Discussion
We examined the physiological phenotyping and chemical responses ofC. reinhardtii treated with ABA and exposed to salt stresses in order to investigate whether ABA enhances salt tolerance in vegetative cells and gametes of this alga in a similar manner to that in higher plants or not.
The first and immediate indicator for increasing tolerance is the growth rate. Chlamydomonas reinhardtii cells are sensitive to high salt stress and therefore have lower growth rate under such conditions compared to unstressed cells (Hema et al., 2007; Mastrobuoni et al., 2012; Meijer et al., 2017). Yoshida et al. (2003) reported that exogenously added ABA enhances the growth of C. reinhardtii , however it was not clear whether this effect was due to the ABA or the interference of its carrier solvent. In this study, by using ethanol as a mock treatment we confirmed that ABA enhances the growth of microalgae and increases their tolerance to high-salinity stress, where ethanol showed no, or even slightly negative effect on algal growth (Figs. 1 & 2), unlike in plants where externally added ethanol was proven to increase salt stress tolerance (Nguyen et al., 2017).
It is known that salinity does not exert hostile effects on the stoichiometry and chlorophyll antenna size of the photosynthetic apparatus. However, salinity-stress increases the susceptibility of cells to photoinhibition in Chlamydomonas (Neale & Melis, 1989). When C. reinhardtii was incubated with 125 mM NaCl for 24 h, as the alga photosynthetic activity recovers up by ~30% of the original activity after 1 h of high salt exposure then it remains during the following 24 h (Vega et al., 2006), a decline in the light-saturated rate of photosynthesis (PEmax) was observed (Fig. 3), indicating for PSII photoinhibition. However, ABA-treated cells showed up to ~2 to 3-fold increase in the photosynthetic activity under salt stress compared to non-treated cells. These results suggest that ABA can participate in protecting PSII against photoinhibition inC. reinhardtii under high salinity, likely through protecting the cells from Na+ toxicity or less Na+ uptake (Gurmani et al. , 2007), as it has been evidenced that Na+ can irreversibly inactivate photosynthesis systems indirectly by promoting a secondary oxidative disruption, or directly by damaging the photosynthetic 32/34 kDa (D1/D2) proteins as in higher plants (Murata et al., 2007; Yang et al., 2014).
As almost all environmental stresses lead an increase in the production of ROS and thereby to oxidative stress in photosynthetic organisms (Mittler, 2002), the imposition of freshwater algae to salinity stress quickly leads to a rise in ROS in their cells (Mallick & Mohn, 2000). Indeed, based on ROS-sensitive fluorescence, ROS appeared to accumulate in the stressed ABA-untreated cells and in the stressed mock treatment, and ROS-sensitive fluorescence was especially prominent in the chloroplast (Figure 4A), where the chloroplasts look degraded. In contrast, ABA-treated cells showed weak ROS-sensitive fluorescence with no sign of chloroplasts degradation. The histograms in Fig. 4B clearly supports the microscope images that salinity stress significantly enhanced the intracellular ROS generation in the untreated cells. However, in ABA-treated cells ROS generation was significantly suppressed. These results suggest that ABA induces the elimination of high salinity ROS-reactions in Chlamydomonas cells.
Under different stress conditions such as phosphate limitation (Olsen et al., 1983), acidic environment (Visviki et al., 2000), the herbicide paraquat (Jamers et al., 2010) and high salinity (Khona et al. , 2016) Chlamydomonas cells have alternative ways to avoid chronic stress by forming stress-resistant life cycle stages undergo abnormal cell division with reduced individual cell size forming “palmelloids”. In the current study, NaCl induced the formation of palmelloids in the stressed untreated-cells unlike in ABA-treated cells where the number of palmelloids was significantly reduced (Fig. 5). These results are evidence that ABA playing a crucial role in protectingChlamydomonas cells from high salinity stress.
In this study, ABA supported the growth of gamete population, as it enhanced their tolerance to high salinity (Figs. 6 & 7). InChlamydomonas, gametogenesis is triggered by N limitation. When N is depleted in the growth medium, the vegetative cells have programs (Beck & Haring, 1996; Goodenough, 1991). The first program is that the cells acclimate to N deprivation through metabolic changes, including synthesis of N-scavenging enzymes (Quesada & Fernández, 1994) and the renewal of ribosomes (Martin et al., 1976). The second program is that the vegetative cells underdo gametogenesis to produce Mt+ and Mt- cells for mating. While N starvation has been extensively studied in Chlamydomonas for producing alternative sources of energy by accumulation more TAG in the cells under N starvation (Montantes et al., 2018; Salas- Siaut et al., 2011; Yang et al., 2020), or as a new tool to create offspring with new traits (Kramer and Lucker, 2020), however, N-starved Chlamydomonas cells can also exhibit various biological pathways for managing photosynthesis to efficiently utilize the absorbed light energy (Saroussi et al., 2017). While underlying these pathways is still in its early stage, it could provide an important direction for developing a more comprehensive understanding of photosynthetic energetics and its control.