Homeostasis Of Cytoplasmic Calcium Involved In The Regulation Of Aluminum-induced Cell Death In Yeast

Aluminum (Al) toxicity has been implicated as a major cause badly threatening crop grown in acidic soils. In recent years, significant progress has been made in understanding the molecular mechanisms of Al toxicity and tolerance in the area of stress phytophysiology. Al exclusion and intracellular resistance are two important mechanisms that promote Al tolerance in plants. It is important to note that internal resistance to Al can also be achieved through negative regulation of programmed cell death (PCD). Al induces apoptosis in yeast (Saccharomyces cerevisiae) concomitant with elevation of cytosolic calcium ion (Ca2+), which can be decreased by overexpressing anti-apoptotic members. It remains unknown, however, whether inhibition of cytoplasmic Ca signal burst is a significant mechanism involved in the negative regulation of Al-induced PCD and internal resistance to Al toxicity. To this end, we investigated the functional roles of vacuolar Ca2+pump Ca2+-ATPase Pmclp, the Ca2+chelator BAPTA-AM and anti-apoptotic members in modulating cytosolic Ca2+and facilitating Al tolerance in yeast. Results obtained in the present study are summarized as follows:1. From Ca sensitivity assay with mutant strains, a yeast mutant, pmcl, lacking the vacuolar Ca2+-ATPase (Pmclp), was found to be more sensitive to Al treatment than the wild-type yeast and other Ca related mutants.2. Although the apoptosis were observed in both wild-type and pmcl mutant strains under Al-treatment, the apoptosis cell number of pmcl mutant was significantly higher than that of the wild-type cells, suggesting that loss of PMC1leads to a failure in pumping Ca2+into the vacuole; thus, the cells fail to regulate cytosolic Ca2+and increase cytosolic Ca2+level. These results indicate that Al-elicited Ca signaling is an early mechanism of Al-induced PCD.3. To ensure that the Al susceptibility of the pmcl mutant was not an artifact, the yeast cells were exposed to diverse stresses that could induce apoptosis in yeast, including sorbitol, copper ion (Cu2+) or cadmium ion (Cd2+). The pmcl mutant displayed enhanced sensitivity to Ca2+and Al3+, but not to any of the other substances.4. The growth properties of wild-type yeast and the pmcl mutant were similar when cells were exposed to different pH values, and Ca2+changed little in response to pH variations. The pmcl mutant was more sensitive to Al than wild-type yeast. These results suggest that the differences between the pmcl mutant and wild-type yeast under Al stress are caused by Al3+rather than pH change.5. To investigate if PMC1assumes an Al tolerance function in complementation tests, the exogenous PMC1gene was transformed into wild-type yeast or back into the pmcl mutant. The Ca2+levels in wild-type cells overexpressing PMC1were distinctly less than that in control (wild-type cells transformed with vector only) in response to Al3+at all concentrations tested. Both wild-type and pmcl strains exhibited increased Ca and Al tolerance in response to PMC1overexpression.6. To provide direct evidence that Ca2+is an important mediator of Al toxicity, we tested yeast cells with the Ca2+chelator BAPTA-AM to inhibit excessive cytosolic free Ca2+and monitored the effects. When incubated with1mM Al3+, BAPTA-AM exposure led to a dose-dependent decrease in cytosolic Ca2+in cells and enhanced the viability of Al-exposed cells. These data suggest that treatment with the cytosolic Ca2+chelator BAPTA-AM can decrease cytosolic free Ca2+and alleviate Al toxicity. It directly supports the idea that Al-induced cell death is mediated by cytosolic Ca signaling flux and Ca2+homeostasis. The inhibition of Al-elicited Ca2+burst can reduce Al toxicity.7. To test whether the expression of anti-apoptotic members restore Al tolerance in the pmcl mutant, the anti-apoptotic genes (Bcl-2, Ced-9or PpBI-1) were transformed into wild-type yeast and the pmcl mutant. Al-induced cytoplasmic Ca2+levels in cells expressing anti-apoptotic members was distinctly decreased compared to the cells transformed with vector only, demonstrating that anti-apoptotic members can complement the function of Ca2+-ATPase PMC1and act upstream of intracellular Ca2+flux in the pathway mediating Al-induced cell death.8. To investigate the molecular mechanisms underlying Ca signaling, quantitative RT-PCR was used to determine the expression levels of certain Ca pathway components. Expression levels of PMC1,CMD1,CNB1and PLC1were changed in response to Al stress. These results indicate that these Ca2+-relevant genes in Ca signaling pathways participate in Al tolerance in yeast.