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.