Sulphur dioxide (SO2) is known as a strongly damaging air pollutant. After conversion to sulphite in aqueous solution, it becomes a strong nucleophilic agent that attacks numerous compounds in the cell. Therefore, plants have developed a mechanism to control sulphite levels. Recently, we have cloned and characterized the enzyme sulphite oxidase (SO) from Arabidopsis thaliana. Yet, its physiological role remained unclear. Here, we describe results demonstrating that SO is essential for detoxifying excessive amounts of sulphite in the cell which is important for the survival of the plant. T-DNA-tagged A. thaliana plants lacking the enzyme showed a decrease in vitality during SO2 fumigation and a change in their S-metabolites. The same was found with RNA-interference (RNAi) plants that were generated for tobacco. On the contrary, over-expression of SO helped the plant to survive SO2 concentrations that are detrimental for non-transformed wild-type (WT) plants, as was shown with poplar plants which are known to be particularly sensitive to SO2. Fumigation induced the expression of the enzyme as demonstrated by promoter-reporter gene fusion, by immunoblot analysis of SO-protein and by induction of enzyme activity. This implies that SO, as an otherwise constitutively expressed protein, is under additional control by SO2 in the environment.

Sulfite oxidizing activities are known since years in animals, microorganisms, and also plants. Among plants, the only enzyme well characterized on molecular and biochemical level is the molybdoenzyme sulfite oxidase (SO). It oxidizes sulfite using molecular oxygen as electron acceptor, leading to the production of sulfate and hydrogen peroxide. The latter reaction product seems to be the reason why plant SO is localized in peroxisomes, because peroxisomal catalase is able to decompose hydrogen peroxide. On the other hand, we have indications for an additional reaction taking place in peroxisomes: sulfite can be nonenzymatically oxidized by hydrogen peroxide. This will promote the detoxification of hydrogen peroxide especially in the case of high amounts of sulfite. Hence we assume that SO could possibly serve as "safety valve" for detoxifying excess amounts of sulfite and protecting the cell from sulfitolysis. Supportive evidence for this assumption comes from experiments where we fumigated transgenic poplar plants overexpressing ARABIDOPSIS SO with SO(2) gas. In this paper, we try to explain sulfite oxidation in its co-regulation with sulfate assimilation and summarize other sulfite oxidizing activities described in plants. Finally we discuss the importance of sulfite detoxification in plants.

Sulfite oxidase (EC 1.8.3.1) from the plant Arabidopsis thaliana is the smallest eukaryotic molybdenum enzyme consisting of a molybdenum cofactor-binding domain but lacking the heme domain that is known from vertebrate sulfite oxidase. While vertebrate sulfite oxidase is a mitochondrial enzyme with cytochrome c as the physiological electron acceptor, plant sulfite oxidase is localized in peroxisomes and does not react with cytochrome c. Here we describe results that identified oxygen as the terminal electron acceptor for plant sulfite oxidase and hydrogen peroxide as the product of this reaction in addition to sulfate. The latter finding might explain the peroxisomal localization of plant sulfite oxidase. 18 O labeling experiments and the use of catalase provided evidence that plant sulfite oxidase combines its catalytic reaction with a subsequent non-enzymatic step where its reaction product hydrogen peroxide oxidizes another molecule of sulfite. In vitro, for each catalytic cycle plant SO will bring about the oxidation of two molecules of sulfite by one molecule of oxygen. In the plant, sulfite oxidase could be responsible for removing sulfite as a toxic metabolite, which might represent a means to protect the cell against excess of sulfite derived from SO 2 gas in the atmosphere (acid rain) or during the decomposition of sulfur-containing amino acids. Finally we present a model for the metabolic interaction between sulfite and catalase in the peroxisome. Sulfite oxidases (SO)3 from vertebrates (published as EC 1.8.3.1) play an essential role in sulfur detoxification by catalyzing the reaction (1), which is the terminal step in the oxidative degradation of cysteine and methionine. Different electron acceptors were reported to interact with vertebrate SO including cytochrome c, ferricyanide, and oxygen (2-4). In mammals, SO is localized in the intermembrane space of mitochondria (5) where electrons derived from sulfite are passed to cytochrome c, the physiological electron acceptor. Vertebrate SO is a homodimeric protein with monomers subdivided into a Moco domain and a heme domain, as verified by the atomic structure of chicken SO (6).Recently we have described plant SO (7) from Arabidopsis thaliana, which is the fourth molybdenum enzyme present in plants in addition to nitrate reductase, xanthine dehydrogenase, and aldehyde oxidase. Cloning and characterization of plant SO was possible by using sequence homologies to the mammalian counterpart. However, in contrast to the animal enzyme plant SO lacks the heme domain, which is evident from the amino acid sequence, its enzymological and spectral properties (7), and the atomic structure (8). Also its subcellular localization differs from that of animals, in plants we showed SO to be localized in peroxisomes (9). SO is wide spread and highly conserved within the plant kingdom; the SO gene is present in higher and lower plants, and the protein encoded seems to be highly conserved because antibodies directed against Arabidopsis SO detect proteins of the correct size...

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HaloTag Interchangeable Labeling Technology (HaloTag) was originally developed for mammalian cell analysis. In this report, the use of HaloTag is demonstrated in plant cells for the first time. This system allows different fluorescent colours to be used to visualize the localization of the non-fluorescent HaloTag protein within living cells. A vector was constructed which expresses the HaloTag protein under the control of the 35S promoter of cauliflower mosaic virus. The functionality of the HaloTag construct was tested in transient assays by (i) transforming tobacco protoplasts and (ii) using biolistic transformation of intact leaf cells of tobacco and poplar plants. Two to fourteen days after transformation, the plant material was incubated with ligands specific for labelling the HaloTag protein, and fluorescence was visualized by confocal laser scanning microscopy. The results demonstrate that HaloTag technology is a flexible system which generates efficient fluorescence in different types of plant cells. The ligand-specific labelling of HaloTag protein was not hampered by the plant cell wall.