This study evaluated the growth and the photosynthetic and antioxidant activities of three phytoplanktonic species under nine experimental conditions. Their biomolecular signatures have confirmed the determination based on morphological traits we did previously (44). The growth of the three species studied was differently affected by increasing salinity. The different levels of salt tolerance measured experimentally were in accordance with the distribution of the three species in the salt marshes. Indeed, previous studies (2) have shown that in ponds with a salinity ranges from 42 and 96, Bacillariophyceae, among which Cylindrotheca closterium, dominate the other taxa since they represent more than 60% of the total phytoplankton; Chlorophyceae, represented mainly by D. salina, and Cyanophyceae, including P. versicolor, represented 13 % and 3 %, respectively. In ponds in which salinity was ranged from 190 to 340, Chlorophyceae and Cyanophyceae were relatively abundant (31% and 70%, respectively) (2). Our results confirmed that NaCl 140 g L-1 decreased at different light levels the growth of D. salina and P. versicolor and inhibited the growth of C. closterium. Moreover, the maximum growth rate in D. salina decreased significantly at NaCl 140 g L-1 when light level increased whereas a significant increase was showed with NaCl 40 and 80 g L-1 under E500 and E1000. Salinity and irradiance were the main determining factors in growth rate variation (45). Our results confirm that D. salina and in P. versicolor resist to salt stress and that the diatom C. closterium is salt sensitive as it is observed in saltworks. Net photosynthesis values of the tree species studied are in accordance with growth curves. Against NaCl stress, each species develop different physiological mechanisms more or less efficient. So, D. salina, except other Chlorophyceae, is devoid of rigid polysaccharide cell wall, giving it the ability to adapt to high NaCl concentrations reaching saturation (46). This adaptability is due to plasma membrane plasticity (membrane reservoir) which prevents break and apoptosis of the cells (6). An increase of the degree of fatty acid saturation and hence, a reduction of the membrane fluidity and permeability of Dunaliella sp. isolated from an Antarctic hypersaline lake were observed (47). Intra-cellular Na+ in D. salina remains unchanged up to 2.0 M NaCl (117 g L-1) and thereafter a significant increase was observed (5). Glycine betaine and glycerol contents increase concomitlantly with salt concentration. Calcium acts as a second messenger in the osmoregulation system of this halotolerant species (4). Other species as Chlamydomonas sp. have depigmentented cells following lipid peroxidation of plasma membrane in the presence of 165 g L-1 NaCl (48). In cyanobacteria, the synthesis of osmolytes depends on their ability to tolerate salt (49): species with low salt tolerance (up to 0.7 M NaCl) accumulate sucrose and trehalose, species such as Synechocystis sp. PCC 6803 with moderate salt tolerance (up to 1.8 M NaCl) accumulate glycosylglycerol (50) and species that tolerate high salt concentration (up to 2.7 M NaCl) such as Synechococcus sp. PCC 7418 and Aphanothece halophytica (51) accumulate glycine betaine or betaine-glutamate. The frustule of Cyclotella meneghiniana contained less silica when cells were exposed to increasing salinity (NaCl 4 to 18 mg L-1) (52). Such demineralization process could contribute to NaCl sensitivity of C. closterium.
On the other hand, carotenoids synthesis is stimulated in adapted cells against high irradiation or high salt concentration (34, 53). These pigments dissipated light energy excess via the xanthophyll cycle and they act as filters that protect photosynthetic apparatus from photo-oxidation (34). Moreover, they have antioxidant properties that avoid lipid peroxidation in the photosynthetic apparatus by scavenging singlet oxygen (54, 55). Our results show that high irradiance and high salinity stimulated carotenoids synthesis, especially in D. salina (15.64 ± 1.46 µg 10-6 cells) and in P. versicolor only in the presence of NaCl 80 g L-1 (Table 1). These results are consistent with those of other authors (5, 46). An enhanced carotenoid production in Nostoc muscorum and Phormidium faveolarum when light and salinity increase whereas Chla were measured and phycocyanin content is significantly affected (56).
