Review of phenotypic response of diatoms to salinization with biotechnological relevance

Salinization is one of the main global environmental issues of the Anthropocene with various consequences for aquatic ecosystems. To understand diatom ecology and evolution from this perspective without knowing the impact of salinity on their physiological and molecular mechanisms is unimaginable. For this reason, we collected the existing knowledge about the intracellular and morphological changes of diatoms induced by salinity. The available studies revealed that salt stress can significantly affect, among others, their photosynthetic activities, pigment contents, growth rate, metabolism, and toxin synthesis. Acclimation capability of diatoms is apparent: they can adjust turgor pressure and ion homeostasis and produce compatible solutes for osmoprotection applying a number of biochemical pathways and complementary mechanisms. Morphological changes like shape resistance, post-auxospore formation, and several micro- and nano-sized sometimes species-specific variations can also be explained by the increasing salinity. Furthermore, abnormal forms indicate the extreme and complex effect of salinity and collateral stress factors. Their salinity tolerance threshold is species specific, which can be exploited by biotechnology. According to studies collected for this review, it is obvious that diatoms have various phenotypic responses to salinity; however, knowledge about their molecular background and long-term adaptation of the species are completely missing.


Introduction
Though even the ancient civilizations experienced the phenomenon of salinization of aquatic ecosystems (Jacobsen & Adams, 1958), it had been considered as a local environmental problem up to the end of the twentieth century (e.g., Kirst, 1989).However, and quite recently, it has grown into a global problem.Natural processes can form saline inland surface waters (known as primary salinization, e.g., Rengasamy, 2006;Herbert et al., 2015), besides, anthropogenic activities such as mining, fertilizers, agricultural, and industrial wastewaters and climate change (Bąk et al., 2020;Liu et al., 2020) contribute considerably to enhancing the salinity of freshwaters (secondary salinization).Since many organisms are glycophytes (e.g., Sudhir & Murthy, 2004;Gupta & Huang, 2014), salinity became a strong evolutionary pressure (Latta et al., 2012) and, being one of the most important environmental constraints, it threatens aquatic biota and indirectly ecosystem services provided by them (Cunillera-Montcusí et al., 2022).Despite the recently increasing scientific interest in salinization (Fig. 1), a number of knowledge gaps exist (Cunillera-Montcusí et al., 2022), which would be keys for understanding and predicting its consequences for aquatic ecosystems, society, and economy.
Depending on qualitative and quantitative changes in salts, aquatic organisms can suffer from osmotic shock in different ways.Therefore, these substances can have different toxicities on the biota.Despite, in salinity regulation and legislation conductivity, thresholds are determined only for irrigation and drinking water, while thresholds which can be lethal for aquatic organisms are generally missing from these standards (Kunz et al., 2013;Cañedo-Argüelles et al., 2016;Schuler et al., 2019) or they are inadequate for their protection (Hintz et al., 2022).
In unfavorable circumstances organisms try to survive, regulate, and maintain their physiological integrity.Salt stress, both osmotic and ionic, influences several physiological processes (Sudhir & Murthy, 2004;Parida & Das, 2005;Gupta & Huang, 2014).Phenotypic alterations, which represent considerable costs for species (Coldsnow et al., 2017), are the basis of and stand behind the changes observed at community and higher levels.Biota are able to live within a certain degree of salinity changes, but different types of organisms can cope with salt stress in different ways (Erdmann & Hagemann, 2001;Ma et al., 2010).Physiological acclimation of aquatic organisms to salts has attracted considerable attention from the second half of the twentieth century, and some reviews were published in this topic over this time, including higher plants (Karsten, 2012;Kumar et al., 2014), animals (Kinne, 1966;Larsen et al., 2014;Pourmozaffar et al., 2020), bacteria (da Costa et al., 1998;Ma et al., 2010), algae (Kirst, 1989), and cyanobacteria (Erdmann & Hagemann, 2001).But none dealt specifically with diatoms, they were only mentioned marginally or in combination with other algae without specific details (in Kirst, 1989;Bisson & Kirst, 1995;Hagemann, 2016).However, this taxonomically separated algal group is among the most successful organisms from ecological and evolutionary point of view.Their silica cell wall and their cell functions are clearly different from other eukaryotic algae (Wilhelm et al., 2006), which assume their distinct phenotypic responses (intracellular and morphological changes) to salinization.
Studies of osmotic stress on diatoms date back to the 1970's (Schobert, 1974;Liu & Hellebust, 1976).These researches were sporadic and were focused only on few species.Detailed and comprehensive study about the physiological processes and acclimation strategies of diatoms as a main algal group of aquatic ecosystems have been lacking, despite their huge importance, for instance, in global primary production (Malviya et al., 2016) and nutrient and biogeochemical cycles (Struyf et al., 2009;Tréguer et al., 2018;Seckbach & Gordon, 2019).Furthermore, their salt acclimation properties can be also interesting for other reasons, since using their salt resistance strategy diatoms can open possibilities for their useful and efficient biotechnological applications (Seckbach & Gordon, 2019).
Therefore, this review is conducted to collect and summarize present ecophysiological knowledge on diatoms, the possible effects of salinity on diatoms at cellular and intracellular level, their potential strategies against osmotic pressure, their tolerance to different ion concentrations, and their applicability in the field of biotechnology.(Diatom species mentioned in this review are collected in Supplement 1 with their authors and currently accepted names.)
Depending on the salt concentration, the photosynthetic electron transport activities, respiration rate, and photosynthetic pigment content can change similarly to growth and reproduction rates (see examples in Brand, 1984).Salinity affects diatom protein synthesis, lipid, and fatty acid metabolism leading changes in membrane instability and permeability and furthermore can affect biosilification (detailed in the chapter of "Morphological response of diatoms") and nutrient dynamics reviewed by Saros & Fritz (2000a).Moreover, salinity can induce production of toxins (e.g., domoic acid; Lelong et al., 2012) and reactive oxygen species (ROS), of which the latter can lead to changes in various cellular components, such as proteins, lipids, and fatty acids (e.g., Mallick & Mohn, 2000).Moreover, it can alter the movement and the cell size of diatoms (Mitra et al., 2012).Table 1 summarizes the observed effects of salinity on diatoms.

