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).
4 DISCUSSION
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.
5 CONCLUSIONS
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.