FIGURE 6 Principal component analysis (PCA). Variables (lines): salinity (S), temperature (T), nutrients (dissolved inorganic nitrogen, DIN and orthophosphate, PO4) and contributions of second monogalactosyldiacylglycerols to total lipids (sMGDG (%)) (a and b) and sMGDG (%), DIN, S, contributions of individual pigments (%) (see Materials and methods for abbreviations) and contributions of individual MGDG fatty acid combinations for the Krka River Estuary (%) (a and c) and the Wenchang River Estuary (b and d).
4DISCUSSION
Phytoplankton assemblages in estuaries are determined by the season and river discharge dynamics (e.g. Chanand and Hamilton, 2001; Cetinić et al., 2006), which also influences succession between marine and freshwater phytoplankton taxa depending on the degree to which seawater intrusion into the estuary is impeded (Chanand and Hamilton, 2001; Zhu et al., 2015). The phytoplankton community in the studied estuaries, the Krka River Estuary and the Wenchang River Estuary, differed both in abundance and in the contribution of the different groups between the estuaries (Figure 3), which is consistent with different environmental conditions (Figure 2). However, a similar response to salinity stress was observed in both estuaries when freshwater phytoplankton met saltwater in terms of MGDG remodeling (Figure 4a).
When the river water reaches the estuary, the freshwater phytoplankton either dies or acclimates to the increased ionic strength caused by mixing with the seawater. The change in ionic strength reflects in the composition of the membrane lipids, as membranes surrounding both the cell and the intracellular organelles, are the first to recognize and respond to the change/s in environmental conditions. The study on the influence of salinity on phospholipids in the same estuaries (Vrana Špoljarić et al., 2020) showed that the fatty acid composition of phospholipids phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylinositol (PI) was similar in both estuaries. This was explained by the importance of these phospholipids for the membrane function(s) in which these phospholipids are involved. In contrast, the fatty acid composition of phospholipids phosphatidylethanolamine (PE), phosphatidic acid (PA) and phosphatidylserine (PS) differed along the salinity gradient and between the two estuaries. This is explained by the adaptability of the plankton to remodel these PL depending on the structure of the plankton community and the environmental conditions.
Photosynthesis is one of the most sensitive cellular processes. It has been shown that the photosynthetic capacity of the freshwater green algaChlamydomonas reinhardtii is suppressed by increased ionic strength (the increase in NaCl concentration) (Husic and Tolbert, 1986). Our results suggest that phytoplankton communities in estuaries have the ability to acquire halotolerance by increasing the MGDG content and its unsaturation (Figures 4 and 5). This led to the conjecture that MGDG and particularly unsaturated MGDGs are crucially involved in the molecular organization and thylakoid membrane function under changing salinity conditions. Osmoregulation in phytoplankton is achieved and stress ameliorated, usually within an hour or two, by regulated ion uptake, by synthesis of osmotically active substances compatible with metabolic processes, by water expulsion via contractile vacuoles or, in species with rigid cell walls, by counterbalancing osmotic pressure with turgor pressure (Rai and Gaur, 2001). In order to respond to stress, sensing and signal transduction are important for the cell survival. Ca2+ is an essential component of signal transduction, which controls a large number of physiological processes (Edel et al., 2017). It is found that the cellular Ca2+ increase is essential for the survival of osmotic shock in the unicellular green alga Chlamydomonas reinhardtii (Bickerton et al., 2016) and the diatom Phaeodactylum tricornutum (Helliwell et al., 2021).
The increase in MGDG unsaturation was observed in the green microalgaeDunaliella tertiolecta exposed to low salinity (Vrana et al., 2022). Although it is generally known that unsaturated fatty acid content decreases with increasing temperature (e.g. Hernando et al., 2022), this was not evident in the much warmer Wenchang River Estuary, where MGDG has a higher unsaturated fatty acid content than in the case of the Krka River Estuary. This could be explained by the phytoplankton ecotypes of the Wenchang River Estuary, which are adapted to high temperatures and also higher DIN content there. We analyzed lipid classes in other seas with freshwater influence, such as the Baltic Sea and the northern Adriatic Sea, and also found elevated sMGDG (%), up to 8% (T. Novak, A. Penezić, pers. comm.).
The data suggest that the main influencing parameter for increased MGDG unsaturation at lowest salinities in estuaries is the increased ionic strength (salinity stress) compared to river waters. The PCA indicates that chlorophytes and prasinophytes, the pigments violaxanthin(the Krka River Estuary) and lutein (the Wenchang River Estuary) are the main groups, probably introduced into the estuaries with the river waters, responsible for increased contributions of sMGDG to cell lipids and for increased MGDG unsaturation in the Krka River Estuary and the Wenchang River Estuary, respectively.