Restoration of the turgor and cell volume
Diatoms can sustain or adjust turgor pressure in only 5-10 s due to their perforated valves and the elastic girdle band.In contrast to the so-called soft algae, which can change their cell volume as a result of the entry or loss of water (Bisson & Kirst, 1995).et al., 2000) and in higher plants (Weig et al., 1997).

Restoration of ion homeostasis
In general, the unbalanced cellular ion homeostasis can be controlled by uptake/export of ions and changes in the permeability of the cell membrane (Erdmann & Hegemann, 2001).Diatoms are able to maintain their ion homeostasis by active transport of Cl − , Na + , and K + ions out from the cell or into the vacuole through antiporter and transporter systems or by passive transport through membrane protein channels (Boyer, 1976;Fujii et al., 1995;Krell et al., 2008;Cheng et al., 2014;Nakov et al., 2020).Moreover, salt-induced alteration in membrane permeability and fluidity can regulate the restoration process of ion homeostasis, since a rigid membranes can lead to a moderately active Na + /H + antiporter system (Allakhverdiev et al., 1999).However, accumulation of these ions in high amounts (~ 100 mM) is disadvantageous for diatoms, since they may inhibit activities of many enzymatic processes (Kirst, 1989) (Fig. 2).

Osmotic adjustment
Osmotic adjustment is a slow process which lasts for 40-120 min in microalgae during which osmoprotection is induced by accumulation of compatible, osmotically active substances as low molecular weight organic molecules, and stress proteins occur until a new steady state is achieved (Kirst, 1989;Erdmann & Hegemann, 2001).Diatoms can produce compatible osmolytes; up-to-date 19 osmolytes are detected in them (Table 1).However, there might be other possible ones (e.g., dulcitol, trehalose), which have been already known in other algae and cyanobacteria (Erdmann & Hegemann, 2001).These compatible solutes can be arranged on the basis of the energetic budget necessary for the biosynthesis and by the degree of their solubility (Oren, 1999;Erdmann & Hegemann, 2001).Accordingly, the highest salt tolerance can be achieved by accumulation of glycerol.Its energetic cost is only 30 ATP equivalent per molecule and it is the most hydrophilic compound.The lowest tolerance level is represented by accumulating of disaccharides, for example, trehalose and sucrose, for which the energetic cost is 109 ATP equivalent per molecule.The most widely compatible solute used by diatoms is proline needing 62 ATP equivalent per molecule, which could be important in short-term acclimation, but might not be maintained in the long term (Nakov et al., 2020).
Additionally, the synthesis of a single osmoprotectant such as dimethylsulfoniopropionate (DMSP) is too slow for short-term acclimation, but their uptake from the surrounding environment (e.g., after release from algal cells due to loss of membrane integrity; Lyon et al., 2016) is a relatively fast process to adjust osmotic pressure.This extracellular uptake is possible by use of their transport proteins, which is, otherwise, more typical for heterotrophic bacteria than phototrophic organisms (Erdmann & Hegemann, 2001).Moreover, they have a wide variety of transport systems, through which osmolytes may be facilitated by the adjustment process (Tuchman, 1996;Welsh, 2000) of DMSP (Van Bergeijk et al., 2003), proline (Schobert, 1980), glycine betaine (Keller et al., 1999), and amino acids (Admiraal et al., 1984;Nilsson & Sundbäck, 1996) (Fig. 2.).
The process of salt response is related to the expression of various genes (e.g., Munns, 2002;Parida & Das, 2005), since genes are responsible for encoding salt stress proteins or for upregulation/ downregulation of RNA (Parida & Das, 2005).Nevertheless, regarding diatoms, small amount of molecular-based approaches (mRNA sequencing, gene knockdown, and whole-genome shotgun) have been applied to explain their salt tolerance mechanisms (Ambrust et al., 2004;Krell, 2006;Krell et al., 2008, Allen et al., 2011;Bussard et al., 2017;Pinseel et al., 2022).These processes seem to be allied to generally multigenic traits and they are likely to act additively and synergistically as in higher plants (Parida & Das, 2005).Based on a genetic study, besides osmolyte production and ion homeostasis, other processes can also play important roles in the salt tolerance mechanisms of diatoms, such as ROS scavenging and protein degradation (Krell et al., 2008).Thus, the simultaneous metabolic processes seem to be also crucial in salt acclimation of diatoms like in higher plants (Parida & Das, 2005).During the acclimation, diatoms may reduce their transcription costs when they decrease the expressions of some genes and/or they change metabolic pathways to ensure the energy demand of their intracellular ion homeostasis (Haimovich-Dayan et al., 2013;Bussard et al., 2017).Furthermore, under high salt content clear intraspecific sequence differences (in Cylindrotheca closterium) can be observed and this differentiation can speed up the speciation processes (Balzano et al., 2011;Glaser & Karsten, 2020).
