Impact on sulphur and glutathione metabolism

Sulphur is an essential element for plant growth. It has been recognized for several decades that foliarly absorbed sulphurous air pollutants may be utilized as sulphur source beneficial for plant growth. It has been estimated that dry sulphur deposition at atmospheric SO2 (and H2S) levels as low as may substantially contribute to the sulphur fertilization of plants (Stulen et al. 1998). From laboratory experiments it is evident that at sufficiently high atmospheric levels plants are able to grow with sulphur gases as exclusive sulphur source. Recently, it has become apparent that in Western Europe, and likely in several other regions, agriculture practice even has co-evolved with the dry and wet deposition of atmospheric sulphur originating from industrial emissions. The recent and ongoing reduction in industrial sulphur emissions have to be compensated by additional sulphur fertilization in order to avoid losses of crop yield and quality by sulphur deficiency (Schnug 1998).

Plant shoots form a sink for S02 and over a wide range there is a linear relation between atmospheric concentration and uptake rate. The deposition is generally directly dependent on the degree of stomatal aperture, since the internal resistance to S02 is low. S02 is highly soluble in the apoplastic water of the mesophyll, where it dissociates under formation of bisulphite (HSO3") and sulphite (S032~). Sulphite may direcdy enter the sulphur assimi-latory pathway and be reduced to sulphide, incorporated into cysteine, and subsequendy into other sulphur compounds including glutathione (Figure 1 gives an overview of the possible impact and on sulphate uptake and assimilation pathways). On the other hand, sulphite may be oxidized to sulphate, extra- and intracellularly by peroxidases, intracellularly by specific sulphite oxidases discovered recently in plants (Mendel & Hansch, unpublished results), or non-enzymatically catalysed by metal ions or superoxide radicals. The resulting sulphate can be assimilated thereafter. Furthermore, the excess of sulphate which is not directly metabolized is translocated into the vacuole, where it appears to be poorly accessible for remobilization and metabolism. The latter explains increased sulphate levels in shoots, which are characteristic for S02 exposed plants.

The pattern of H2S uptake by plants differs distinctly from that of S02. The mesophyll (internal) resistance of the shoots to H2S and its deposition appears to be direcdy dependent on the rate of H2S metabolism into cysteine and subsequently into other sulphur compounds (De Kok et al. 1998, 2000). In contrast to S02, the uptake rates of H2S show a saturation curve (Figure 2) which indicates that the rate limiting step for uptake is a metabolic process, probably the incorporation of sulphide into cysteine (Figure 1). There is strong evidence that O-acetyl-serine (thiol)lyase is directly responsible in the sible in the active fixation of atmospheric H2S by plants. Carbonyl sulphide (COS) may also be metabolized after hydrolysis to H2S and C02, however, its atmospheric concentration is probably too low to have any significance as plant sulphur source (Kesselmeier and Merk 1993).

Part of the atmospheric sulphur, which is metabolized in excess of the normal requirement, may appear in the thiols (-SH groups) fraction in plant tissues (Grill et al. 1979, De Kok 1990, De Kok et al. 1998). In general, S02 and H2S exposure result in enhanced thiol levels in shoots and in roots, which can already be observed within a few hours after the start of the exposure. In coincidence with the differences in metabolism between H2S and S02 (Figure 1), the thiol accumulation is generally higher upon H2S than upon S02 exposure at equal atmospheric concentrations. For H2S it may be substantial at ambient concentrations as low as 0.03 (il l"1. The thiol accumulation depends on the atmospheric S02 and H2S level, but it may increase up to 5-fold and 2-fold in shoots and roots, respectively. The composition ofthe thiol pool may also be strongly modified upon S02 and H2S exposure. The thiol pool in plants is dynamic and its level may strongly be affected by physiological and environmental factors, e.g. sulphur nutrition or stress impacts (Rennenberg and Lamoureux 1990, De Kok and Stulen 1993). The variation in the thiol concentration is mainly due to changes in glutathione content (or its homologues), the predominant thiol present in plant tissue

(typically > 90 % of total low molecular weight thiols). Upon exposure to S02 and H2S also high levels of cysteine and, in darkness, of y-glutamyl-cysteine may accumulate in the shoot, though the impact of S02 and H2S on the composition of the thiol pool strongly varies between species. In roots, the thiol accumulation is generally exclusively due to enhanced glutathione levels (De Kok et al. 1998). After cessation of the exposure, the thiol level decreases very rapidly and normal values are reached again within one or two days.