In microalgae like Chlamydomonas reinhardtii and Dunaliella tertiolecta the light harvesting antenna size is adjusted according to light and salinity (57). Our results showed that the number of photosystems increased significantly in D. salina when light level and NaCl increased, their size remaining unchanged. On the contrary, Chla and Chlc contents tended to decrease in C. closterium leading to a decrease of photosynthesis rate and a lower growth under salt stress. The photosynthetic apparatus adjusts not only the number of photosystems but also its activity according to the light level. (9) showed that the photosynthetic apparatus in D. salina was stimulated by high irradiance (2,000 µmol photons m−2 s−1). We only observed this trend between E300 and E500. On a Chla basis, the net photosynthesis rate in D. salina was about 2 fold than that in P. versicolor and in C. closterium. Antenna truncation in the cyanobacterium Synechocystis sp. strain PCC6803 results in decreased productivity (58). The photosynthetic activity also depends on salt concentration. It appears that the photosynthetic apparatus of D. salina and P. versicolor is more protected against salt stress than in C. closterium. NaCl increasing from 0.5 to 1M (from 29 to 58 g L-1) leads to the decrease of photosynthesis in Spirulina platensis under different light levels (80, 100, 200 and 3,500 µmol photons m-2 s-1) (59, 60). This decrease is a regulation of the photosynthetic activity rather than a real damage (59). Berry et al. (61) suggested that Spirulina platensis adapts itself under high salinity by different mechanisms in thylakoid and cytoplasmic membranes like the regulation of intracellular Na+ concentration via a Na+-ATPase, ATP being generated by respiration and the cyclic electron transport around PSI (62). Na+-ATPases belonging to the family of P-type ATPases have also been found in marine microalgae, Tetraselmis viridis (63), Heterosigma akashiwo (64) and D. maritima (65). Adaptation of Synechocystis to light and salt stress can be associated to the balance between the rate at which damage was induced and the rate of repair of PSII (3). To estimate the state of photosystems, especially PSII, fluorescence of Chla was measured with a modulated fluorometer.
The ratio Fv/Fm has been widely used to assess the extent of the photo-inhibition in microalgae (66). A decrease of Fv/Fm can both be an indicator of PSII damage or a regulation index of electron transport at the PSII level, which leads to heat dissipation of light energy exces. Fv/Fm was almost constant (about 0.7) in both microalgae but it was lower (about 0.55) in P. versicolor (67). This ratio was defined as an index of maximum photochemical efficiency of PSII (68) which depends on both F0 and Fv. In cyanobacteria, phycobiliprotein fluorescence interfers with chlorophyll fluorescence which leads to an increase in F0 value. As a consequence Fv/Fm value decreases (69). Moreover, the saturating flash detaches phycobiliproteins from the photosynthetic apparatus causing fluorescence decrease (70). This reaction is considered as a photo-protective mechanism that protects photosynthetic apparatus against high light levels in cyanobacteria. Aquaporins in the cytoplasmic membrane of Synechocystis sp PCC6803 might be necessary for the repair of PSII and PSI photodamage (71).
When photochemistry is working, the effective quantum yield (ΦPSII) decreased since a part of PSII centres are reduced (or closed). Under salt stress, the reduction of PSII activity in D. maritima leads to an immediate reduction of ΦPSII values (72). Under our experimental conditions, a decrease of ΦPSII in D. salina was measured when irradiance increased and in C. closterium when it was summitted to a high salinity and a high light level. We can notice that PN and PSII did not always have the same trend in the diatom and the cyanobacterium (for example: C. closterium NaCl 80 g L-1, E1000). This absence of positive correlation between these two parameters is due to salt and/or light impacts on the other components of photosynthetic activity. (3) showed that Synechocystis sp. (PCC 6803) cells exposition to light (E500) or salt stress (NaCl 29 g L-1) leaded to partial inactivation of PSII. Moreover, the combination of these two stresses induced a complete PSII inhibition. We observed a similar phenomenom with C. closterium that was unable to survive in the presence of NaCl 140 g L-1 beyond E500. According to Zakhozhii et al. (72), the reduction of PSII activity is due to structural as well as functional disturbances of PSII and electron transport chain in D. maritima. Despite these disruptions, photosynthetic apparatus continued to operate and produce energy required for physiological and bio-chemical processes (5). Bukhov and Carpentier (73) showed that PSI has a crucial role by producing the energy needed for defence mechanisms against stress. Net photosynthesis as ΦPSII decreased in both the microalgae while ΦPSII values increased and net photosynthesis decreased in response to NaCl rising under the three light levels in P. versicolor. In this latter species, PSII could be less affected by NaCl than carbohydrate synthesis. Liska et al. (74) showed that photosynthesis activity was over 2-fold (from 96.8 to 193.6 µM O2 mg-1 Chla h-1) in cells grown in 3 M NaCl than in 0.5 M NaCl in D. salina. According to these authors, this improvement serves the synthesis of organic solutes and osmolytes. So, cyanobacteria like Aphanothece sp., Phormidium or Oscillatoria sp hilled up glycine betaine or betaine glutamate in the presence of NaCl 156 g L-1 (75). The effect of salt stress on PSII in cyanobacteria could be attributed to a direct interaction between salt and PSII via cellular components still unknown (69). Zeng and Vonshak (60) observed that ΦPSIIin Spirulina platensis decreases by 15% after a 25 h-exposition to NaCl 29 g L-1 under 100 µmol m-2 s-1, whereas ΦPSII decreased by about 75% under 200 µmol m-2 s-1 at the same salinity. However, PSII activity regained its original level after an 80 h-exposition showing that, after an initial acclimation phase during which photosynthetic activity was inhibited, a new steady state was established with a recovery of the photosynthetic activity. Our results showed that light level had no significant effect on P. versicolor PSII activity.