In summary, beyond the salinity limits, algal growth may be sacrificed to maintain osmotic adjustments for guaranteeing survival (Kirst, 1989).It is obvious that diatoms can apply many salt tolerance mechanisms.However, there are other possible strategies like role of stress hormones (Stirk et al., 2018), Ca 2+ regulation (Bisson & Kirst, 1995), water channels (Allakhverdiev et al., 2000), and supplying extra energy by alternative pathways in photosynthesis (Satoh et al., 1983, Erdmann & Hegemann, 2001), which are known for other algae but still not for diatoms.Furthermore, since other physical and chemical parameters, such as nutrient enrichment (Saros & Fritz, 2000a), temperature (Lengyel et al., 2020), or different ionic compositions (Ziemann, 1967), may expand the tolerance range and their combined investigation has been an urgent need.

Morphological response of diatoms
Diatom species can adapt well to constraints, like salinity (Leterme et al., 2010), which induces several phenotypic changes of diatoms.This variation of morphological features explained by salinity was demonstrated already at the beginning of the twentieth century by Richter (1909) for Nitzschia putrida.The phenotypic changes of diatoms can be conspicuous in light microscopy or can be discovered only on nanoscales.In some cases, diatoms maintain their normal outline and in other cases they change it completely.This morphological plasticity is determined by the gene pool diversity (Cox, 2006) and can explain their ecological success in different, sometimes extreme environments (Leterme et al., 2013).Vice versa, these altered morphological features can indicate the changes of environmental drivers (Trobajo et al., 2004).

Shape resistance
Specific gravity of silica cell walls (2.4 g cm −3 ) is more than twice as high as that of the protoplasm.Therefore, diatoms are too heavy to remain in suspension in waterbody without easily adjustable mechanisms.For minimizing sedimentary loss of species the following evolutionary mechanisms are available: to decrease size, to decrease specific gravity, and to increase form resistance (Naselli-Flores et al., 2021).
From the point of view of shape resistance, presence of Chaetoceros, a primarily marine genus, with only few species adapted to inhabit continental saline waters, is especially interesting.Its species exploit almost all evolutionary mechanisms to remain entrained in the euphotic layers.Cells are small, weakly silicified, and contain lipids as storage materials (Miller et al., 2014) (Fig. 3.) making the cells relatively light compared to other diatoms.Additionally, cells often form chains and/or long spines on the valve, which increases form resistance (Padisák et al., 2003) (Fig. 3.).These features are largely shared with species of Thalassiosira and Acanthoceras.Because of their extended area and relatively stable environment, oceans represent diversity hot spots for diatoms (Potapova, 2011).Therefore, the above-mentioned diatoms are, presumably, did not adapted to increased salinization of freshwaters, but "backward."They evolved under "average" marine conditions and progressively inhabited hyposaline regions, finally entering into inland waters.This required not only adaptation to relatively low salinity but also to the different ionic compositions of these habitats.Why do not we have more marine species in continental saline lakes?
The answer is that waters with intermediate salt content are intermittent and isolated in space and time; therefore, they constitute migration barriers through both directions (Potapova, 2011).The separated evolution of freshwater and marine diatoms probably lay in their physiological thresholds (Potapova, 2011), which is a task to explore using laboratory experiments and ecophysiological tools.

