Plants cultivated with Cd can produce large amounts of phytochelatins. Since these compounds contain much cysteine, these plants should have an increased rate of assimilatory sulfate reduction, the biosynthetic pathway leading to cysteine. To test this prediction, the effect of Cd on growth, sulfate assimilation in vivo and extractable activity of two enzymes of sulfate reduction, ATP-sulfurylase (EC 2.7.7.4) and adenosine 5'-phosphosulfate sulfotransferase were measured in maize (Zea mays L.) seedlings. For comparison, nitrate reductase activity was determined. In 9-day-old cultures, the increase in fresh and dry weight was significantly inhibited by 50 micromolar and more Cd in the roots and by 100 and 200 micromolar in the shoots. Seedlings cultivated with 50 micromolar Cd for 5 days incorporated more label from 35SO42-into higher molecular weight compounds than did controls, indicating that the predicted increase in the rate of assimilatory sulfate reduction took place. Consistent with this finding, an increased level of the extractable activity of both ATP-sulfurylase and adenosine 5'-phosphosulfate sulfotransferase was measured in the roots of these plants at 50 micromolar Cd and at higher concentrations. This effect was reversible after removal of Cd from the nutrient solution. In the leaves, a significant positive effect of Cd was detected at 5 micromolar for ATP-sulfurylase and at 5 and 20 micromolar for adenosine 5'-phosphosulfate sulfotransferase. At higher Cd concentrations, both enzyme activities were at levels below the control. Nitrate reductase (EC 1.6.6.1) activity decreased at 50 micromolar or more Cd in the roots and was similarly affected as ATP-sulfurylase activity in the primary leaves.In 1957 a cysteine-rich, Cd-binding protein was isolated from equine kidney (10) and subsequently called metallothionein. In higher plants the inducible Cd-binding compounds showed properties distinctly different from metallothioneins: in Ouchterlony two-dimensional immunodiffusion tests, no cross-reaction could be observed (7), and attempts to establish the primary structure by Edman degradation failed (5). Similarities were a high cysteine content, A254:A280> 1, and comparable circular dichroism spectra (5), indicating the lack of aromatic residues and the possible binding of heavy metals by mercaptide complexes.

In roots and shoots of pea plants (Pisum sativum L.) cultivated with CdCI2 concentrations up to 50 micromolar, growth, the content of total acid soluble thiols, and the activity of glutathione synthetase (EC 6.3.2.3) and of adenosine 5'-phosphosulfate sulfotransferase were measured. In addition, the occurrence of Cdbinding peptides (phytochelatins) and the contents of glutathione and cysteine were determined in roots of plants exposed to 20 micromolar Cd and/or 1 millimolar buthionine sulfoximine, an inhibitor of glutathione synthesis. An appreciable increase in activity of glutathione synthetase at 20 and 50 micromolar Cd and of adenosine 5'-phosphosulfate sulfotransferase at 5 micromolar and higher Cd concentrations was detected in the roots. Most of the additional thiols formed due to Cd treatment were eluted from a gel filtration HPLC column together with Cd, indicating the presence of phytochelatins. In plants treated with buthionine sulfoximine and Cd, no phytochelatins could be detected but the cysteine content increased 21-fold. Additionally, a larger increase in both enzyme activities occurred than with Cd alone. Taken together, our results are consistent with the hypothesis that glutathione is a precursor for phytochelatin synthesis.In plants, the general principle for complexing heavy metal ions is the formation of peptides with the structure (y-glucys)n = 2-11), called phytochelatins (8, 10), poly(y-glutamylcysteinyl)glycines [(yEC)nG] (25), Cd-binding peptides (22), y-glutamyl metal-binding peptides (23), or class III metallothioneins (7). The capacity to produce such peptides due to Cd-treatment seems to be widespread in the plant kingdom (8). Identical peptides, called cadystins, have been found in Cd-treated fission yeast (14). Yeast mutants unable to synthesize phytochelatins (17) and tomato (16,29) and tobacco (21) cell cultures supplied with BSO2, a potent and specific inhibitor of the enzyme catalyzing the first step of glutathione biosynthesis, y-glutamylcysteine synthetase (EC 6.3.2.2), were hypersensitive towards Cd. This indicates that phytochelatins play a role in the detoxification of heavy metal ions (5,8,12). Because of the y-glutamic acid bonds in the peptide chains, phytochelatins cannot be primary gene products, and glutathione (y-glu-cys-gly), with its structure closely related to the one of phytochelatins, could be used as a precursor for the

The intercellular distribution ofassimilatory sulfate reduction enzymes between mesophyll and bundle sheath cells was analyzed in maize (Zea mays L.) and wheat (Triticum aestivum L.) leaves. In maize, a C4 plant, 96 to 100% of adenosine 5'-phosphosulfate sulfotransferase and 92 to 100% of ATP sulfurylase activity (EC 2.7.7A) was detected in the bundle sheath cells. Sulfite reductase (EC 1.8.7.1) and O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) were found in both bundle sheath and mesophyll cell types. In wheat, a C3 species, ATP sulfurylase and adenosine 5'-phosphosulfate sulfotransferase were found at equivalent activities in both mesophyll and bundle sheath cells. Leaves of etiolated maize plants contained appreciable ATP sulfurylase activity but only trace adenosine 5'-phosphosulfate sulfotransferase activity. Both enzyme activities increased in the bundle sheath cells during greening but remained at negligible levels in mesophyll cells. In leaves of maize grown without addition of a sulfur source for 12 d, the specific activity of adenosine 5'-phosphosulfate sulfotransferase and ATP sulfurylase in the bundle sheath cells was higher than in the controls. In the mesophyll cells, however, both enzyme activities remained undetectable. The intercellular distribution of enzymes would indicate that the first two steps of sulfur assimilation are restricted to the bundle sheath cells of C4 plants, and this restriction is independent of ontogeny and the sulfur nutritional status of the plants.C4 plants are characterized by an intercellular compartmentation of CO2 and NO3-assimilation between mesophyll and bundle sheath cells (2,3,6,16). Recently, a predominant localization ofenzymes ofassimilatory sulfate reduction in the bundle sheath cells was proposed (7,8,14). This pathway begins with the formation ofAPS2 from ATP and S042-via ATP sulfurylase (EC 2.7.7.4) (1,18 Kolibri) were imbibed for 24 h in aerated tap water at room temperature and then transferred to moist filter paper. After 3 d in the dark, the seedlings were cultivated on quartz sand watered regularly with nutrient solution (23). Nutrient solution deficient in S was composed by replacing the SO42-salts by equimolar amounts of the corresponding Cl-salts. The plants were grown at 26°C, with a RH of 40% and a light intensity of 46 ,uE m-2 s-' provided by incandescent lamps (Philips TL 40 w).Cell Isolation. Mesophyll cells and bundle sheath strands were obtained either by a mechanical or by an enzymic isolation procedure. For both procedures, leaves were cut diagonally in approximately 1-mm-wide strips with a razor blade and then transferred to a medium for protoplast isolation according to Mills and Joy (12), containing 2% cellulase and 2% pectinase. Ten ml of medium were used per g leafstrips. The plant material was infiltrated by applying a vacuum. The incubation time was 15 min in the light for the mechanical procedure. The plant material was then homogenized using a Servall Omni Mixer (Ivan Sorvall, Norwalk, CT) for 10 s at 100 v (6,700 rpm) an...

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