ABA enhances the growth and photosynthesis of C.
Reinhardtii under salt stress
To investigate the sensitivity of Chlamydomonas to NaCl, cells
were grown in TAP medium supplemented with increasing concentrations of
NaCl from 100-300 mM. After 72 h of incubation the growth was reduced in
which ~ 60% of the cells survived at 125 mM NaCl,
compared to the unstressed control (Supplementary Fig. S3). The negative
effect of the salt on cell growth was more pronounced at higher
concentrations such as 250 mM NaCl. However, Chlamydomonas is not
fully arrested by NaCl at higher concentration, after longer incubation
period we still detected slow growth of the cells (Supplementary Fig.
S3). Therefore, the concentration of 125 mM NaCl was considered for salt
stress treatments in this study.
Dimethylsulfoxide (DMSO) is widely used as a carrier solvent for small
molecules in biological assays, with low toxicity at concentrations
<10% (Szmant, 1975). In previous research DMSO was used in
ABA experiments on C.
reinhardtii (Yoshida et al. , 2003; Yoshida et al. , 2004),
however its interfering influence on the grown was not evaluated. In
this study, DMSO was used as a carrier solvent for ABA in low
concentration of 50 µM. After 3 d of cultivation the growth increased
40% compared to control (Supplementary Fig. S4). Therefore, and
instead, we decided to use methanol as a solvent for ABA for the
experiments. To test whether ethanol has a positive impact on growth or
not, we ran a mock treatment that contains only ethanol of a final
concentration of 50 µM and C. reinhardtii cells, along with the
other treatments.
We next set out to confirm whether ABA enhances the growth of C.
reinhardtii under optimal and salt stress conditions or not. Fig. 1
shows that the addition of 50 μM and 500 μM ABA to dense cultures ofC. reinhardtii increases about 10% of the growth without a lag
phase compare to the untreated control, while adding ethanol as a mock
treatment reduced the growth 20% after 48 h of incubation under
favorable conditions (Fig. 1A). However, when ABA was added to cultures
grown on TAP medium containing 125 mM NaCl, the growth increased 30%,
with culture optical density 1.1 ± 0.1 and 1.3 ± 0.1, respectively,
compared to untreated stressed cultures (0.8 ± 0.1), after 48 h (Fig.
1B). Since the alleviation of the negative impact of salinity was
comparable between 50 μM and 500 μM ABA treatments, we used the lower
concentration (50 μM) in the next experiments.
For a deeper investigation, it is critical to determine if the salt
stress caused cellular death rather than cell inhibition, to understand
the mechanisms involved. Using unstressed cultures as a reference,
~ 60% of C. reinhardtii cells treated with salt
survived and formed colonies. In comparison, the percent of cell
vitality increased up to ~90% when ABA was externally
added (Fig. 1C). These results were in coherence with the optical
density values.
The photosynthetic activities of the cultures were monitored by
measuring O2 production at different actinic light
intensities in the P v E curve. C. reinhardtii cells grown under
125 mM NaCl displayed a lower light-saturated rate of photosynthesis
(PE max) of 4.8 µmol. mmol Chl–1 s–1compared to the unstressed control of 9.2 µmol . mmol
Chl–1 s–1 (Fig. 3), indicating
that a salinity-induced decline in the rate of photosynthesis is
directly responsible for the decrease in cell growth. Surprisingly, when
the cultures were incubated with 50 μM ABA the PEmax value increased to 20.1 µmol .mmol Chl–1 s–1 and 12.3 µmol. mmol Chl–1s–1, with and without NaCl, respectively, which is
~ 2‒3 fold higher than the ABA-untreated controls. This
result suggests that ABA does not only protect photosynthesis, but also
enhance its rate.