Despite an increasing literature on biochemical events in response to heavy metals stress in higher plants, little is known about unicellular algae. Microalgae play an important role in the equilibrium of aquatic ecosystems and represent highly suitable biological indicators of environmental changes.
We utilized a strain of Chlorella sorokiniana, an unicellular chlorophyte, as a model system for physiological, biochemical and morphological studies on heavy metal stress response. The algal cells were exposed for a short and a long period (2 and 24 h) to heavy metal such as Cd and Pb (250 μM).
Cd and Pb inhibited the specific growth rate. In Pb-treated samples we observed most of the algal cells grew as colonies and contained plenty of cytoplasm lipid droplets. Probably, Chlorella cells, after first mitosis, continue to divide, forming colonies of four- and more cells with a common cell wall, and only some algae live independently.
The occurrence of adipocyte forms deriving from starch granules and lipids, even after a short time treatment, is regarded as stress symptoms (Lebsky 2004).
Concentric multilamellar/multivesicular bodies were well distinct in Chlorella cells Cd-polluted. These organelles were regarded as accumulation of membranes correlated with incomplete digestion of endocytosed material: double-membrane-bound vesicles arising from endoplasmic reticulum tubes enclose cytoplasm or organelles and coalesce to seal the region in a double-membrane-bound compartment.
We suggest that the repeated phenomena of encircling and sealing cytoplasmic regions by endoplasmic reticulum tubes might generate the concentric multilamellar bodies in our cadmium exposed samples.
Thompson and Vierstra (2005) reported the autophagic activity of cell vacuoles to destroy the cytoplasm and nucleus. Autophagocytosis phenomena have been reported also for unicellular algae (Reunova et al. 2007).
We suggest that the cytoplasm vesicles, and concentric multilamellar/multivesicular bodies were induced by heavy metal treatment and they might be involved in the transport of metal ions to vacuoles or vesicles and/or in the elaboration of massive newly formed or damaged material.
In Chlorella, Cd had a much higher effect than Pb on physiological functions such as photosynthesis and respiration. Particularly, photosynthetic activity resulted strongly compromised by Cd treatment. According to Neelam and Rai (2003), a possible explanation for Cd effect on photosynthesis could be the inhibition of the PS II as a result of damage of thylakoid membranes and reaction centers. In fact, Chlorella cells showed an ultrastructural alteration in the shape and organization of thylakoids that confirm a damage of the photosynthetic apparatus. Chloroplasts and their arrangement represent a common target of toxic substances in algae and higher plants (Carginale et al. 2004; Nacorda et al. 2007; Basile et al. 2008). The altered chloroplast shape could be a consequence of a perturbation of cation exchange induced by heavy metal-treatment (Basile et al. 2008), while the structural alterations of the thylakoid system could depend on the ability of heavy metals to bind to proteins and to interfere with their normal functions, also inducing oxidative damage (Heumann 1987). Moreover, the chloroplast alterations in leaves of tomato exposed to heavy metals were related to an increase in the production of ROS (Gratao et al. 2009). In our experiments, associated to ultrastructural alterations of plastids, we noted a significant reduction of the photosynthetic rate related to a decrease of total Chl and Chl a contents that both pollutants Pb and Cd caused. The decreases of Chl a and total Chl indicated a decline in the antenna size of the photosynthetic reaction center complexes. Qiu et al. (2006) showed that in Chlorococcum sp. AZHB the decrease of Chl a correlated to the increasing concentrations of Cu or Cd treatment. The decrease of chlorophyll, accompanied by the degradation of the chloroplast structure, in Chlorella heavy metal polluted indicates that the photosynthetic apparatus in these cells could be disrupted.
Therefore, the decrease of growth occurring in the algae Cd or Pb treated could be ascribed to the reduction of the photosynthetic activity.
A decrease of soluble protein content was detected in C. sorokiniana cells Pb- or Cd-treated for 24 h by 61 and 65% of the control, respectively. Probably, chlorophylls and proteins, and even the chloroplast proteins, represented an emergency source of nitrogen and sulfur to ensure cell growth. Moreover, the reduction of protein content might also be attributed to the shortage of carbon skeleton resulting from low photosynthetic rate.
The respiratory rate of the alga, under Pb or Cd exposure significantly increased. Little is known in literature about heavy metal-induced cellular respiration. Desouky (2011) reported that the respiration of pollutant Chlorella vulgaris cultures was considerably increased by heavy metal exposure: but these studies were conducted in algae treated with high (6 and 8 ppm) and low (2 and 4 ppm) concentrations of CoCl2 or NiCl2.
The remarkable enhancement of respiratory oxygen consumption in C. sorokiniana cells exposed to Pb or Cd could be an adaptive developed strategy to compensate the energy depletion caused by a low photosynthetic activity. C. sorokiniana cells seemed to use respiration to provide more energy to sustain the metabolism as previously reported (Vona et al. 1999).
Most studies regarding oxidative stress responses indicate Cys and GSH as the main antioxidants in the plant cell apart from ascorbate (Foyer and Noctor 2009).
In this study, we reported that in C. sorokiniana cells Cd-treated, OASTL activity enhanced up to 2.2-fold after 24 h, respect to control cells. However, the activity of this enzyme seems to be not influenced by Pb treatment. The different results on OASTL activity between Cd and Pb treatment could depend on the toxicity level of the single metal. Like in the epiphytic moss Scorpiurum circinatum (Carfagna et al. 2011b), the positive relationship concerning OASTL and metal exposure may suggest that in Chlorella cells exists a link between heavy metal tolerance and Cys synthesis. Increased Cys synthesis associated with heavy metals appears to be a necessary response for biosynthesis of GSH and of the other ligands involved in the heavy metal binding.