β-Cyano-alanine synthase (CAS; EC 4.4.1.9) plays an important role in cyanide metabolism in plants. Although the enzymatic activity of β-cyano-Ala synthase has been detected in a variety of plants, no cDNA or gene has been identified so far. We hypothesized that the mitochondrial cysteine synthase (CS; EC 4.2.99.8) isoform, Bsas3, could actually be identical to CAS in spinach (Spinacia oleracea) and Arabidopsis. An Arabidopsis expressed sequence tag database was searched for putative Bsas3homologs and four new CS-like isoforms, ARAth;Bsas1;1,ARAth;Bsas3;1, ARAth;Bsas4;1, andARAth;Bsas4;2, were identified in the process. ARAth;Bsas3;1 protein was homologous to the mitochondrial SPIol;Bsas3;1 isoform from spinach, whereas ARAth;Bsas4;1 and ARAth;Bsas4;2 proteins defined a new class within the CS-like proteins family. In contrast to spinach SPIol;Bsas1;1 and SPIol;Bsas2;1 recombinant proteins, spinach SPIol;Bsas3;1 and Arabidopsis ARAth;Bsas3;1 recombinant proteins exhibited preferred substrate specificities for the CAS reaction rather than for the CS reaction, which identified these Bsas3 isoforms as CAS. Immunoblot studies supported this conclusion. This is the first report of the identification of CAS synthase-encoding cDNAs in a living organism. A new nomenclature for CS-like proteins in plants is also proposed.

SummaryThe two-electron reduction of sulfate to sulfite in plants is mediated by 5 0 -adenylylsulfate (APS) reductase, an enzyme theorized to be a control point for cysteine synthesis. The hypothesis was tested by expression in Arabidopsis thaliana under transcriptional control of the CaMV 35S promoter of the APS reductase from Pseudomonas aeruginosa (PaAPR) fused with the rbcS transit peptide for localization of the protein to plastids. PaAPR was chosen for the experiment because it is a highly stable enzyme compared with the endogenous APS reductase of A. thaliana, and because PaAPR is catalytically active in combination with the plant thioredoxins m and f indicating that it would likely be catalytically active in plastids. The results indicate that sulfate reduction and O-acetylserine (OAS) production together limit cysteine synthesis. Transgenic A. thaliana lines expressing PaAPR accumulated sulfite, thiosulfate, cysteine, c-glutamylcysteine, and glutathione. Sulfite and thiosulfate increased more than did cysteine, c-glutamylcysteine and glutathione. Thiosulfate accumulation was most pronounced in flowers. Feeding of OAS to the PaAPRexpressing plants caused cysteine and glutathione to increase more rapidly than in comparably treated wild type. Both wild-type and transgenic plants accumulated sulfite and thiosulfate in response to OAS feeding. The PaAPR-expressing plants were slightly chlorotic and stunted compared with wild type. An attempt to uncover the source of thiosulfate, which is not thought to be an intermediate of sulfate reduction, revealed that purified b-mercaptopyruvate sulfurtransferase is able to form thiosulfate from sulfite and b-mercaptopyruvate, suggesting that this class of enzymes could form thiosulfate in vivo in the presence of excess sulfite.

Different field surveys have shown that sulphur (S) fertilization can increase the resistance of agricultural crops against fungal pathogens. The mechanisms of this sulphur-induced resistance (SIR) are, however, not yet known. Volatile S compounds are thought to play an important role because H(2)S is toxic to fungi. A field experiment was conducted to analyse the influence of S fertilization and the activity of H(2)S-releasing enzymes on fungal infections. Two levels of N and S fertilizers and two varieties of oilseed rape were investigated with respect to their potential to release H(2)S by the enzymatic activity of L-cysteine desulphydrase (LCD) and O-acetyl-L-serine(thiol)lyase (OAS-TL). LCD releases H(2)S during cysteine degradation, while OAS-TL consumes H(2)S during cysteine synthesis and free H(2)S is only released in a side reaction. All plots of the field trial showed an infection with Pyrenopeziza brassicae and leaf disc samples were taken from visibly infected leaf areas and apparently uninfected areas to investigate the reaction to the infection in relation to the treatments. Different S fractions and the activities of LCD and OAS-TL were measured to evaluate the potential to release H(2)S in relation to S nutrition and fungal infection. S fertilization significantly increased the contents of total S, sulphate, organic S, cysteine, and glutathione in the plants, but decreased LCD activity. Infection with P. brassicae increased cysteine and glutathione contents, as well as the activity of LCD. Therefore crops were able to react to a fungal infection with a greater potential to release H(2)S, which is reflected by an increasing LCD activity with fungal infection.

