Production of low-molecular weight thiols as a response to cadmium uptake by tumbleweed (Salsola kali).

Tumbleweed (Salsola kali) is a desert plant species that has shown to be a potential Cd hyperaccumulator. In this study, the production of low-molecular weight thiols (LMWT) as a response to cadmium stress was determined in hydroponically grown seedlings exposed to 0, 45, 89, and 178 microM Cd(2+). The treatment of 89 microM Cd(2+) was tested alone and supplemented with an equimolar concentration of ethylenediaminetetraacetic acid (EDTA) to determine the effect of this chelating agent on Cd uptake and thiols production. After 6 days of growth, the Cd concentration in plant tissues was determined by using inductively coupled plasma/optical emission spectroscopy (ICP/OES). Results indicated that Cd uptake by plants was concentration-dependent. Plants treated with 178 microM Cd(2+), had 10+/-0.62, 9.7+/-1.4, and 4.3+/-0.83 mmol Cd kg(-1) dry tissue in roots, stems, and leaves, respectively. The production of thiols was dependent on Cd concentration in tissues. According to the stoichiometry performed, plants treated with Cd concentrations up to 178 muM produced 0.131+/-0.02, and 0.087+/-0.012 mmol SH per mmol Cd present in roots and stems. In leaves, the production of thiols decreased at the highest Cd concentration tested. Thus, up to 89 microM Cd in the media, 0.528+/-0.004 mmol SH per mmol Cd in leaf tissues were produced. EDTA equimolar to Cd reduced both Cd uptake and thiols production. Catalase activity (CAT) (EC 1.11.1.6) was significantly depressed at the lowest Cd concentration. None of the conditions tested affected biomass or plant elongation.

p53-independent checkpoint controls in a plant cell model.

Allium cepa L. meristems were used as a plant model to study the p53-independent control of S and G2 phases by checkpoint pathways, in eukaryotic cells. Checkpoint blocks were induced at early and mid S by hydroxyurea. After their spontaneous override, cells became accumulated in G2-prophase, giving rise later on to a delayed mitotic wave. Cell growth was maintained during the checkpoint blocks, as the delayed mitoses were larger in size than the control ones. Under continuous hydroxyurea treatment, the delayed mitotic was formed by two subpopulations: normal mitoses corresponding to cells having properly recovered from the checkpoint block, and abnormal ones resulting from checkpoint adaptation. These latter cells displayed broken chromatids as they had unduly overriden the G2 checkpoint block, without completing DNA repair. The frequency of the checkpoint-adapted mitoses increased with the hydroxyurea concentration from 0.25 to 1.0 mM. However, from 1 mM hydroxyurea upwards, some of the cells lost their competence for checkpoint adaptation. Therefore, the dose of a genotoxic agent that still allows G2 checkpoint adaptation should always be applied in order to get rid of uncontrolled proliferating cells. This is specially suitable for cells lacking a functional p53 protein.

A plant cyclin B2 is degraded early in mitosis and its ectopic expression shortens G2-phase and alleviates the DNA-damage checkpoint.

Mitotic progression is timely regulated by the accumulation and degradation of A- and B-type cyclins. In plants, there are three classes of A-, and two classes of B-type cyclins, but their specific roles are not known. We have generated transgenic tobacco plants in which the ectopic expression of a plant cyclin B2 gene is under the control of a tetracycline-inducible promoter. We show that the induction of cyclin B2 expression in cultured cells during G2 phase accelerates the entry into mitosis and allows cells to override the replication checkpoint induced by hydroxyurea in the simultaneous presence of caffeine or okadaic acid, drugs that are known to alleviate checkpoint control. These results indicate that in plants, a B2-type cyclin is a rate-limiting regulator for the entry into mitosis and a cyclin B2-CDK complex might be a target for checkpoint control pathways. The cyclin B2 localization and the timing of its degradation during mitosis corroborate these conclusions: cyclin B2 protein is confined to the nucleus and during mitosis it is only present during a short time window until mid prophase, but it is effectively degraded from this timepoint onwards. Although cyclin B2 is not present in cells arrested by the spindle checkpoint in metaphase, cyclin B1 is accumulating in these cells. Ectopic expression of cyclin B2 in developing plants interferes with differentiation events and specifically blocks root regeneration, indicating the importance of control mechanisms at the G2- to M-phase transition during plant developmental processes.

p53-independent checkpoint controls in a plant cell model

Allium cepa L. meristems were used as a plant model to study the p53-independent control of S and G2 phases by checkpoint pathways, in eukaryotic cells. Checkpoint blocks were induced at early and mid S by hydroxyurea. After their spontaneous override, cells became accumulated in G2-prophase, giving rise later on to a delayed mitotic wave. Cell growth was maintained during the checkpoint blocks, as the delayed mitoses were larger in size than the control ones. Under continuous hydroxyurea treatment, the delayed mitotic was formed by two subpopulations: normal mitoses corresponding to cells having properly recovered from the checkpoint block, and abnormal ones resulting from checkpoint adaptation. These latter cells displayed broken chromatids as they had unduly overriden the G2 checkpoint block, without completing DNA repair. The frequency of the checkpoint-adapted mitoses increased with the hydroxyurea concentration from 0.25 to 1.0 mM. However, from 1 mM hydroxyurea upwards, some of the cells lost their competence for checkpoint adaptation. Therefore, the dose of a genotoxic agent that still allows G2 checkpoint adaptation should always be applied in order to get rid of uncontrolled proliferating cells. This is specially suitable for cells lacking a functional p53 protein.