SummaryThe effect of externally applied L-cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5¢-phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined in Arabidopsis thaliana root cultures. Addition of increasing L-cysteine to the nutrient solution increased internal cysteine, g-glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and extractable activity. An effect on APR could already be detected at 0.2 mM L-cysteine, whereas ATP sulphurylase was signi®cantly affected only at 2 mM L-cysteine. APR mRNA, protein and activity were also decreased by GSH at 0.2 mM and higher concentrations. In the presence of L-buthionine-S, Rsulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mM L-cysteine had no effect on APR activity, indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO and 0.5 mM GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP sulphurylase activity was not affected by this treatment. Tracer experiments using 35 SO 4 2± in the presence of 0.5 mM L-cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity and the¯ux of label into cysteine, GSH and protein, but had no effect on the activity of all other enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent with the hypothesis that thiols regulate the¯ux through sulphate assimilation at the uptake and the APR step. Analysis of radioactive labelling indicates that the¯ux control coef®cient of APR is more than 0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of external sulphate is inhibited by GSH to a greater extent than the¯ux through the pathway, and that the¯ux control coef®cient of APR for the pathway, including the transport step, is proportionately less, with a signi®cant share of the control exerted by the transport step.

Using Arabidopsis, we analyzed the effect of omission of a nitrogen source and of the addition of different nitrogen-containing compounds on the extractable activity and the enzyme and mRNA accumulation of adenosine 5-phosphosulfate reductase (APR). During 72 h without a nitrogen source, the APR activity decreased to 70% and 50% of controls in leaves and roots, respectively, while cysteine (Cys) and glutathione contents were not affected. Northern and western analysis revealed that the decrease of APR activity was correlated with decreased mRNA and enzyme levels. The reduced APR activity in roots could be fully restored within 24 h by the addition of 4 mM each of NO 3 ؊ , NH 4 ؉ , or glutamine (Gln), or 1 mM O-acetylserine (OAS).35 SO 4 2؊ feeding showed that after addition of NH 4 ؉ , Gln, or OAS to nitrogen-starved plants, incorporation of 35 S into proteins significantly increased in roots; however, glutathione and Cys labeling was higher only with Gln and OAS or with OAS alone, respectively. OAS strongly increased mRNA levels of all three APR isoforms in roots and also those of sulfite reductase, Cys synthase, and serine acetyltransferase. Our data demonstrate that sulfate reduction is regulated by nitrogen nutrition at the transcriptional level and that OAS plays a major role in this regulation.

The N ratio of nitrogen oxides (NO) emitted from vehicles, measured in the air adjacent to a highway in the Swiss Middle Land, was very high [δN(NO) = +5.7‰]. This high N abundance was used to estimate long-term NO dry deposition into a forest ecosystem by measuring δN in the needles and the soil of potted and autochthonous spruce trees [Picea abies (L.) Karst] exposed to NO in a transect orthogonal to the highway. δN in the current-year needles of potted trees was 2.0‰ higher than that of the control after 4 months of exposure close to the highway, suggesting a 25% contribution to the N-nutrition of these needles. Needle fall into the pots was prevented by grids placed above the soil, while the continuous decomposition of needle litter below the autochthonous trees over previous years has increased δN values in the soil, resulting in parallel gradients of δN in soil and needles with distance from the highway. Estimates of NO uptake into needles obtained from the δN data were significantly correlated with the inputs calculated with a shoot gas exchange model based on a parameterisation widely used in deposition modelling. Therefore, we provide an indication of estimated N inputs to forest ecosystems via dry deposition of NO at the receptor level under field conditions.

Summary Adenosine 5′‐phosphosulphate reductase (APR) is considered to be a key enzyme of sulphate assimilation in higher plants. We analysed the diurnal fluctuations of total APR activity and protein accumulation together with the mRNA levels of three APR isoforms of Arabidopsis thaliana. The APR activity reached maximum values 4 h after light onset in both shoots and roots; the minimum activity was detected at the beginning of the night. During prolonged light, the activity remained stable and low in shoots, but followed the normal rhythm in roots. On the other hand, the activity decreased rapidly to undetectable levels within 24 h of prolonged darkness both in shoots and roots. Subsequent re‐illumination restored the activity to 50% in shoots and to 20% in roots within 8 h. The mRNA levels of all three APR isoforms showed a diurnal rhythm, with a maximum at 2 h after light onset. The variation of APR2 mRNA was more prominent compared to APR1 and APR3. 35SO42– feeding experiments showed that the incorporation of 35S into reduced sulphur compounds in vivo was significantly higher in light than in the dark. A strong increase of mRNA and protein accumulation as well as enzyme activity during the last 4 h of the dark period was observed, implying that light was not the only factor involved in APR regulation. Indeed, addition of 0.5% sucrose to the nutrient solution after 38 h of darkness led to a sevenfold increase of root APR activity over 6 h. We therefore conclude that changes in sugar concentrations are also involved in APR regulation.

Glutathione is an important component of the ascorbate-glutathione cycle, which is involved in the regulation of hydrogen peroxide (H2O2) concentrations in plants. During chilling and cold acclimation, i.e. exposure to temperatures between 0 and 15 degrees C, H2O2 may accumulate. Excess electrons from the photosynthetic and respiratory electron transport chains can be used for the reduction of oxygen, thus producing superoxide radicals (O2.-); these are subsequently transformed to H2O2 via superoxide dismutase (SOD; EC 1.15.1.1). During the removal of excess H2O2, reduced glutathione (GSH) is converted to its oxidised form (GSSG), and GSH is regenerated by the activity of NADPH-dependent glutathione reductase (GR; EC 1.6.4.2). At low non-freezing temperatures, high GSH content and GR activity were detected in several plant species, indicating a possible contribution to chilling tolerance and cold acclimation. Changes in H2O2 concentration and GSH/GSSG ratio alter the redox state of the cells and may activate special defence mechanisms through a redox signalling chain. The finding that several defence genes have antioxidant responsive elements or GSSG binding sites in their regulatory regions supports the idea that redox signalling is involved in regulating gene expression in response to low temperature.