Characterization of the decline and recovery of heat-treated Scenedesmus vacuolatus

Abstract

Background: To find out how algal cells cope with and recover from heat stress, the small vegetative cells of the synchronous Scenedesmus vacuolatus culture were subjected to a heat pretreatment (46.5°C for 1 h) followed by dark recultivation. The changes in physiological activities and morphology of Scenedesmus cells were continuously monitored throughout the course of decline and recovery.

Results: It was found that the heat treatment, though completely inhibited photosynthesis, did not kill Scenedesmus cells. These cells, during dark recultivation, could make a fast repair and regained the ability of proliferation. We suggest that they entered a 'stand-by' state, which was characterized by condensed chromatin, partially functional but morphologically altered chloroplasts, disappeared vacuoles, slightly shrunk protoplast and intact plasma membranes. These stressed cells, on the surface, seemingly were undergoing some kind of disintegration, could readily and quickly return to normal cells upon illumination. Cell death occurred only after a long period of darkness (>48 h).

Conclusions: Our results suggest that the recovery of algal cells from stress damage may actually proceed in two steps. The middle "stand-by' stage normally is gone through too rapidly to be detected unless cells are kept in the dark.

Keywords: Chloroplast; Heat stress; Programmed cell death (pcd); Scenedesmus vacuolatus; TEM.

Figure 1

The variation of the ability of cell proliferation. The relative numbers of green colonies formed from seeded cells that had either been heat-treated (○) or not been treated (●) are plotted as functions of the time of dark cultivation. All the data are normalized with respect to the value of untreated cells at the beginning of dark cultivation. Each data point represents the mean ±SD of three independent measurements with two replicates each.

Figure 2

The variation of photosynthetic activity (Fv/Fm). (A) The decay of Fv/Fm ratio as a function of the time of heat treatment. (B) The variations of the Fv/Fm of heat-treated cells are plotted as functions of the time of recultivation in the dark (○), and those with light (150 μmol photon m^-2 s^-1) turned on at 12 (∇), 24 (■), 36 (Δ), 48 (□), 60 (◊) and 72 h (♦), respectively. The Fv/Fm of untreated cells as a function of the time of dark cultivation is also shown (●). Each data point represents the mean ±SD of three independent measurements with two replicates each. The dotted line indicates the Fv/Fm (0.74 ± 0.02) of untreated cells at the beginning of dark cultivation.

Figure 3

The variation of chlorophyll content. (A) The relative chlorophyll contents of heat-treated (○) and untreated (●) cells are plotted as functions of the time of dark cultivation. All the data are normalized with respect to that of untreated cells at the beginning of dark cultivation. Each data point represents the mean ±SD of three independent measurements with two replicates each. (B) The frequency distribution histograms of Scenedesmus cells analyzed for chlorophyll a fluorescence from (i) untreated cells at the beginning of dark cultivation and cells subjected to a heat treatment followed by cultivation in the dark for (ii) 0, (iii) 48 and (iv) 72 h, respectively.

Figure 4

The MTT assays for the succinate dehydrogenase activity of Scenedesmus mitochondria. The relative activities of heat-treated (○) and untreated (●) cell cultures are plotted as functions of the time of dark cultivation. All the data are normalized with respect to that of untreated cells at the beginning of dark cultivation. Each data point represents the mean ±SD of three independent measurements with two replicates each.

Figure 5

Representative transmission electron micrographs. (A) Untreated cell before dark cultivation (control) and the cells that had been heat treated and then cultured in the dark for (B) 0, (C) 2, (D) 6, (E) 12, (F) 24, (G) 48 and (H) 72 h, respectively. Bars = 1 μm. Abbreviations: c, chloroplast; m, mitochondrion; n, nucleus; nl, nucleolus; s, starch granule; v, vacuole. Arrows in (B): condensation of chromatin on the inner side of the nuclear membrane. Arrows in (E-G): exudation of organic substances between the cell wall and the cell membrane. Arrow in (H): rupture of cell membrane.

Figure 6

The transmission electron micrographs of thylakoid. (A) Untreated cell before dark cultivation (control) and the cells that had been heat treated and then cultured in the dark for (B) 0, (C) 2, (D) 12 h, (E) 24 h and (F) 48 h, respectively. Bars = 100 nm.