The physiological background of the altered composition of the thiol pool in the shoot upon exposure to and is not yet solved. Apparently, if the sulphur is directly supplied to the shoot and the regulation of sulphate uptake by the roots is by-passed, then there is no strict regulation of composition of the thiol pool in the shoot (De Kok 1990, De Kok et al. 1998). The absorbed atmospheric sulphur may even be metabolized outside of the chlo-roplast, wherein sulphur assimilation is located under normal conditions (Hell 1997). In the cytosol there might be a substrate shortage for the metabolism of the cysteine to glutathione. It has been observed that the accumulation of in the dark can be prevented by adding the substrate glycine directly to the leaf tissue, which results in glutathione accumulation (Buwalda et al. 1990). A similar shortage of glycine for glutathione synthesis was observed in plants wherein the level of y-glutamyl-cysteine synthetase was overexpressed (Noctor et al. 1997).

Thiol compounds such as glutathione are assumed to fulfil signal functions in the regulation of sulphur uptake and sulphur reduction in plants (Rennenberg and Lamoureux 1990, De Kok and Stulen 1993). Under normal conditions, sulphate uptake is in tune with the metabolic need for growth. The uptake may be regulated by changes in activity and/or the expression of sulphate transporter protein by negative feedback from sulphate itself or modulated by changes in concentrations of reduced sulphur compounds including glutathione (Davidian et al. 2000). S02 and H2S impact studies are a promising tool to elucidate the signals involved in the regulatory aspects of the uptake, transport and reduction of sulphate in plants, and in the interactions between shoots and roots. Exposure of plants to sulphurous air pollutants may repress the uptake of sulphate by the roots (Figure 1; Brunold and Erismann 1974, Herschbach et al. 1995, De Kok et al. 1998) and its further transport to the shoots (Herschbach et al. 1995). It is still unclear to what extent an enhanced glutathione level in the roots is the trigger of the repression of sulphate uptake. Without regard of a normal sulphur supply to the roots, plants are able to switch in part to atmospheric taken up by the shoots as sulphur source for plant growth. However, the pattern of increase in glutathione levels in roots upon exposure (see above) appeared to be not in tune with the repression of sulphate uptake (Westerman et al. 2000).

Some plants may already accumulate sulphate in the shoot upon exposure to relative low levels of S02 or H2S (De Kok 1990). It still needs to be resolved whether the accumulated sulphate originates from the oxidation of absorbed atmospheric sulphur gases in the plant foliage, or it reflects a poor shoot/root interaction in the regulation of the sulphate uptake by the roots and its transport to the shoot.

The plant foliage is the predominant site of sulphate reduction. It has been observed that S02 or H2S exposure may result in a decrease of the activity of ATP-sulphurylase and adenosine 5'-phosphosulphate (APS) reductase (Brunold and Erismann 1974, Westerman et al. 2001), with sulphide, O-acetylserine or cysteine being the most likely regulators (Brunold 1990, Hawkesford and Wray 2000).

Glutathione and phytotoxicity of sulphurous air pollutants

Reactive oxygen species (ROS) can be formed at various sites in the plant cell, and even have important functions in plant metabolism, e. g. in lignin synthesis. The chloroplast is a major site of ROS formation, especially under conditions where the photosynthetic carbon fixation (Calvin cycle) is not in tune with photosynthetic electron transport (see Tausz in this volume). Under such conditions molecular oxygen may be photo-reduced at the site of photosystem I yielding superoxide (Asada 1999). Glutathione and glutathione reductase are of great significance in the protection of plants against the harmful effects of reactive oxygen species and free radicals. Glutathione may react directly with ROS, or it may stabilize and protect protein thiol groups by acting as a thiol buffer. Furthermore it plays a role as reductant in the enzymatic detoxification of in the chloroplast in the ascorbate per-oxidase-dehydroascorbate reductase-glutathione reductase cycle (De Kok and Stulen 1993, Kunert and Foyer 1993). Glutathione reductase catalyses the reduction of the formed oxidized glutathione, with NADPH as reductant (for more details see Tausz in this volume).

