Growth of three strains of Tetracladium marchalianum was inhibited by Cd-, and, to a lesser extent, by Cu-and Zn-chloride. In the presence of 50 µM Cd(II), all strains increased total thiol and glutathione production to 6, 11, and 21 µmoles · mg -1 dry mass, respectively. Cd(II) also induced the synthesis of one to several compounds reacting with 5,5'-dithio-bis-(2-nitrobenzoic acid). In order to identify buffer-soluble thiolic compounds other than cysteine, γ-EC and γ-ECG (glutathione) were analyzed and confirmed by mass spectrometry. No water soluble sulfides were detectable in any of the culture filtrates, but Cd(II) exposure at a concentration of 50 µM raised sulfide levels in the mycelia of two of the strains between 3 and 7-fold, Cu(II) and Zn(II) had no effect. Energy Dispersive X-ray-analysis (EDX) and Electron Spectrometry-Images (ES-I) of one strain revealed increased levels of Cu and Zn in the cytoplasm and even higher levels in vacuolar precipitates. Zn and Cu are accumulated in the vacuoles as polyphosphates, identified by Electron Energy Loss-Spectrometry (EELS). Cd was found only in the vacuoles.
Despite their ubiquitous distribution, the exact functions of metallothioneins (MTs) are still not known. We analyzed the expression of four novel barley (Hordeum vulgare L.) genes coding for MTs, comparing two different situations: (i) leaf senescence, and (ii) heavy metal stress. Physiological analysis of chlorophyll content and photosystem II efficiency revealed similar stress response during natural leaf senescence and short‐term heavy metal treatment with toxic concentrations of Cu and Cd. Gene expression patterns of the newly identified MTs (HvMT‐1a, HvMT‐2a, HvMT‐2b and HvMT‐3a) clearly vary under these different conditions, suggesting specific roles for the barley MTs during leaf senescence and heavy metal stress. While HvMT‐1a is induced during leaf senescence and after heavy metal treatment but not during metal deficiency, expression of HvMT‐2a is not affected under any of conditions tested. The MT HvMT‐3a is only induced in response to Cu deficiency and HvMT‐2b is downregulated in response to Cd stress.
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