Holger Hesse

Sulfur plays a pivotal role in the cellular metabolism of many organisms. In plants, the uptake and assimilation of sulfate is strongly regulated at the transcriptional level. Regulatory factors are the demand of reduced sulfur in organic or non-organic form and the level of O-acetylserine (OAS), the carbon precursor for cysteine biosynthesis. In plants, cysteine is synthesized by action of the cysteine–synthase complex (CSC) containing serine acetyltransferase (SAT) and O-acetylserine-(thiol)-lyase (OASTL). Both enzymes are located in plastids, mitochondria and the cytosol. The function of the compartmentation of the CSC to regulate sulfate uptake and assimilation is still not clearly resolved. To address this question, we analyzed Arabidopsis thaliana mutants for the plastidic and cytosolic SAT isoenzymes under sulfur starvation conditions. In addition, subcellular metabolite analysis by non-aqueous fractionation revealed distinct changes in subcellular metabolite distribution upon short-term sulfur starvation. Metabolite and transcript analyses of SERAT1.1 and SERAT2.1 mutants [previously analyzed in Krueger et al. (Plant Cell Environ 32:349–367, 2009)] grown under sulfur starvation conditions indicate that both isoenzymes do not contribute directly to the transcriptional regulation of genes involved in sulfate uptake and assimilation. Here, we summarize the current knowledge about the regulation of cysteine biosynthesis and the contribution of the different compartments to this metabolic process. We relate hypotheses and views of the regulation of cysteine biosynthesis with our results of applying sulfur starvation to mutants impaired in compartment-specific cysteine biosynthetic enzymes.

A systems approach has previously been used to follow the response behaviour of Arabidopsis thaliana plants upon sulphur limitation. A response network was reconstructed from a time series of transcript and metabolite profiles, integrating complex metabolic and transcript data in order to investigate a potential causal relationship. The resulting scale-free network allowed potential transcriptional regulators of sulphur metabolism to be identified. Here, three sulphur-starvation responsive transcription factors, IAA13, IAA28, and ARF-2 (ARF1-Binding Protein), all of which are related to auxin signalling, were selected for further investigation. IAA28 overexpressing and knock-down lines showed no major morphological changes, whereas IAA13- and ARF1-BP-overexpressing plants grew more slowly than the wild type. Steady-state metabolite levels and expression of pathway-relevant genes were monitored under normal and sulphate-depleted conditions. For all lines, changes in transcript and metabolite levels were observed, yet none of these changes could exclusively be linked to sulphur stress. Instead, up- or down-regulation of the transcription factors caused metabolic changes which in turn affected sulphur metabolism. Auxin-relevant transcription factors are thus part of a complex response pattern to nutrient starvation that serve as coordinators of the metabolic shifts driving sulphur homeostasis rather then as direct effectors of the sulphate assimilation pathway. This study provides the first evidence ever presented that correlates auxin-related transcriptional regulators with primary plant metabolism.

The availability of the carbon backbone O-phosphohomoserine (OPHS) is critical to methionine (met) and threonine (thr) synthesis. OPHS derives from homoserine and is formed by homoserine kinase (HSK). To clarify the function of HSK in cellular metabolism, the E. coli HSK ortholog thrB was expressed in potato plants targeting the EcHSK protein to chloroplasts and to the cytosol. Both approaches resulted in up to 11 times increased total HSK enzyme activity. Transgenic plants exhibited reduced homoserine levels while met and thr did not accumulate significantly. However, the precursor cysteine and upstream intermediates of met such as cystathionine and homocysteine did indicating an accelerated carbon flow towards the end products. Coincidently, plants with elevated cytosolic levels of EcHSK exhibited a reduction in transcript levels of the endogenous HSK, as well as of threonine synthase (TS), cystathionine β-lyase (CbL), and met synthase (MS). In all plants, cystathionine γ-synthase (CgS) expression remained relatively unchanged from wild type levels, while S-adenosylmethionine synthetase (SAMS) expression increased. Feeding studies with externally supplied homoserine fostered the synthesis of met and thr but the regulation of synthesis of both amino acids retained the wild type regulation pattern. The results indicate that excess of plastidial localised HSK activity does not influence the de novo synthesis of met and thr. However, expression of HSK in the cytosol resulted in the down-regulation of gene expression of pathway genes probably mediated via OPHS. We integrated these data in a novel working model describing the regulatory mechanism of met and thr homeostasis.

The establishment of technologies for high-throughput DNA sequencing (genomics), gene expression (transcriptomics), metabolite and ion analysis (metabolomics/ionomics) and protein analysis (proteomics) carries with it the challenge of processing and interpreting the accumulating data sets. Publicly accessible databases and newly development and adapted bioinformatic tools are employed to mine this data in order to filter relevant correlations and create models describing physiological states. These data allow the reconstruction of networks of interactions of the various cellular components as enzyme activities and complexes, gene expression, metabolite pools or pathway flux modes. Especially when merging information from transcriptomics, metabolomics and proteomics into consistent models, it will be possible to describe and predict the behaviour of biological systems, for example with respect to endogenous or environmental changes. However, to capture the interactions of network elements requires measurements under a variety of conditions to generate or refine existing models. The ultimate goal of systems biology is to understand the molecular principles governing plant responses and consistently explain plant physiology.

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.

Plants can provide most of the nutrients for the human diet. However, the major crops are often deficient in some of the nutrients. Thus, malnutrition, with respect to micronutrients such as vitamin A, iron, and zinc, but also macronutrients such as the essential amino acids lysine and methionine, affects more than 40% of the world's population. Recent advances in molecular biology, but also the grasp of biochemical pathways, metabolic fluxes, and networks can now be exploited to produce crops enhanced in key nutrients to increase the nutritional value of plant-derived foods and feeds. Some of the predictions appear to be accurate, while others not, reflecting the fact that plant metabolism is more complex than presently understood. A good example for a complex regulation is the methionine biosynthetic pathway in plants. The nutritional importance of Met and cysteine has motivated extensive studies of their roles in plant molecular physiology, especially regarding to their transport, synthesis, and accumulation in plants. Recent studies have demonstrated that Met metabolism is regulated differently in various plant species.

Since cysteine is the first committed molecule in plant metabolism containing both sulphur and nitrogen, the regulation of its biosynthesis is critically important. Cysteine itself is required for the production of an abundance of key metabolites in diverse pathways. Plants alter their metabolism to compensate for sulphur and nitrogen deficiencies as best as they can, but limitations in either nutrient not only curb a plant’s ability to synthesize cysteine, but also restrict protein synthesis. Nutrients such as nitrate and sulphate (and carbon) act as signals; they trigger molecular mechanisms that modify biosynthetic pathways and thereby have a profound impact on metabolite fluxes. Cysteine biosynthesis is modified by regulators acting at the site of uptake and throughout the plant system. Recent data point to the existence of nutrient‐specific signal transduction pathways that relay information about external and internal nutrient concentrations, resulting in alterations to cysteine biosynthesis. Progress in this field has led to the cloning of genes that play pivotal roles in nutrient‐induced changes in cysteine formation.