Subcellular localization of cadmium and cadmium-binding peptides in tobacco leaves : implication of a transport function for cadmium-binding peptides.

The synthesis of Cd-binding peptides (CdBPs) was induced upon addition of 20 micromolar CdCl(2) (nonphytotoxic level) to the nutrient solution of hydroponically grown tobacco seedlings (Nicotiana rustica var Pavonii). Amino acid analysis showed that the main components were gamma-(Glu-Cys)(3)-Gly and gamma-(Glu-Cys)(4)-Gly. Seedlings exposed to the metal for 1 week contained similar glutathione levels as found in the controls (about 0.18 micromole per gram fresh weight). If, as has been proposed, CdBPs are involved in Cd-detoxification by chelation, both metal and ligand must be localized in the same cellular compartment. To directly determine the localization of Cd and CdBPs, protoplasts and vacuoles were isolated from leaves of Cd-exposed seedlings. Purified vacuoles contained virtually all of the CdBPs and Cd found in protoplasts (104% +/- 8 and 110% +/- 8, respectively). CdBPs were associated with the vacuolar sap and not with the tonoplast membrane. Glutathione was observed in leaves and protoplasts but not in vacuoles. The probability that CdBPs are synthesized extravacuolarly and our finding that they and Cd are predominantly located in the vacuole suggest that these molecules might be involved in transport of Cd to the vacuole. Our results also suggest that a simple cytoplasmic chelator role for CdBPs in Cd tolerance cannot be assumed.

[1]  G. Wagner,et al.  Relationships between Cadmium, Zinc, Cd-Peptide, and Organic Acid in Tobacco Suspension Cells. , 1989, Plant physiology.

[2]  Z. C. Li,et al.  Soluble and Bound Apoplastic Activity for Peroxidase, beta-d-Glucosidase, Malate Dehydrogenase, and Nonspecific Arylesterase, in Barley (Hordeum vulgare L.) and Oat (Avena sativa L.) Primary Leaves. , 1989, Plant physiology.

[3]  R. Reese,et al.  Sulfide stabilization of the cadmium-gamma-glutamyl peptide complex of Schizosaccharomyces pombe. , 1988, The Journal of biological chemistry.

[4]  E. Tel-or,et al.  Localization and toxic effects of cadmium, copper, and uranium in azolla. , 1988, Plant physiology.

[5]  A. B. Tomsett,et al.  Molecular biology of metal tolerances of plants , 1988 .

[6]  P. Goldsbrough,et al.  Phytochelatin synthesis and glutathione levels in response to heavy metals in tomato cells. , 1987, Plant physiology.

[7]  C. Latus,et al.  Localization of Glutathione Synthetase and Distribution of Glutathione in Leaf Cells of Pisum sativum L. , 1987 .

[8]  R. Reese,et al.  Effects of buthionine sulfoximine on cd-binding Peptide levels in suspension-cultured tobacco cells treated with cd, zn, or cu. , 1987, Plant physiology.

[9]  E. Takahashi,et al.  Sodium, Potassium, Chloride, and Betaine Concentrations in Isolated Vacuoles from Salt-Grown Atriplex gmelini Leaves. , 1987, Plant physiology.

[10]  C. Ackerley,et al.  Localization of cadmium in granules within differentiating and mature root cells , 1987 .

[11]  R. Reese,et al.  Properties of tobacco (Nicotiana tabacum) cadmium-binding peptide(s). Unique non-metallothionein cadmium ligands. , 1987, The Biochemical journal.

[12]  D. Hunt,et al.  Accumulation of non-protein metal-binding polypeptides (gamma-glutamyl-cysteinyl)n-glycine in selected cadmium-resistant tomato cells. , 1986, The Journal of biological chemistry.

[13]  K. Schumaker,et al.  Calcium transport into the vacuole of oat roots. Characterization of H+/Ca2+ exchange activity. , 1986, The Journal of biological chemistry.

[14]  M. Venis,et al.  ATP-Dependent Ca2+ Transport in Tonoplast Vesicles from Apple Fruit , 1986 .

[15]  E. Blumwald,et al.  Na/H Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris. , 1985, Plant physiology.

[16]  A. Bariaud,et al.  Mechanism of Cd2+ resistance in Euglena gracilis , 1985 .

[17]  A. Stobart,et al.  The effect of Cd2+ on the biosynthesis of chlorophyll in leaves of barley , 1985 .

[18]  D. A. Cataldo,et al.  Cadmium distribution and chemical fate in soybean plants. , 1981, Plant physiology.

[19]  Z. Tynecka,et al.  Energy-dependent efflux of cadmium coded by a plasmid resistance determinant in Staphylococcus aureus , 1981, Journal of bacteriology.

[20]  H. Weigel,et al.  Subcellular distribution and chemical form of cadmium in bean plants. , 1980, Plant physiology.

[21]  T. Boller,et al.  Hydrolytic enzymes in the central vacuole of plant cells. , 1979, Plant physiology.

[22]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[23]  E. Grill,et al.  Phytochelatins, a class of heavy-metal-binding peptides from plants, are functionally analogous to metallothioneins. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[24]  W. E. Rauser.  Changes in glutathione content of maize seedlings exposed to cadmium , 1987 .

[25]  B. Halliwell,et al.  Purification and properties of glutathione synthetase from spinach (Spinacia oleracea) leaves , 1986 .

[26]  H. Woolhouse.  Toxicity and Tolerance in the Responses of Plants to Metals , 1983 .

[27]  P. Benveniste,et al.  Sterol biosynthetic capability of purified membrane fractions from maize coleoptiles , 1978 .

[28]  T. Hodges,et al.  Purification of a plasma membrane-bound adenosine triphosphatase from plant roots. , 1974, Methods in enzymology.

[29]  W. A. Svec,et al.  [42] Analytical procedures for the isolation, identification, estimation, and investigation of the chlorophylls☆ , 1971 .