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Sulfur deficiency-induced proteins SDI1 and SDI2 play a fundamental role in sulfur homeostasis under sulfate-deprived conditions (−S) by downregulating glucosinolates. Here, we identified that besides glucosinolate regulation under –S, SDI1 downregulates another sulfur pool, the S-rich 2S seed storage proteins in Arabidopsis (Arabidopsis thaliana) seeds. We identified that MYB28 directly regulates 2S seed storage proteins by binding to the At2S4 promoter. We also showed that SDI1 downregulates 2S seed storage proteins by forming a ternary protein complex with MYB28 and MYC2, another transcription factor involved in the regulation of seed storage proteins. These findings have significant implications for the understanding of plant responses to sulfur deficiency., Sulfur Deficiency Induced1 (SDI1) upregulates S-poor seed storage proteins in favor of S-rich seed storage proteins.

Vegetative growth requires the systemic coordination of numerous cellular processes, which are controlled by regulatory proteins that monitor extracellular and intracellular cues and translate them into growth decisions. In eukaryotes, one of the central factors regulating growth is the serine/threonine protein kinase Target of Rapamycin (TOR), which forms complexes with regulatory proteins. To understand the function of one such regulatory protein, Regulatory-Associated Protein of TOR 1B (RAPTOR1B), in plants, we analyzed the effect of raptor1b mutations on growth and physiology in Arabidopsis (Arabidopsis thaliana) by detailed phenotyping, metabolomic, lipidomic, and proteomic analyses. Mutation of RAPTOR1B resulted in a strong reduction of TOR kinase activity, leading to massive changes in central carbon and nitrogen metabolism, accumulation of excess starch, and induction of autophagy. These shifts led to a significant reduction of plant growth that occurred nonlinearly during developmental stage transitions. This phenotype was accompanied by changes in cell morphology and tissue anatomy. In contrast to previous studies in rice (Oryza sativa), we found that the Arabidopsis raptor1b mutation did not affect chloroplast development or photosynthetic electron transport efficiency; however, it resulted in decreased CO2 assimilation rate and increased stomatal conductance. The raptor1b mutants also had reduced abscisic acid levels. Surprisingly, abscisic acid feeding experiments resulted in partial complementation of the growth phenotypes, indicating the tight interaction between TOR function and hormone synthesis and signaling in plants.

Developmental senescence is a coordinated physiological process in plants and is critical for nutrient redistribution from senescing leaves to newly formed sink organs, including young leaves and developing seeds. Progress has been made concerning the genes involved and the regulatory networks controlling senescence. The resulting complex metabolome changes during senescence have not been investigated in detail yet. Therefore, we conducted a comprehensive profiling of metabolites, including pigments, lipids, sugars, amino acids, organic acids, nutrient ions, and secondary metabolites, and determined approximately 260 metabolites at distinct stages in leaves and siliques during senescence in Arabidopsis (Arabidopsis thaliana). This provided an extensive catalog of metabolites and their spatiotemporal cobehavior with progressing senescence. Comparison with silique data provides clues to source-sink relations. Furthermore, we analyzed the metabolite distribution within single leaves along the basipetal sink-source transition trajectory during senescence. Ceramides, lysolipids, aromatic amino acids, branched chain amino acids, and stress-induced amino acids accumulated, and an imbalance of asparagine/aspartate, glutamate/glutamine, and nutrient ions in the tip region of leaves was detected. Furthermore, the spatiotemporal distribution of tricarboxylic acid cycle intermediates was already changed in the presenescent leaves, and glucosinolates, raffinose, and galactinol accumulated in the base region of leaves with preceding senescence. These results are discussed in the context of current models of the metabolic shifts occurring during developmental and environmentally induced senescence. As senescence processes are correlated to crop yield, the metabolome data and the approach provided here can serve as a blueprint for the analysis of traits and conditions linking crop yield and senescence.

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 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.

cDNAs encoding a high-affinity sulfate transporter and an adenosine 5′-phosphosulfate reductase from potato (Solanum tuberosum L. cv Désirée) have been cloned and used to examine the hypothesis that sulfate uptake and assimilation is transcriptionally regulated and that this is mediated via intracellular O-acetylserine (OAS) pools. Gas chromotography coupled to mass spectrometry was used to quantify OAS and its derivative, N-acetylserine. Treatment with external OAS increased sulfate transporter and adenosine 5′-phosphosulfate reductase gene expression consistent with a model of transcriptional induction by OAS. To investigate this further, the Escherichia coli gene cysE (serine acetyltransferase EC 2.3.1.30), which synthesizes OAS, has been expressed in potato to modify internal metabolite pools. Transgenic lines, with increased cysteine and glutathione pools, particularly in the leaves, had increased sulfate transporter expression in the roots. However, the small increases in the OAS pools were not supportive of the hypothesis that this molecule is the signal of sulfur (S) nutritional status. In addition, although during S starvation the content of S-containing compounds decreased (consistent with derepression as a mechanism of regulation), OAS pools increased only following extended starvation, probably as a consequence of the S starvation. Taken together, expression of these genes may be induced by a demand-driven model, via a signal from the shoots, which is not OAS. Rather, the signal may be the depletion of intermediates of the sulfate assimilation pathway, such as sulfide, in the roots. Finally, sulfate transporter activity did not increase in parallel with transcript and protein abundance, indicating additional posttranslational regulatory mechanisms.

