**1. Introduction**

Diatoms represent one of the most widespread and diversified groups of unicellular photosynthetic eukaryotes, widely distributed in all aquatic environments. Due to their relevant abundance in marine ecosystems, they contribute to one-fifth of the photosynthesis carried out on Earth [1] and play a crucial role in biogeochemical cycles [2]. Moreover, diatoms can provide a rich source of bioactive products, including carotenoids, vitamins and polyunsaturated fatty acids [3–5], thus representing promising "biofactories" for biotechnological applications [6,7].

Due to the complex evolutionary history and multiple horizontal gene transfer events from bacteria and viruses [8,9], diatoms possess unique metabolic capabilities, allowing them to adapt to a plethora of ecological niches and to efficiently cope with the environmental forcing variability [10–14]. Among such environmental variables, light is a key factor influencing diatom growth and physiology. Indeed, diatoms are passively transported along the water column, and thus are exposed to very fast changes in light intensity and spectral composition. In a short time frame, the same species can switch from darkness or a very low light intensity environment—with dominance of blue and/or green as well as absence of red wavelength—to very high light intensity characterized by the full range of visible wavelengths (400–700 nm) [14]. Diatoms evolved an efficient ability to adapt to different light conditions by modulating the levels of photosynthetic and photoprotective pigments, as well as of antioxidant compounds, especially carotenoids [15–19]. Among carotenoids, pigments forming the xanthophyll cycle (XC) are responsible for most of the short-term photoprotection, including the non-photochemical quenching of chlorophyll α fluorescence (NPQ) [18,20–22]. High light intensity, or more generally light induced stress, also promotes the synthesis of other antioxidants, such as ascorbic and phenolic acid, as well as the activation of antioxidant enzymes [18,19].

Recently, we have discovered that the coastal centric diatom *Skeletonema marinoi*, grown under moderate light condition, produces micromolar concentrations of ovothiol B [23], hitherto identified only in clams [24].

Ovothiol B belongs to the π-methyl-5-thiohistidines class (ovothiols), considered powerful antioxidants and mainly found in marine invertebrates, proteobacteria and protists, e.g., microalgae [25]. Due to the aromaticity of the imidazole ring, ovothiols possess a highly acidic thiol group (pKa = 1.4) compared to other cellular thiols, such as glutathione (pKa = 8.75) [26–28]. This chemical feature is related to the ability of ovothiols to act as efficient scavengers of radicals and peroxides [29]. Ovothiols have been receiving grea<sup>t</sup> interest for their biological activities in *in vitro* and *in vivo* human model systems. Indeed, they exhibited pleiotropic beneficial properties, revealing antifibrotic activities in a murine model of liver fibrosis [30,31], antiproliferative action in human cancer cell lines [32–34] and anti-inflammatory effects in endothelial cells derived from women affected by gestational diabetes [35]. Ovothiols are synthesized *in vivo* by two key enzymes, the sulfoxide synthase OvoA and the β-lyase OvoB [36,37], in three different forms, A, B and C, differing in the methylation state of the α-amino group. Ovothiol B is a monomethylated form, while ovothiol A lacks this methyl and ovothiol C possesses two methyl groups [25].

Only few studies performed on microalgae have highlighted some connections between ovothiol biosynthesis and light-dependent processes. Indeed, ovothiol was reported to be a redox regulator, controlling the activity of the chloroplast-coupling factor in *Dunaliella salina* [38]. Moreover, different ovoA transcripts were identified in *Euglena gracilis* under dark or sunlight conditions [39].

The present study is intended to explore more systematically the possible role of ovothiol in the light-dependent response of the diatom *S. marinoi* and to understand if this poorly known antioxidant could contribute to defend diatoms from light stress. To this aim, *S. marinoi* cells were exposed to different light regimes, varying in photon flux density (PFD), velocity of intensity increase (sinusoidal *versus* square-wave distribution) and light:dark photoperiod cycle. We tested the hypothesis that changes in light conditions could affect the expression of the gene *ovoA* encoding the key ovothiol biosynthetic enzyme. Moreover, to investigate whether ovothiol biosynthesis may be part of the cellular antioxidant response, we monitored the cellular stress status by measuring the concentration of reactive oxygen species (ROS) and nitric oxide (NO), important messengers of stress response [40–43], as well as the expression of the enzyme responsible for NO biosynthesis, e.g., NO synthase, recently identified in diatoms [44]. We also tested the possibility that light-induced upregulation of *ovoA* gene expression could result in an increased ovothiol production, activating the complete biosynthetic machinery necessary for its biosynthesis. The outcomes of the present work open new perspectives on the possible exploitation of diatoms for a large-scale production of this compound. Indeed, diatoms' biomass

enriched with antioxidant molecules (ovothiol, carotenoids, polyphenols, etc.) may represent an ecofriendly solution for biotechnological purposes, especially using light as a manipulating factor [45].
