Programmed cell death following sugar and phosphate starvation in Pinus pinaster suspension cultured cells

Abstract

Examples of programmed cell death (PCD) have been shown to occur in plants at the levels of development and cell differentiation, as well as during the defence responses to abiotic and biotic stress. Suspension cells of maritime pine (Pinus pinaster) do not exhibit a stationary phase at the end of the batch cell culture cycle. Immediately following the end of exponential growth, the cultures evidence a quick decrease of biomass and a loss in cell viability. This is simultaneous with the depletion in the extracellular medium of two of the main nutrients: sugar and phosphate. In the present work we wanted to differentiate the determining nutrient behind the induction of cell death, and also ascertain the involvement of a programmed cell death event. P. pinaster suspension cell cultures were transferred to medium containing different combinations of sugar and phosphate and analysed for biomass production. Results evidenced that carbon depletion was the sole responsible for the induction of cell death. Still, the simultaneous depletion of sugar and phosphate had a synergistic effect, anticipating the induction of cell death. The analysis of gDNA fragmentation through the TUNEL assay suggested that sugar depletion induced programmed cell death in maritime pine suspension cell cultures. Further evidences for the involvement of PCD were obtained through the analysis of the marker enzyme acid phosphatase (APase). The observed increase in APase activity during sugar starvation suggests the involvement of an autophagic type of PCD. This is coherent with the increase in free phosphate observed after sugar starvation, which is likely the result of the hydrolytic degradation of cellular components. The utilization of non-metabolizable analogs of both nutrients provided further insight into the mechanism of PCD. Growth of maritime pine suspensions cells in the presence of the phosphate analog phosphite confirmed that sugar levels were responsible for the triggering of PCD. In addition, results considering the utilization of the glucose analogs 3-O-methyl-D-glucose and 2-desoxiglucose, suggest that perception of the sugar depletion signal that is capable of triggering PCD does not occur at the plasma membrane level, but at the intracellular level.