The isolation and characterization of type II metallothionein-like genes from tomato (Lycopersicon esculentum L.).

Two cDNAs encoding putative metallothionein (MT)-like peptides have been isolated from tomato (L. esculentum L.). The predicted protein products of these two cDNAs (LeMT(A) and LeMT(B)) are 72 and 83 amino acids respectively and both encode peptides with arrangements of cysteine residues characteristic of type II plant MTs. In other plants which possess more than one gene expressing MT proteins of the same type, the products are closely related or identical, but LeMT(A) and LeMT(B) constitute two different classes of message, and encode two different protein products. Northern blot analysis of LeMT(A) and LeMT(B) showed that transcripts of both MT-like genes were more abundant in leaves than roots in tomato plants grown without addition of extra metal ions, a characteristic of type II MTs. A genomic clone corresponding to LeMT(B) (LeMT(B)) was isolated and sequenced. The 5'-flanking region of LeMT(B) was shown to contain a putative metal regulatory element (MRE) which suggests the possibility of metal-regulated transcription. In addition, the upstream region also contains a G-box like motif (CACGTG) and an 8 bp sequence (AATTCAAA) found within the promoters of genes shown to be ethylene-responsive.

Metallothionein genes from the flowering plant Mimulus guttatus.

In response to excess metal, higher plants produce metal-binding peptides [( gamma EC]nG) whose biosynthesis is believed to be mediated by enzymes involved in glutathione (gamma ECG) metabolism. In contrast, animals synthesize metallothioneins, gene-encoded low molecular weight cysteine-rich metal-binding proteins. In an investigation of copper-regulated genes in the copper-tolerant flowering plant Mimulus guttatus, we have isolated a series of cDNA clones identifying two genes which encode a protein with class I metallothionein domains. This represents the first description of a metallothionein gene in a flowering plant.

Isolation of a copper complex and its rate of appearance in roots of Mimulus guttatus.

A Cu-complex was isolated from the roots of copper-tolerant Mimulus guttatus. The elution volume of the complex determined by gel permeation chromatography was similar to that of rat-liver cadmium thionein. The complex was heat stable, had a relatively high ratio of absorbance at 254 nm: 280 nm and incorporated (35)S. The complex, purified using a combination of gel permeation chromatography and anion-exchange chromatography, contained more glutamine/glutamic acid and glycine residues than mammalian metallothioneins. The amount of the complex in roots increased after 5 h growth in a solution containing 16 µM Cu. Induction was preceded by an increase in the concentrations in root tissue of unknown compounds containing sulphur which may serve as precursors. The availability of these compounds appeared to regulate the rate of synthesis of this Cu-complex.

Accumulation of zinc and Organic Acids in Roots of Zinc Tolerant and Non-tolerant Ecotypes of Deschampsia caespitosa.

A much higher zinc level was necessary to inhibit root elongation in the zinc tolerant ecotype as compared to the non-tolerant ecotype of Deschampsia caespitosa. In the presence of a range of high levels of zinc, zinc accumulated to a much higher concentration in the roots of the tolerant ecotype, especially in the root sap. Accumulation of citrate in the root sap was highly correlated to the accumulation of zinc. Gel filtration chromatography of the root sap showed zinc to be mainly present as zinc-citrate. This was the only zinc complex found. The malate concentration of the root sap was much lower than the concentration of citrate. However the malate content of aqueous root homogenates was comparable or even greater than the content of citrate, suggesting that malate and citrate are located in different compartments within the cell. The results are consistent with a model of zinc tolerance in which zinc is complexed with citrate in the vacuole.

Metal tolerance in tissue cultures of anthoxanthum odoratum.

Tissue cultures were initiated from one non-tolerant (S20) and two zinc and lead tolerant (T92 and T94) clones of Anthoxanthum odoratum. Growth of callus from the non-tolerant clone was reduced by the presence of zinc, lead, copper and nickel, whereas callus from the two tolerant clones showed no reduction of growth in the presence of zinc and lead but growth was reduced by copper and nickel. The specificity of metal tolerance shown by the parental material was maintained in the callus. Tolerant and non-tolerant callus accumulated similar amounts of zinc and lead.

The degradation of canavanine by jack bean cotyledons.

Germinating jack bean cotyledons liberated (14)CO2 when fed (14)C-guanidoxy-canavanine but did not accumulate any (14)C-compounds other than the applied canavanine. This suggested that the canavanine was being degraded by the action of canavanase to canaline and urea, the urea then being converted to ammonia and carbon dioxide by the action of urease. Hydroxyurea and acetohydroxamic acid (both inhibitors of urease activity) strongly inhibited the liberation of (14)CO2 from (14)C-guanidoxy-canavanine by the cotyledons but neither compound induced the accumulation of (14)C-urea within the tissues. This inhibitory action of hydroxyurea on (14)CO2 output was thought to be due at least in part, to this inhibition of canavanase activity.