Plant cysteine (Cys) synthesis can occur in three cellular compartments: the chloroplast, cytoplasm, and mitochondrion. Cys formation is catalyzed by the enzyme O-acetylserine(thiol)lyase (OASTL) using O-acetylserine (OAS) and sulfide as substrates. To unravel the function of different isoforms of OASTL in cellular metabolism, a transgenic approach was used to down-regulate specifically the plastidial and cytosolic isoforms in potato (Solanum tuberosum). This approach resulted in decreased RNA, protein, and enzymatic activity levels. Intriguingly, H2S-releasing capacity was also reduced in these lines. Unexpectedly, the thiol levels in the transgenic lines were, regardless of the selected OASTL isoform, significantly elevated. Furthermore, levels of metabolites such as serine, OAS, methionine, threonine, isoleucine, and lysine also increased in the investigated transgenic lines. This indicates that higher Cys levels might influence methionine synthesis and subsequently pathway-related amino acids. The increase of serine and OAS points to suboptimal Cys synthesis in transgenic plants. Taking these findings together, it can be assumed that excess OASTL activity regulates not only Cys de novo synthesis but also its homeostasis. A model for the regulation of Cys levels in plants is proposed.

In plants, metabolic pathways leading to methionine (Met) and threonine diverge at the level of their common substrate, O-phosphohomoserine (OPHS). To investigate the regulation of this branch point, we engineered transgenic potato (Solanum tuberosum) plants affected in cystathionine γ-synthase (CgS), the enzyme utilizing OPHS for the Met pathway. Plants overexpressing potato CgS exhibited either: (a) high transgene RNA levels and 2.7-fold elevated CgS activities but unchanged soluble Met levels, or (b) decreased transcript amounts and enzyme activities (down to 7% of wild-type levels). In leaf tissues, these cosuppression lines revealed a significant reduction of soluble Met and an accumulation of OPHS. Plants expressing CgS antisense constructs exhibited reductions in enzyme activity to as low as 19% of wild type. The metabolite contents of these lines were similar to those of the CgS cosuppression lines. Surprisingly, neither increased nor decreased CgS activity led to visible phenotypic alterations or significant changes in protein-bound Met levels in transgenic potato plants, indicating that metabolic flux to Met synthesis was not greatly affected. Furthermore, in vitro feeding experiments revealed that potato CgS is not subject to feedback regulation by Met, as reported for Arabidopsis. In conclusion, our results demonstrate that potato CgS catalyzes a near-equilibrium reaction and, more importantly, does not display features of a pathway-regulating enzyme. These results are inconsistent with the current hypothesis that CgS exerts major Met metabolic flux control in higher plants.

Methionine (Met) and threonine (Thr) are members of the aspartate family of amino acids. In plants, their biosynthetic pathways diverge at the level of O-phosphohomo-serine (Ser). The enzymes cystathionine gamma-synthase and Thr synthase (TS) compete for the common substrate O-phosphohomo-Ser with the notable feature that plant TS is activated through S-adenosyl-Met, a metabolite derived from Met. To investigate the regulation of this branch point, we engineered TS antisense potato (Solanum tuberosum cv Désirée) plants using the constitutive cauliflower mosaic virus 35S promoter. In leaf tissues, these transgenics exhibit a reduction of TS activity down to 6% of wild-type levels. Thr levels are reduced to 45% wild-type controls, whereas Met levels increase up to 239-fold depending on the transgenic line and environmental conditions. Increased levels of homo-Ser and homo-cysteine indicate increased carbon allocation into the aspartate pathway. In contrast to findings in Arabidopsis, increased Met content has no detectable effect on mRNA or protein levels or on the enzymatic activity of cystathionine gamma-synthase in potato. Tubers of TS antisense potato plants contain a Met level increased by a factor of 30 and no reduction in Thr. These plants offer a major biotechnological advance toward the development of crop plants with improved nutritional quality.

Plant response mechanisms to deficiency of a single nutrient, such as sulfur (S) or iron (Fe), have been described at agronomic, physiological, biochemical, metabolomics, and transcriptomic levels. However, agroecosystems are often characterized by different scenarios, in which combined nutrient deficiencies are likely to occur. Soils are becoming depleted for S, whereas Fe, although highly abundant in the soil, is poorly available for uptake because of its insolubility in the soil matrix. To this end, earlier reports showed that a limited S availability reduces Fe uptake and that Fe deficiency results in the modulation of sulfate uptake and assimilation. However, the mechanistic basis of this interaction remains largely unknown. Metabolite profiling of tomato (Solanum lycopersicum) shoots and roots from plants exposed to Fe, S, and combined Fe and S deficiency was performed to improve the understanding of the S-Fe interaction through the identification of the main players in the considered pathways. Distinct changes were revealed under the different nutritional conditions. Furthermore, we investigated the development of the Fe deficiency response through the analysis of expression of ferric chelate reductase, iron-regulated transporter, and putative transcription factor genes and plant sulfate uptake and mobilization capacity by analyzing the expression of genes encoding sulfate transporters (STs) of groups 1, 2, and 4 (SlST1.1, SlST1.2, SlST2.1, SlST2.2, and SlST4.1). We identified a high degree of common and even synergistic response patterns as well as nutrient-specific responses. The results are discussed in the context of current models of nutrient deficiency responses in crop plants.