We assume that chlorophytes are mainly responsible for this, which is explained in more detail in the following discussion. Green algae (chlorophytes) are known to have a high MGDG concentration in the thylakoids, e.g., compared to diatoms (Garab et al., 2016). According to our findings on the dominance of chlorophytes at low salinities, D’ors et al. (2016), who studied the short-term effects of low salinity on chlorophytes, a diatom and two dinoflagellates and found that chlorophytes were best able to adapt to low salinity in terms of growth rate and photosynthetic activity. Bharathia et al. (2022) also found that green algae are an important component of the phytoplankton community in the upper low-salinity estuaries, as found in the 26 estuaries along the Indian coast. The chlorophyte D. tertiolecta(Vrana et al., 2022) was also shown to increase the unsaturation of MGDG by lowering salinity from 38 to 3. However, the contribution of other groups, including marine groups, cannot be excluded, as their contribution to increased MGDG unsaturation is probably smaller and therefore not confirmed by the PCA analysis. By increasing the unsaturation of the fatty acids in the membrane lipids,Synechococcus shows a greater tolerance to salt stress in the form of less damage to the photosynthetic apparatus compared to the more saturated fatty acids (Allakhverdiev et al., 2001). Changes in the proportion of MGDG are often observed in plants in response to changing environmental conditions (Harwood, 1998).
The decreasing unsaturation of MGDG fatty acids with increasing salinity could be explained by the disappearance of freshwater chlorophytes when salinity is too high in the lower estuary. Indeed, Zhu et al. (2015) found that chlorophytes in the Wenchang River make the largest contribution to Chl a , while their contribution in the estuary decreases with increasing salinity. We assume that freshwater chlorophytes cannot adapt to too high salinity because the time span for successful adaptation is too short. Long-term adaptation mechanism has been observed in the response of phytoplankton to increased temperature, in which phytoplankton resume their unsaturated fatty acid synthesis over a longer period of time after their initial loss (Jin et al., 2029). The higher MGDG unsaturation in the Wenchang River Estuary than in the Krka River Estuary could be explained by the positive role of the abundance of nitrogen nutrients on unsaturated MGDG fatty acid synthesis (Figures 6a and b).
Thylakoid membranes show remarkable structural flexibility, which plays an important role in various short-term adaptive mechanisms in response to rapidly changing environmental conditions (Garab, 2014). The most abundant lipid (~50%) in the thylakoid membrane is MGDG (Douce and Joyard, 1990). MGDG may have several functions in the cellular response to salinity stress. In response to various abiotic and biotic stressors, marine phytoplankton and cyanobacteria synthesize oxylipins (oxidation products of unsaturated FA), the bioactive metabolites (Mosblech et al., 2009). For example, jasmonic acid (oxylipin) has been shown to play a role in salinity tolerance (Zhao et al., 2014). We hypothesize that polyunsaturated fatty acids from MGDG may be at least partially converted to oxylipins in salinity-stressed cells, which then play a role in phytoplankton adaptation to elevated salinity. In addition, MGDG strongly promote membrane stacking and increase the mechanical stability of the large light-harvesting complex (protein LHCII) located in the thylakoid (Seiwert et al., 2017; 2018). Therefore, we can speculate that lipid homeostasis of the thylakoid membrane of the phytoplankton in the estuary is promoted by an increased degree of MGDG unsaturation during salinity-stress.
The MGDG fatty acids that contributed to increased MGDG unsaturation in the Krka River Estuary (6+6, 5+6, 1+4 and 0+5) and in the Wenchang River Estuary (4+5, 3+4 and 1+3) were highly unsaturated. It is not easy to assign the mentioned combinations of double bonds in MGDG to chlorophytes, as there are almost no published articles providing data on the fatty acid composition of MGDG in chlorophytes. Our study on the green microalga D. tertiolecta (Vrana et al., 2022) has shown that the major fatty acids in MGDG are 18:3/16:4, the proportion of which increases with a decrease in salinity from 38 to 3, and 18:2/16:3, the proportion of which decreases with a decrease in salinity from 38 to 3.
The unsaturation of fatty acids in phytoplankton is crucial for maintaining survival and high growth reproduction rates of many aquatic organisms (Brett and Müler-Navarra, 1997). Since polyunsaturated fatty acids are synthesized exclusively by phytoplankton, the accumulation of more unsaturated MGDG in estuarine phytoplankton described here provides an advantage to organisms that feed on them and thus to higher trophic levels.
5CONCLUSIONS
Although phytoplankton from temperate and subtropical estuaries live in completely different climate and environmental conditions, the mechanism of adaptation to salinity stress is similar in both cases as far as the composition of thylakoid MGDG glycolipids is concerned. Here we demonstrate a mechanism of salinity stress tolerance utilized by phytoplankton in estuaries, namely the accumulation of the more unsaturated MGDG (sMGDG) to control thylakoid membrane function and finally to protect photosynthetic machinery. The unsaturation of MGDG is highest at the lowest salinity. This is likely a response of the river phytoplankton to the initial shock of exposure to salt, i.e. higher ionic strength at the upper estuary. Higher availability of DIN also plays a positive role in the synthesis of more unsaturated fatty acids in MGDG. Overall, the results of our studies have shown that the reorganization of MGDG fatty acids is an important survival strategy of phytoplankton in challenging environments such as estuaries where salinity changes are constant. The results of our research should be incorporated into predictions and modelling of the effects of global warming and the resulting changes to the future ocean existence. This is because the salinity in the seas and oceans is changing: reduced river inflows, e.g. in the Mediterranean Sea, lead to more saline estuaries and coastal seas, while the melting of glaciers leads to a lower salinity in the nearby ocean.