Salinity and growth effects on dimethylsulfoniopropionate (DMSP) and dimethylsulfoxide (DMSO) cell quotas of Skeletonema costatum, Phaeocystis globosa and Heterocapsa triquetra

Highlights

• DMSOp increase at the end of exponential growth phase for Heterocapsa triquetra and Phaeocystis globosa.

• DMSP is upregulated by increasing salinity for the 3 species.

• Salinity-induced oxidative stress causes DMSP oxidation into DMSO in Phaeocystis globosa.

• DMSP/DMSO decrease in case of oxidative stress (late exponential phase and salinity stress).

Abstract

The effects of growth stage and salinity on dimethylsulfoniopropionate (DMSP) and dimethylsulfoxide (DMSO) cellular content were investigated in laboratory batch cultures of three phytoplankton species (Skeletonema costatum, Phaeocystis globosa and Heterocapsa triquetra). DMSP and DMSO cell quotas of the three microalgae were measured at three salinities (20, 27, 35) and in three growth phases at salinity 35. DMSP and DMSO cell quotas varied along the growth for all species with an increase of DMSP for S. costatum and a decrease of the DMSP to DMSO ratio (DMSP/DMSO) for P. globosa and H. triquetra in late exponential-stationary phase. We hypothesized that the oxidative stress caused by light and/or nutrients limitation induced the oxidation of DMS or DMSP to DMSO. DMSP cell quotas increased with salinity, mostly in S. costatum and H. triquetra, for which DMSP is supposed to be an osmoregulator. In H. triquetra, DMSO cell quotas stayed constant with increasing salinity. DMSO was near detection limits in S. costatum experiments. In P. globosa, DMSP and DMSO concentrations increased at low and high salinity. DMSO showed higher increase at low salinity presumably as the result of a salinity-induced oxidative stress which caused DMSP oxidation into DMSO in hyposaline conditions. We concluded that DMSP acts as an osmoregulator for the three studied species and DMSO acts as an antioxidant for P. globosa at low salinity. In P. globosa and H. triquetra, DMSP/DMSO increase with salinity in response to salinity stress.

Introduction

Salinity can be one of the major limiting factor for growth and productivity of plants and algae depending on their salt-tolerance (Parida and Das, 2005; García et al., 2012). Salinity stress has many physiological effects on the cell such as ion toxicity, damage of photosynthetic apparatus, decreased growth, lower cell volume, increased respiration, disruption of enzyme activity causing shifts in metabolites, oxidative stress and changes in membrane permeability (Kirst, 1990; Sudhir and Murthy, 2004; García et al., 2012; Lyon et al., 2016). Even the most euryhaline phytoplankton species are affected in their chemical composition (protein, lipid and carbohydrate contents) by low or high salinity levels (García et al., 2012).

The immediate effect of salinity changes on plant cells is rapid water fluxes due to osmotic gradients that lead to volume changes and severe disturbance of the metabolism due to changes in the cellular water potential. Cells respond to these immediate effects by processes of osmotic acclimation to maintain a constant cell turgor. It is done firstly by the regulation of internal inorganic ions such as K⁺, Na⁺ and Cl⁻ (“non compatible” osmolytes), and secondly by the regulation of compatible solutes (osmolytes) such as DMSP, proline and glycine betaine (Stefels, 2000) which are characterized by similar structure (Vairavamurthy et al., 1985). DMSP can be accumulated or released from the cell in case of salinity up- or down-shock respectively (Kirst, 1996; Yang et al., 2011; Niki et al., 2007). Compatible osmolytes are mainly used in case of long term salinity upshock because the concentration of ions needed to counterbalance external hyperosmotic potential may be toxic for the cell (Kirst, 1996).

Effects of salinity stress on the induction of oxidative stress has been observed in plants and algae (Jahnke and White, 2003; Parida and Das, 2005; Liu et al., 2007; Tammam et al., 2011). Under environmental stresses including salinity stress, Calvin cycle activity is reduced causing an inhibition of nicotinamide adenine dinucleotide phosphate (NADP⁺) regeneration and a consequent over-reduction of the electron transport chain. Under such conditions, excess electrons are transferred to oxygen generating reactive oxygen species (ROS), responsible of an immediate response of plants and algae (Apel and Hirt, 2004; Dring, 2005; Tammam et al., 2011; Ahmad, 2014). To detoxify ROS, plants (including algae) possess low-molecular weight antioxidants (ascorbate, glutathione, phenolic compounds, tocopherols), antioxidant enzymes (superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT)) and compatible solutes with regulatory roles to alleviate damaging effects (Cavalcanti et al., 2007; Cuin and Shabala, 2007; Sekmen et al., 2007; Tammam et al., 2011). A greater salinity tolerance and resistance to oxidative damage comes from high levels of antioxidants (Parida and Das, 2005; Tammam et al., 2011). Oxidative stress occurs when the accumulation of ROS exceeds the scavenging capacity of the cell (Apel and Hirt, 2004; Kreslavski et al., 2007; Tammam et al., 2011; Ahmad, 2014). In case of a long term stress, ROS cause damages in photosystems through desoxyribonucleic acid (DNA) mutation, protein denaturation, lipid peroxidation, chlorophyll bleaching and loss of membrane integrity (Leshem et al., 2007; Tammam et al., 2011).

