KEYWORDS
salinity stress, phytoplankton resilience, lipids,
monogalactosyldiacylglycerol, fatty acids, estuaries
1 INTRODUCTION
Estuaries are highly dynamic systems, extremely important in providing a
wealth of scientific information as they are exposed to a variety of
stressors of natural (variable salinity, light attenuation, notable
changes in nutrient availability and temperature) and anthropogenic
origin (pollutants, various commercial activities in coastal areas,
changes in water circulation due to construction, the effects of global
warming, which are more easily felt in coastal areas than in the open
oceans) (Miller et al., 2009; Sheela and Dhinagaran, 2023), They are
also characterized by high productivity (Corell, 1978) and are often
exploited as mariculture sites (Yang et al., 2017). In such
environments, plankton develop mechanisms to acclimate or adapt to rapid
environmental changes through physiological and/or community responses.
Much of the productivity and microbiological diversity of estuaries is
related to phytoplankton and its composition. Studies of phytoplankton
communities in estuaries have shown that phytoplankton communities along
the salinity gradient consist of marine phytoplankton at higher
salinities, freshwater species at low salinity and species that develop
at intermediate salinity (Lancelot and Muylaert, 2011). The osmotic
shock caused by the increase in salinity influences the decline in
abundance of freshwater phytoplankton downstream (Burić et al., 2007).
The phytoplankton community is dependent on the flushing rate in the
estuary (Lionard et al., 2008). Their studies in the Scheldt Estuary
(Belgium) have shown that diatoms dominate the community at low flushing
rates, while chlorophytes from the Scheldt are more important at high
flushing rates. Diatoms have also been reported to develop in the lower
parts of the Krka River Estuary, while the upper estuary due to the
constant salinity changes short-lived nanoflagellates tend to develop
(Burić et al., 2007).
Autotrophic plankton is the most important lipid producer in the seas
(Gašparović et al., 2014). Lipid concentrations in seawater are
relatively low, although they are involved in many important biological
processes (Arts et al., 2001). Their quantity and quality depend on
environmental factors and the stage in the life cycle of the primary
producers (Zhukova and Aizdaicher, 2001). One of the most important
functions of lipids is the formation of membrane lipid bilayers for
cells and organelles. The adaptability and flexibility of the membrane
structure imposed by the nature of environment are only possible with a
broad spectrum of lipid mixtures (Dowhan et al., 2008). Increased
synthesis of glycolipids by phytoplankton has been observed under
conditions of P scarcity, high temperatures and high light intensities
(Gašparović et al. 2013; Novak et al., 2019).
The thylakoid membranes of chloroplasts consist mainly of glycolipids,
monogalactosyldiacylglycerols (MGDG), digalactosyldiacylglycerols and
sulfoquinovosyldiacylglycerols , with a small proportion of
phosphatidyldiacylglycerols (Douce and Joyard, 1990). They are highly
unsaturated (Selstam, 1998). These membranes are crucial for plant cell
metabolism (Douce and Joyard, 1990), reflecting the importance of their
adaptability to environmental changes. The effect of different
stressors, such as changes in temperature and salinity, drought, and
exposure to pollutants, on thylakoid membrane lipid remodelling has
often been studied in higher plants (e.g. Ristic and Cass, 1991;
Stefanov et al., 1995; Zheng et al., 2011; Omoto et al., 2016), but
rarely in autotrophic plankton population. The literature indicates that
different photosynthetic organisms use different strategies to cope with
stress in terms of the quantity and quality of thylakoid membrane
lipids.
Phytoplankton in estuaries are exposed to constant stress due to
salinity changes. To maintain membrane homeostasis, the composition of
membrane lipids is expected to change. The scientific question we tried
to answer is: Does the change in salinity lead to membrane lipid
remodeling in the estuarine phytoplankton to acclimate/adapt to such
changes? Since photosynthesis is one of the most sensitive cellular
processes, we hypothesized that regulating the state of the thylakoid
membrane would contribute to the maintenance of photosynthesis under
osmotic stress and thus to cells
survival. To address this hypothesis, we analyzed the lipid profiles of
particles from the subtropical, eutrophic Wenchang River Estuary and the
temperate, mesotrophic Krka River Estuary. We used thin–layer
chromatography–flame ionization detection (TLC/FID) to investigate the
changes in the composition of lipid classes. To explore the stress
responses, in which fatty acids play an important role, the
redistribution of fatty acids within a single lipid class, was
investigated, using high-performance liquid chromatography/electrospray
ionization tandem mass spectrometry (HPLC/ESI/MS/MS). In addition,
phytoplankton pigments were analyzed to gain insight into the
phytoplankton community responses within the salinity gradients along
the estuaries. This allowed us to propose a mechanism for cell stress
acclimation through thylakoid membrane remodeling.
2 MATERIALS AND METHODS
2.1 Study sites and sample collection
Two estuaries, the Krka River Estuary and the Wenchang River Estuary,
were selected because of their multiple differences, including
temperature and riverine nutrient loading. We only sampled surface water
to avoid other possible influencing factors on lipid synthesis, e.g.
attenuated light at different depths.
The Krka River is a karst river
that forms a 25 km long salt wedge estuary spreading from the Skradinski
Buk waterfalls to the Šibenik Channel (Figure1a). Because of the
physical barrier of the Skradinski Buk, riverine water, rich in
phytoplankton developing at the Visovac lake flows into the estuary by
the waterfall contributing to the estuarine phytoplankton community
(Cetinić et al 2006, Šupraha et al 2016). The main sources of nutrients
in this estuary are the Krka River and numerous submarine groundwater
discharges connected to the karst aquifer. The Krka River is the most
pristine and organic matter–poor river with a dissolved organic carbon
(DOC) of only 0.5 mg L-1 (Louis et al., 2009; Hao et
al, 2021), while the DOC concentration in the Krka River Estuary
averages 1 mg L-1 (Lechtenfeld et al., 2013). The
Wenchang and Wenijao rivers flow into the Wenchang River Estuary (Figure
1b). Input from the rivers, groundwater discharge and aquaculture
wastewater are the main sources of nutrients entering the Wenchang River
Estuary (Liu et al., 2011). The DOC content in the Wenchang River
Estuary reaches values of up to 3.8 mg L-1 (Hao et
al., 2021) and even up to 20 mg L-1 in ponds (Herbeck
et al., 2013), which indicates eutrophic character.