NPQ increase acquaints about the dissipation of light excess energy as heat when cells are subjected to stress (26). Our results are in accordance with those of other works (20, 66, 76) who reported that NPQ increases when microalgae are subjected to salt and / or light stress. Under the most stressful condition, NPQ was 24-fold the value measured in D. salina under control condition, 80-fold in C. closterium and 10 fold in P. versicolor. In C. closterium that was the most NaCl sensitive species, NPQ reached the value of 21 in the presence of NaCl 80 g L-1 and E1000. The xanthophyll-dependant NPQ appeared as an efficient photoprotective mechanism in diatoms (24) since the net photosynthesis of C. closterium was stimulated under E1000. Thaipratum et al. (77) precised that NPQ in D. salina is a multi-component process as it was also shown in the diatom Phaeodactylum tricornutum (7).
Reactive oxygen species (ROS) generated by abiotic stresses are scavengered by antioxidative molecules and antioxidative enzyme activities in species having physiological mechanisms to cope with ROS (78). APX, CAT and SOD activities were only detected when the three species were cultivated under E1000, except the APX activity in the cyanobacterium. Nostoc flagelliforme (79) and Cyanobium bacillare (80) are also devoided of APX activity. The salinity rise stimulated ROS production and the three enzyme activities studied. Similar results were obtained in Ulva fasciata after a 12 h exposure to NaCl 90 g L-1 since CAT, Fe-SOD, Mn-SOD and APX activities were stimulated (81). Rijstenbil (82) showed that salt stress (60 PSU) stimulates the production of ROS in C. closterium regardless of light irradiance since SOD and APX activities attained 400 and 35 enzyme units per mg proteins, respectively. These values are clearly higher than those obtained in the strain isolated from the Sfax saltern under the most stressful condition (NaCl 140 g L-1 and E1000). It is probable that strains living in salt marshes have acquired adaptative mechanisms to salt that are more efficient than in marine strains. Among the salt adaptative mechanisms, species living in saltern can have a non-enzymatic antioxidative system particularly active and / or an efficient NaCl exclusion system and / or an efficient photoprotective system. We also noticed enhanced SOD and CAT activities in P. versicolor when the salinity increased whereas antioxidative enzyme activities in D. salina weakly varied when salinity increased. In this latter species, the carotenoid accumulation could play a major role in the antioxidative defence. The raise of SOD, CAT and APX activities in relation with salt concentration was higher in C. closterium than in the two other species. This biochemical response could be related to growth inhibition as in Chlamydomonas reinhardtii and Peridinium gatunense in which a highest antioxidative activity preceds cell death (83), these authors suggest that the high antioxidative activity or a metabolite generated by stress triggers cell death cascade.
Despite the stimulation of antioxidative enzyme activities in C. closterium, this diatom was more affected by NaCl 140 g L-1 than D. salina and P. versicolor. Salt and high irradiance triggered protective mechanisms that were more efficient in D. salina and P. versicolor than in C. closterium. The maintain of photosynthetic activity allowed the production of energy required for physiological and bio-chemical processes necessary for cell survival (eg. osmolytes and carotenoids synthesis, antioxidative enzyme activities). In C. closterium, antioxidative enzyme activities were triggered but this defence mechanism was not sufficient to cope with NaCl and light stress.