The cellular content of dimethylsulfoniopropionate (DMSPp) and dimethylsulfoxide (DMSOp) in marine microalgae is specific and its regulation linked to their physiological functions. DMSP and DMSO productions are constitutively species-specific (Keller, 1989; Hatton and Wilson, 2007; Caruana and Malin, 2014) and even strain-specific (Shen et al., 2011). Dinophyceae, Prymnesiophyta and Chrysophyta being the major producers (Keller, 1989; Hatton and Wilson, 2007; Caruana and Malin, 2014); and Chlorophyta and diatoms low producers with a few exceptions (Keller, 1989; Spielmeyer et al., 2011). Several authors have also shown that the intracellular DMSPp to DMSOp ratio (DMSPp/DMSOp) ratio decrease during the growth of A.carterae (dinoflagellate) from log phase to stationary phase as a result of an increase of the DMSO intracellular pool due to limiting conditions inducing oxidative stress (Simó et al., 1998; Hatton and Wilson, 2007). Other authors observed a DMSP and DMS increase until late stationary and senescent stages of growth in different taxa (dinoflagellates, prasinophytes, coccolithophorids and diatoms) with a positive correlation with cell density (Zhuang et al., 2011; Liu et al., 2014).

The DMS(P,O) content of algae are also affected by many abiotic and biotic variables, such as salinity, light, temperature, nutrients and growth phase. Several physiological functions have been attributed to DMSP and its derivatives (DMS, acrylate and DMSO). DMSP functions as an osmoregulator (Vairavamurthy et al., 1985), cryoprotectant (Kirst et al., 1991), methyl donor (Kiene et al., 2000), and potentially as a ballast mechanism in aflagellate phytoplankton (Lavoie et al., 2015, 2016). DMSP and its derivatives are grazing deterrent (Wolfe and Steinke, 1996). Intracellular DMSO is exclusively formed by the oxidation of DMSP (or DMS) by ROS (Foote and Peters, 1971; Amels et al., 1997; Sunda et al., 2002; Spiese, 2010) and only acts as an osmolyte in cold environments; it is a cryo-osmoregulator (Lee et al., 1999). In addition, these molecules take part of an intracellular antioxidant cascade as ROS scavengers (Sunda et al., 2002). Phytoplankton cells may probably use DMSP and derivatives for different functions, they are not necessarily exclusive (Harada and Kiene, 2011). Indeed, multifunctional osmolytes like DMSP are more likely to be selected by phytoplankton species (Welsh, 2000). For example, DMSP may serve a dual role for salinity stress tolerance in sea-ice diatoms; as compatible solute that lesser inhibits enzyme activity than equimolar concentration of NaCl (Gröne and Kirst, 1991) and as part of the antioxidant cascade (Sunda et al., 2002; Deschaseaux et al., 2014) with DMSO, DMS, acrylate and methane-sulfinic acid.

An increase of DMSP and/or DMS production with salinity has been observed for different micro- and macroalgae species belonging to diatoms (including Skeletonema costatum), prymnesiophytes (including Phaeocystis spp.) and dinoflagellates (Vairavamurthy et al., 1985; Dickson and Kirst, 1986; Karsten et al., 1992; Zhang et al., 1999; Zhuang et al., 2011; Yang et al., 2011; Kettles et al., 2014). The increase is exponential for the Haptophyceae Phaeocystis sp. (Stefels, 2000). Field studies have also revealed positive correlations between DMSP or DMS and salinity from the estuarine to the coastal and shelf environments but with differences in the phytoplankton community with mainly diatoms in estuarine waters and Prymnesiophyceae in coastal and shelf waters (Iverson et al., 1989; Sciare et al., 2002). The accumulation or release of DMSP in response to the extreme environmental salinity gradients encountered by sea-ice diatoms is well documented (Lyon et al., 2016). They can accumulate DMSP in higher concentrations than their low DMSP producers temperate counterparts (Keller et al., 1989). Lyon et al. (2016) observed DMSO elevations and low ROS levels above and below 35-salinity controls supporting the dual role of DMSP.

Oxidation of DMSP (or DMS) by ROS produce DMSO, which seems to be exclusively formed this way. No direct biological pathways for DMSO synthesis are known so far (Kinsey et al., 2016). Therefore, concentrations of DMSO increase under oxidative stress with the increasing of ROS production (Sunda et al., 2002; Kinsey et al., 2016) and the intracellular DMSPp/DMSOp ratio is a good indicator of an oxidative stress (Hatton and Wilson, 2007). In some high DMSP producers, mainly dinoflagellates and prymnesiophytes such as Phaeocystis spp. (Keller et al., 1989; Stefels and van Leeuwe, 1998; Steinke et al., 1998; Hatton and Wilson, 2007), cellular DMSP is high enough to better control ROS levels than other antioxidants such as ascorbate and glutathione (Spiese, 2010). DMSO can act, in turn, as an antioxidant against hydroxyl radical but DMSP and DMS are more effective for that reaction because of their higher cellular concentrations with equivalent rate constants (Spiese, 2010; Kinsey et al., 2016). Salinity-induced oxidative stress in hyposaline conditions can lead to the increasing production of antioxidant enzymes and other antioxidant molecules in the macroalgae Ulva prolifera (Luo and Liu, 2011). This effect also exists in hypersaline conditions such as those encountered at low tide in the intertidal zone by macro- and microalgae (Rijstenbil, 2005; Liu and Pang, 2010; Luo and Liu, 2011; Kumar et al., 2011; Pancha et al., 2015).

In this study, we investigate cell quotas of DMSP and DMSO in acclimated batch cultures of Phaeocystis globosa (P.globosa, Prymnesiophyceae), Skeletonema costatum (S. costatum, Coscinodiscophyceae) and Heterocapsa triquetra (H. triquetra, Dinophyceae) in different growth phases and at different salinities. We test the hypothesis for these species that the intracellular DMSPp/DMSOp ratio is an indicator of a salinity stress potentially inducing an oxidative stress (Simó and Vila-Costa, 2006; Hatton and Wilson, 2007).