis plants

Abstract

Sulfur is an essential macro-element 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 sulfur-related 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.

Figure 1.

Analysis of general biosynthetic activity in control and sulfur-starved plants. Exp, Experiment. A, Values ± sd characterize the average of five independent repetitions; asterisks indicate pairs of significantly different values with P < 0.05, as determined by Student's t test. B, The decline in the internal protein and chlorophyll levels, depicted as a percentage to the level in sulfur-sufficient conditions, which was assigned to 100%. Slopes depict the rate of the decline. The decline of the internal sulfur level was reported earlier (Nikiforova et al., 2003) and is given here for comparison.

Figure 2.

A, Chromatogram fragments derived from GC-MS analysis containing OAS and putrescine peaks. B, Portion of ion peaks belonging to nonidentified metabolites. C, Independent Component Analysis (ICA) applied for annotated metabolites (exp, experiment; c1 and c2, constitutive starvation time points 1 and 2; i1 and i2, induced starvation time points 1 and 2).

Figure 3.

Proportional changes in the levels of some metabolites under sulfur deficiency, measured by LC-MS, depicted as percentage of the sum of the metabolites in the respective chemical class (c1 and c2, constitutive starvation time points 1 and 2; i1 and i2, induced starvation time points 1 and 2). A, Lipids (PG, PE, MGDG, DGDG, and DAG). B, SAM and SAH.

Figure 4.

Mapping of measured metabolite concentrations onto plant biosynthetic pathways, with those showing increased levels under sulfur deficiency depicted in red and decreased levels in blue; standard amino acids are framed.

Figure 5.

Integrated analysis of transcriptome and metabolome. A, Mapping of the transcript and metabolite ratios (sulfur-starved plants to control plants) on metabolic pathways from the AraCyc database, using an Omics Viewer tool. Those pathways are depicted, which show the strongest and the most consistent changes on transcript level, and thus are considered to be mostly regulated transcriptionally. B, Functional categorization of the genes, correlating significantly (P < 0.05) to the sulfur-responding metabolites anthocyanins (Anth), tryptophan (TRP), OAS, Ser, putrescine, glutathione (GSH), allantoin, and SAM, depicted as percentage of genes from the corresponding functional category among the whole set of genes, significantly correlating to the corresponding metabolite. These results are put together in comparison to the percent representation of the corresponding functional category in the total set of annotated genes (dark columns).

Figure 6.

Summary scheme of the revealed mutual influences of physiological processes in sulfur-starved plants that lead to the rebalancing of the system.