Polymorphism
Salinity-caused osmotic pressure can also influence the chain morphology of diatoms.The distance between two adjacent cells of Skeletonema subsalsum and S. potamos Hasle (Fig. 4c) and the average cell number in a chain can increase with increasing salinity (S. subsalsum).At high salinity (35 psu) diatom species can enlarge their cell size resulting in shorter chains (Fig. 4c); moreover, the number of the chloroplasts in these cells can also increase (Hasle & Evensen, 1975;Paasche et al., 1975;Sarno et al., 2007;Torgan et al., 2009;Balzano et al., 2011;Falasco et al., 2021).
Salinity may have a direct effect on cell morphogenesis (Roubeix & Lancelot, 2008) by affecting the thickness of the silica wall.Namely, the external ionic strength affect the uptake of silicic acid and other ions and their ratio determine the silica polymerization.Under high salinities the aggregation of small silica particles inside the silica deposition vesicle is less expressed, resulting in thicker but more hydrated biosilica (Vrieling et al., 2007).In case of, e.g., Cyclotella meneghiniana and Thalassiosira pseudonana (Olsen & Paasche, 1986), these thin valves were observed with poorly developed spines and costae and sometimes missing silica granules along the valve mantle (Cyclotella meneghiniana) (Tuchman et al., 1984).
Complete cell size reduction (Thalassiosira pseudonana and T. weissflogii) (Fig. 4c) or only the height of the valve is demoted (Cyclotella meneghiniana) at higher salinity levels (Hildebrand et al. 2006;García et al., 2012).Height reduction is driven by turgor pressure during the interphase before the division.At high salinity, freshwater diatoms may not be able to produce so high intracellular osmolarity to reach the similar turgor pressure as at low salinity levels (Roubeix & Lancelot, 2008), which results in changes of size.Furthermore, smaller than average cell size of Stephanodiscus minutulus, Brachysira vitrea, Asterionella formosa, Achnanthes minutissima, and Tabellaria flocculosa is also characteristic at enhanced heavy metal concentration (Lynn et al., 2000;Cattaneo et al., 2004;Su et al., 2018).
Although these external morphological changes presumably caused by intracellular changes in response to salinity, studies about the links between intracellular processes and cell wall morphogenesis are rare (as you can see above) just as there are minimal explanations of the gene-level regulation of the valve morphology (Bussard et al., 2016.).However, change in the morphology might be explained with the trouble in uptaking silica (Cattaneo et al., 2004;Vrieling et al., 2007), which is regulated by sulfhydryl (-SH) groups on the cell surface (Lewin, 1954) as they have a high affinity to heavy metals and other toxic compounds.The increasing -SH binders reduce the silica uptake and inhibit ATPs as active sites of the -SH groups (De La Rocha et al., 2000).An another explanation might be that at high salinity level cytoskeletal genes are down-regulated, which cause changes in the gene expressions resulting in modification of the position of the silica deposition vesicle with the consequent modification of valve morphology (Bussard et al., 2016.)To understand these morphological changes in more detail, the extension of knowledge about molecular, physiological processes, and cell cycle regulation is necessary (Leterme et al., 2013).This knowledge would allow us to understand the plastic and genetic component of the salinity tolerance and the potential response on evolutionary time scale along increasing salinity (Castillo et al., 2018).

Salt tolerance of diatoms
Elevated salinity can cause sublethal or even lethal effects for a variety of organisms (e.g., Hart et al., 1991;Hintz & Relyea, 2019) depending on their tolerance ranges.The number of experimental studies analyzing the toxic effect of salinity or its different components on freshwater diatoms is limited.Most of the available studies are focusing on brackish and marine species; however, some of them are common in freshwaters with elevated ion content.The salt tolerance of Nitzschia species is often examined using different endpoints and units of salinity (Clavero et al., 2000;Trobajo et al., 2011;Lengyel et al., 2015Lengyel et al., , 2020;;Bagmet et al., 2017).
The hypothetical salinity threshold limits of diatoms are assumed from 0.2 to 18 g l −1 (Potapova, 2011); however, the above-mentioned studies revealed that this barrier is highly exceeded under natural conditions and is species specific.Further experiments and long-term mesocosm experiments would be needed to identify precisely these thresholds (Cañedo-Argüelles et al., 2016) and the examination of the impacts of complex chemical mixtures ("chemical cocktails") is also urgent (Kaushal et al., 2021) to develop the recent water quality guidelines to protect aquatic ecosystems from secondary salinization (Hintz et al., 2022).