It is well documented that, in general, amino acids are used in the l-form, and enzymes involved in their metabolism are stereospecific for the l-enantiomers. However, d-amino acids are widely distributed in living organisms [1]. Examples of the natural occurrence of d-amino acids include d-amino acid-containing natural peptide toxins [2], antibacterial diastereomeric peptides [3], and the presence of d-amino acids at high concentrations in human brain [4]. In plants d-amino acids were detected in gymnosperms as well as monoand dicotyledonous angiosperms of major plant families. Free d-amino acids in the low percentage range of 0.5-3% relative to their l-enantiomers are principle constituents of plants [5]. The functions of d-amino acids and their metabolism are largely unknown. Various pyridoxal-5¢-phosphate (PLP)-dependent enzymes that catalyse elimination and replacement reactions of amino acids have been purified and characterized [6]. In several organisms d-cysteine desulfhydrase (d-CDes) activity (EC 4.1.99.4) was measured; this enzyme decomposes d-cysteine into pyruvate, H 2 S, and NH 3 . A gene encoding a putative d-CDes protein was identified in Arabidopsis thaliana (L) Heynh. based on high homology to an Escherichia coli protein called YedO that has d-CDes activity. The deduced Arabidopsis protein consists of 401 amino acids and has a molecular mass of 43.9 kDa. It contains a pyridoxal-5¢-phosphate binding site. The purified recombinant mature protein had a K m for d-cysteine of 0.25 mm. Only d-cysteine but not l-cysteine was converted by d-CDes to pyruvate, H 2 S, and NH 3 . The activity was inhibited by aminooxy acetic acid and hydroxylamine, inhibitors specific for pyridoxal-5¢-phosphate dependent proteins, at low micromolar concentrations. The protein did not exhibit 1-aminocyclopropane-1-carboxylate deaminase activity (EC 3.5.99.7) as homologous bacterial proteins. Western blot analysis of isolated organelles and localization studies using fusion constructs with the green fluorescent protein indicated an intracellular localization of the nuclear encoded d-CDes protein in the mitochondria. d-CDes RNA levels increased with proceeding development of Arabidopsis but decreased in senescent plants; d-CDes protein levels remained almost unchanged in the same plants whereas specific d-CDes activity was highest in senescent plants. In plants grown in a 12-h light ⁄ 12-h dark rhythm d-CDes RNA levels were highest in the dark, whereas protein levels and enzyme activity were lower in the dark period than in the light indicating post-translational regulation. Plants grown under low sulfate concentration showed an accumulation of d-CDes RNA and increased protein levels, the d-CDes activity was almost unchanged. Putative in vivo functions of the Arabidopsis d-CDes protein are discussed.Abbreviations

Due to the clean air acts and subsequent reduction of emission of gaseous sulfur compounds sulfur deficiency became one of the major nutrient disorders in Northern Europe. Typical sulfur deficiency symptoms can be diagnosed. Especially plants of the Cruciferae family are more susceptible against pathogen attack. Sulfur fertilization can in part recover or even increase resistance against pathogens in comparison to sulfur-deficient plants. The term sulfur-induced resistance (SIR) was introduced, however, the molecular basis for SIR is largely unknown. There are several sulfur-containing compounds in plants which might be involved in SIR, such as high levels of thiols, glucosinolates, cysteine-rich proteins, phytoalexins, elemental sulfur, or H2S. Probably more than one strategy is used by plants. Species- or even variety-dependent differences in the development of SIR are probably used. Our research focussed mainly on the release of H2S as defence strategy. In field experiments using different BRASSICA NAPUS genotypes it was shown that the genetic differences among BRASSICA genotypes lead to differences in sulfur content and L-cysteine desulfhydrase activity. Another field experiment demonstrated that sulfur supply and infection with PYRENOPEZIZA BRASSICA influenced L-cysteine desulfhydrase activity in BRASSICA NAPUS. Cysteine-degrading enzymes such as cysteine desulfhydrases are hypothesized to be involved in H2S release. Several L- and D-cysteine-specific desulfhydrase candidates have been isolated and partially analyzed from the model plant ARABIDOPSIS THALIANA. However, it cannot be excluded that H2S is also released in a partial back reaction of O-acetyl-L-serine(thiol)lyase or enzymes not yet characterized. For the exact determination of the H2S concentration in the cell a H2S-specific microsensor was used the first time for plant cells. The transfer of the results obtained for application back on BRASSICA was initiated.