It has been postulated that an increase in glutathione concentrations and glutathione reductase (and superoxide dismutase) activities would have an adaptive value in the protection of plants against the toxic effects of S02 (Mehlhorn et al. 1986, Madamanchi and Alscher 1991, Soldatini et al. 1992). Part of the absorbed would initiate a superoxide mediated free radical chain oxidation in the chloroplast, resulting in a burst of reactive oxygen species and other free radicals which could be the basis for the injurious effects of S02. Indeed, in vitro, in isolated chloroplasts the addition of relatively high levels of sulphite or sulphide resulted in a superoxide-triggered oxidation of these reduced sulphur compounds upon illumination (Asada and

Kiso 1973, De Kok et al. 1983, Ghisi et al. 1990, Dittrich et al. 1992). Light-induced oxidation of sulphite by chloroplasts was only substantial in broken chloroplasts (Ghisi et al. 1990) and was diminished by the addition of the electron acceptor of photosystem I (ferredoxin and NADP; Asada and Kiso 1973) or by the addition of superoxide dismutase or other scavengers of reactive oxygen species, glutathione included (Asada and Kiso 1973, De Kok et al. 1983, Ghishi et al. 1990, Dittrich et al. 1992). It is likely that sulphite/sulphide-induced superoxide mediated free radical chain reactions may have significance at acute high levels of sulphurous air pollutants, when the deposition substantially exceeds the plant potential to metabolize the absorbed sulphur and/or the oxidation potential of the extracellular peroxidases in the apoplast (Pfanz et al. 1990) and the antioxidative defence systems in the apo- and symplast. It may be one of the causes of the development of visible injury. Still it is unclear whether light is an essential factor for the development of plant injury by S02 (Olszyk and Tingey 1984). At normal ambient pollutant levels it is unlikely that high levels of sulfite occur in the chloroplast, since it would directly and with high affinity be metabolized as substrate in the assimilatory sulphate reduction (Figure 1). Even if significant amounts of sulphite entered the chloroplast, sulphite-induced radical formation in the light would be very unlikely. Intact chloroplasts are likely to contain sufficient active oxygen scavenging capacity to prevent light-triggered sulphite oxidation (Ghisi et al. 1990, Dittrich et al. 1992). Furthermore, the impact of S02 exposure on glutathione reductase levels are rather inconsistent (Tanaka et al. 1982, Grill et al. 1982, Madamanchi and Alscher 1991, Soldatini et al. 1992).

Chronically enhanced glutathione levels have even been suggested as one of the causes of the phytotoxicity of S02 (Grill et al. 1979). In this view it may result in a deregulation of cellular metabolism. The role of glutathione in its interaction with proteins is well known (Kunert and Foyer 1993). The glutathione redox system may also have an important role in environmental sensing and stress signalling within cells, within or between tissues, or even between organs (May et al. 1998). Enhanced glutathione levels beyond the normal metabolic control can have serious effects on the antioxidant system, which was demonstrated in transgenic plants with constitutively increased GSH concentration in chloroplasts. This did not stimulate antioxidant defence but, quite in contrast, promoted oxidative stress in chloroplasts (Creis-sen et al. 1999).

Another phenomenon that may involve glutathione is the development of chromosomal damages in root tissues of trees after the canopy has been exposed to sulphurous pollutants. This has been observed repeatedly upon both S02 and H2S exposure (Müller et al. 1997, Wonisch et al. 1999a, b). The signalling pathway leading to this effect is enigmatic, but since glutathione concentrations are enhanced in the needles, and glutathione is easily translocated in the phloem, it has been suggested as a likely candidate. Furthermore, the feeding of glutathione to fine roots of spruce trees indeed caused an increase in chromosomal damages in the meristematic tissues and abnormalities in the cell ultrastructure therein (Zellnig et al. 2000). A direct link between sulphurous air pollutants, enhanced tissue glutathione levels, and chromosomal damages in root meristems could not be established measuring tissue concentrations of GSH in the roots, because chromosomal damages were observed before GSH concentrations increased in the roots (Wonisch et al. 1999a). However, since average tissue concentrations may mask changes in certain cell types or even sub-cellular compartments, this result might not necessarily prove this hypothesis wrong. With the help of laser scanning microscopy and fluorescence labelling of glutathione it was shown that the meristematic region of root tips contain specifically high GSH concentrations which are important for the cell divisions therein (Sanchez-Fernanedz et al. 1997). The application of fluorescence microscopy in combination with digital image analysis also showed that the nuclei contain particularly high concentrations of glutathione (Müller et al. 1999, see image on the front cover). Changes of the glutathione system in the nucleus may be sufficient to cause the observed effects, and these changes can be easily overlooked when average tissue concentrations are investigated.

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