Rüdiger Hell

Isolation and characterization of two cDNAs encoding for compartment specific isoforms of O-acetylserine (thiol) lyase from Arabidopsis thaliana.

cDNAs encoding for two isoforms of O‐acetylserine (thiol) lyase (OAS‐TL), which catalyzes the synthesis of cysteine, have been isolated from Arabidopsis thaliana. Secondary structure together with expression patterns derived during photomorphogenesis indicate cellular localizations in the cytosol and plastids, thus allowing a direct comparison of compartment‐specific forms within one species. The cytosolic OAS‐TL complemented an E. coli auxotrophic mutant lacking cysteine synthesis. Both isoforms are represented by small gene families. They are expressed under all conditions investigated and were observed to increase in expression in plants grown with limited sulfate supply.

ISOLATION AND CHARACTERIZATION OF A GENE FOR ASSIMILATORY SULFITE REDUCTASE FROM ARABIDOPSIS THALIANA

Sulfite reductase (SIR) represents a key enzyme in sulfate assimilation in higher plants. The genomic DNA sequence of the sir gene from Arabidopsis thaliana including regulatory and structural regions was isolated and characterized. The sequence of a 6 kb fragment encoding SIR revealed a coding region of 2891 basepairs (bp) that consists of eight exons separated by seven introns between 83 and 139 bp in length. The transcription start point was determined 272 bp upstream of the translation start site. Southern analysis indicates a single locus for the sir gene that gives rise to a 2.4 (kb) mRNA in leaves and in roots. The promoter region was verified by functional expression of the gusA reporter gene in transgenic A. thaliana plants and was shown to provide correct expression in root and leaf.

Cysteine biosynthesis in plants: isolation and functional identification of a cDNA encoding a serine acetyltransferase from Arabidopsis thaliana

A cDNA encoding for serine acetyltransferase which catalyzes the committing step of cysteine biosynthesis has been cloned from Arabidopsis thaliana. The plant protein has a predicted molecular weight of 32.8 kDa and shows up to 43% of amino acid homology to bacterial serine acetyltransferases. It complements a serine acetyltransferase negative E. coli mutant and can be enzymatically determined in the heterologous host. The corresponding mRNA is predominantely expressed in light exposed tissue and represents one of at least two related genes.

Photosynthesis and fluorescence quenching, and the mRNA levels of plastidic glutamine synthetase or of mitochondrial serine hydroxymethyltransferase (SHMT) in the leaves of the wild-type and of the SHMT-deficient stm mutant of Arabidopsis thaliana in relation to the rate of photorespiration

Abstract.The regulation by photorespiration of the transcript level corresponding to plastidic glutamine synthetase (GS-2) was investigated in the leaves of Arabidopsis thaliana (L.) Heynh.. Photorespiration was suppressed by growing the plants in an atmosphere containing 300 Pa CO2. Suppression of photorespiration was demonstrated by the ability of the conditionally lethal serine hydroxymethyltransferase (SHMT)-deficient stm mutant of A. thaliana to grow normally under these conditions. In contrast to previous studies with bean or pea that were performed at very high CO2 partial pressure (2–4 kPa; Edwards and Coruzzi, 1989, Plant Cell 1: 241–248; Cock et al., 1991, Plant Mol Biol 17: 761–771), suppression of photorespiration during growth of A. thaliana in an atmosphere with 300 Pa CO2 had no effect on the leaf GS-2 transcript level. In the short term, neither suppression of photorespiration induced by the transfer of air-grown A. thaliana plants into a CO2-enriched atmosphere, nor an increase in the rate of photorespiration achieved by the transfer of high-CO2-grown A. thaliana plants into air resulted in a change in the GS-2 mRNA level. The absence of photorespiratory ammonium release in leaves of the stm mutant had no effect on the GS-2 transcript level. Overall, our data argue against a control by photorespiration of the A. thaliana leaf GS-2 mRNA pool. In contrast, regulation of the leaf SHMT mRNA level may involve a negative feedback effect of at least one metabolite derived from the glycine/serine conversion during photorespiration, as indicated by the overexpression of SHMT transcripts in the leaves of the stm mutant.

The Plant Sulphur Cycle

Sulphur represents one of the essential macronutrients of plants. It is usually available to plants as soluble sulfate from soil, but is mainly required by cellular processes in its reduced form, i.e. as sulfide. Assimilatoy sulfate reduction by photoautotrophic plants therefore acts as the counterpart to oxidation and dissimilation, respectively, performed by microorganisms and animals (Schiff 1983). Up to 90% of the total sulphur is present in most plants as cysteine and methionine that inturn are predominantely bound in protein (Giovanelli 1990). The biochemical versatility of sulphur allows a participation in very different cellular functions. Especially cysteine is crucial in proteins as structural element and target in redox modulation of activity (Buchanan 1991), in catalytic centers, electron transport in Fe-S clusters, and many more functions. Reduced sulphur is required in the function of cofactors such as acetyl coenzyme A, thiamine, biotine, and lipoic acid. Accordingly, these sulphur compounds are essential in the diet of humans and monogastric mammals. The sulphur-containing tripeptide glutathione is involved in the regulation of protein synthesis (Kranner et al. 1996), and in the compensation of various forms of stress (Rennenberg and Brunold 1994). Both functions of glutathione are highly significant for the survival of plants in a stressful environment. Sulphur-containing plant secondary compounds like alliin and isothiocyanate derivatives can be important for phytomedical applications (Fahey et al. 1995, Sendl 1995, Stoner 1995).

Verhältnis von Sproß und Wurzel bei der Expression von Genen der Sulfatassimilation in Arabidopsis thaliana

The relationship of shoots and roots in sulfate assimilation has been investigated both at the biochemical and molecular level. The model plant Arabidopsis thaliana provides for the first time the possibility to analyse the expression patterns of almost all the genes of sulfate assimilation and cysteine biosynthesis in plants. Thus the troublesome assay of the notoriously low activities and labile enzymes of the pathway is avoided. Quantification of steady-state mRNA levels of genes of sulfate transporters, ATP sulfurylase, APS-kinase, APS reductase, sulfite reductase, serine acetyltransferases and O-acetylserine (thiol)layses in rosette leaves and roots from A. thaliana plants was performed using Northern hybridization. Genes for sulfate reduction were predominantely expressed in leaves, however, substantial mRNA levels were also detected in roots for all genes tested. Similarly, genes that encode iso-enzymes of cysteine and methionine biosynthesis exhibited strong expression in roots comparable to the amounts in leaves. In addition, stored sulfate from roots contributed about one third of the sulfur required for metabolic processes during early stages of external sulfate deprivation. It is concluded that the shoot is not entirely responsible for storage of sulfate and provision of reduced sulfur in A. thaliana. Instead, the root system plays a significant role in sulfate storage and reduction and may even be independent from import of reduced sulfur compounds from the shoot.