Biotechnology
Diatoms are a widely distributed group of microalgae with capabilities that make them ideal for multiple biotechnological applications.Although most of their biotechnological use is not specifically linked to their salt tolerance (B-Béres et al., 2022), there are some examples for those linked to salinity (Marella et al., 2020a(Marella et al., , 2020b)).One of them is to find a way using diatoms to obtain drinking water from unconventional water supplies as brackish groundwater or reclaimed water (Ikehata et al., 2018), although this ability of diatoms has only recently been used in water reuse and desalination (Ikehata et al., 2017(Ikehata et al., , 2018;;Alsar et al., 2020).It is well known that silica is essential for building the cell wall of diatoms, thus these algae efficiently extract dissolved silica from water.An up-to-date study pointed out that a diatom consortium dominated by Nitzschia, Pseudostaurosira and Halamphora species successfully removed more than 95% of reactive silica from brackish agricultural drainage water within 28 h (Ikehata et al., 2017).In addition, Nitzschia and Pseudostaurosira species were effectively applied in reverse osmosis methods, in which 95% of concentrate aqueous silica (78 mgl −1 ) was removed under suboptimal conditions within 72 h (Ikehata et al., 2018).Not only diatom cells but also deionized diatomite (Diatomaceous Earth) can be used in desalinization processes as the pretreated diatomite was able to remove even more than 50% of NaCl content of waters (Alsar et al., 2020).
It is also known that saline lakes host diatoms, like Nitzschia palea, which can produce considerable amount of lipids for alternative fuel production (Abdel-Hamid et al., 2013).This production of diatoms are also characteristic during the removal of nutrients from natural and wastewater (Adey et al., 2011(Adey et al., , 2013)), when significant amount of biomass are produced with high percentage of various unsaturated or saturated fatty acids and lipids (20-30% of dry cell weight).These compounds are known to be widely used as raw material of cosmetics, medicines, biofuel precursors, and aquaculture food implementers (Mishra et al., 2017;Marella et al., 2020aMarella et al., , 2020b)).
In the last decades, we witness the significant development of biotechnology.Diatoms seem to be a promising source (Seckbach & Gordon, 2019) as their special morphological features and intracellular processes may provide several unexplored potential for further industrial application especially in connection with salinization (Ishika et al, 2018;Navarro et al., 2021).However, there are many unanswered questions in this area.Research and a deeper understanding of the processes at both genotypic and phenotypic levels in diatoms would further accentuate its applicability in biotechnology (Mishra et al., 2017).

Conclusion
Diatoms have developed a number of salt tolerance mechanisms in order to ensure their success in continental salty environments.However, our knowledge has been limited concerning the intracellular changes and its consequences, similar to understanding the detailed process of biomineralization and the meaning of the morphological variations of these beautiful microscopic creatures.Although salinity tolerances are driven by genes and infraspecific genetic diversity, genomic, transcriptomic, proteomic, and metabolic studies and their combinations are almost completely missing; however, they would be crucial to discover the relevant processes and their energy costs.Saltresponsive genes, salt-sensible mutants, and the gene regulation of the morphological changes induced by salinity have been also sparsely explored.Besides short-term acclimation studies, further, long-term studies would be necessary to reveal the adaptation of diatoms to salinization.This knowledge would be an effective key for understanding the salinity induced processes also at higher (population, community, ecosystem, and above up to biogeochemistry) levels and to manage the ecological and economic consequences of salinization.Furthermore, these results could open the way for more and more biotechnological applications as ecosystem services provided by diatoms.

Fig. 2
Fig. 2 Schematic figure of intracellular processes in diatoms induced by salinity

Fig. 3
Fig. 3 Shape resistance of diatoms to changing salinity in the phytoplankton: a common forms of planktic diatoms in freshwaters.Diatom cells b with spines, chains, and c lipid materials in saline waters

Table 1
List of the experimentally studied diatom species in relation to salinity, the applied salinity range, their observed physiological responses, acclimation strategies, and