1. Introduction
Aerobic organisms need to deal with reactive oxygen species (ROS) which are harmful to their metabolism since high ROS concentrations can damage cellular machinery ultimately threatening cell survival; simultaneously ROS play also a role as secondary messengers. In all cells, mitochondria, NADPH oxidase (NOX) complexes and the enzyme lipoxygenase are major ROS sources. Photosynthetic eukaryotic cells possess, in addition, the chloroplasts, in which ROS are formed via energy transfer from chlorophyll or via electron transfer. Indeed, ROS intracellular concentration controls the photosystem II (PSII) activity and therefore photosynthesis and defense strategies [
1]. The balance between toxicity, when ROS are in excess, and the signaling action requires cells to finely tune the ROS concentration [
2,
3,
4], thanks to an efficient intracellular network composed by antioxidant molecules and enzymes. Antioxidants include molecules such as ascorbic acid (AsA), carotenoids, glutathione (GSH), tocopherols as well as phenolic compounds. AsA is a strong antioxidant component of the cell plasma [
5]. AsA is also substrate of antioxidant enzymes such as peroxidases and violaxanthin de-epoxidase, thus contributing to dissipate excess energy [
6]. Carotenoids occur in the chloroplast membranes interacting directly where photosynthesis-derived ROS are generated. Besides their role as photosynthetic pigments, carotenoids can efficiently quench peroxides and singlet oxygen thus preventing the formation of ROS [
5,
7,
8,
9]. GSH buffers the redox equilibrium of the cell by undergoing oxidation or reduction reactions, according to the redox potential of the cell. Specifically, GSH can act as an electron donor inactivating free radicals. Moreover, GSH is also a cofactor of several antioxidant enzymes [
10]. Tocopherols are only produced by photoautotrophic taxa, they are lipophilic and can use resonance energy transfer to scavenge singlet oxygen, thus protecting PSII [
11]. Phenolic compounds are present in all plants and derive from the shikimate-phenylpropanoids-flavonoids pathways [
12]. They include a wide range of molecules with several phenol structural units. The most important phenolic compounds are flavonoids, which can donate both electrons or hydrogen atoms directly to ROS [
13]. A wide range of flavonoids are present in photosynthetic microorganisms [
14]. A recent study highlighted the diversity of flavonoids in phytoplankton and found that ferulic acid and apigenin are the dominant flavonoids in both cyanobacteria and eukaryotic microalgae [
14]. In contrast with higher plants, their distribution and functions in microalgae are not fully clear [
14,
15]. Antioxidant enzymes are located in various cell compartments and include catalase (CAT), superoxide dismutase (SOD), and several peroxidases. Ascorbate peroxidase (APX) and glutathione peroxidase (GPX) accept, as substrates, AsA and GSH, respectively, in order to detoxify ROS. Furthermore, three additional enzymes, the monodehydroascorbate reductase (MDHAR), the dehydroascorbate reductase (DHAR), and the glutathione reductase (GR) contribute to regenerate the antioxidant substrates.
With the exception of carotenoids [
16,
17,
18], the knowledge on antioxidant molecules and enzymes from marine microalgae is still scarce. Few studies focused on the concentration and composition of antioxidants in marine microalgae [
17,
19,
20,
21,
22,
23,
24], and even fewer on the mechanisms for antioxidant defense in these microorganisms [
24]. Indeed, both the photoprotective and antioxidant network appeared strongly controlled by light spectral composition and intensity, resulting in a complex regulation system, which allows planktonic diatoms to survive in their highly fluctuating light environment they naturally inhabit [
24].
While the photoprotective mechanisms have been investigated in diatoms [
25,
26,
27,
28,
29], only few studies investigated the role of the antioxidant network as a second defense line able to reduce the light stress [
30,
31]. Being light a crucial ecological axis in ruling the metabolism of photosynthetic organisms, and thus modulating the growth, the objective of this study was to investigate the impact of light intensity, photoperiod, and wave light distribution on the cellular concentrations of antioxidant molecules such as AsA and flavonoids, and total phenolic content. This knowledge can thus to be exploited to improve microalgal culturing with a productivity-driven purpose. Indeed, marine microalgae are gaining increasing attention for ecofriendly production of new antioxidant compounds. The ultimate aim of this study is to clarify the role of microalgal antioxidants in modulating light stress.
4. Discussion
Current results highlighted that
Skeletonema marinoi turned out to be rich in phenolic compounds, which are the most widespread antioxidant substances in photosynthetic organisms [
48,
49]. Phenolic compounds are able to act directly against radical species as well as indirectly via the inhibition of pro-oxidant enzymes such as lipoxygenase or through metal chelation, preventing the occurrence of the Haber–Weiss and the Fenton reactions, which are important sources of radical species [
50]. Although some studies reported that phenolic compounds are the main contributors to microalgal antioxidant capacity [
17,
24,
48,
51], the microalgal phenolic content is studied little [
14,
17,
20]. The most abundant phenolic compounds in phytoplankton are phloroglucinol,
p-coumaric acid as well as flavonoids such as ferulic acid and apigenin [
14]. Among them, few studies explored their modulation in response to environmental forcing changes [
44,
49,
52,
53]. As reported previously [
17,
24], the content of phenolic compounds in microalgae is higher than in macroalgae and many higher plants. Assuming a dry weight per cell equivalent to 55 pg as in
Skeletonema costatum [
54], we estimate an average phenolic content ≈ 5.5 mg GAE g
−1 DW, with values up to 12.7 mg GAE g
−1 DW in some conditions. These values are in the range of previous estimations on the same species object of the present study [
24] and in the higher range of results from different studies [
17,
44,
48,
51]. Yet, another study [
20] reported high phenolic content (8–17.5 mg GAE g
−1 DW) in four microalgae from different taxa:
Nannochloropsis oceanica (Eustigmatophyceae),
Chaetoceros calcitrans (diatom),
Skeletonema costatum (diatom), and
Chroococcus turgidus (cyanophyte).
Among the phenolic compounds family, recent findings demonstrated diatoms’ ability to produce flavonoids [
49], which display relevant antioxidant activity and act as signaling molecules able to up-regulate the defense strategies [
13,
49]. In most of the light conditions tested in our study, flavonoids’ concentration generally shows the same trend observed for the phenolic compounds. Flavonoids are located in different organelles, including chloroplasts where they play a key photoprotective role [
55,
56,
57]; in particular, they can scavenge radical species and stabilize membranes containing non-bilayer lipids, such as monogalactosyldiacylglycerol (MGDG) [
58].
Our study shows that flavonoids are strictly related to ABTS scavenging activity under unnatural light stress, such as continuous (0:24 h, light: dark) and very high light (600 μmol photons m
−2 s
−1; Quad 600), conversely to AsA. This might confirm the powerful capacity of flavonoids to act as defense against stress as photoprotector [
59]. Their concentration ranges from circa 50 to 400 fg quercetin equivalent (QEq) cell
−1, corresponding to ≈ 1 to 8 mg quercetin equivalent (QEq) g
−1 DW. Interestingly, these values correspond to concentrations reported in a wide range of vegetables, fruits or higher plants [
60,
61,
62].
AsA concentration in
S. marinoi is also high, with values spanning from 10 to 300 fg AsA cell
−1 (≈1.8–5.5 mg AsA g
−1 DW) in the range of the values previously reported for the same species [
24], as well as other phytoplankters [
32,
63,
64]. The latter study highlighted the high variability of AsA concentration among different groups and between exponential and stationary growth phases, with concentrations up to 16 mg AsA g
−1 DW. Our results highlight the huge potential of
S. marinoi, the diatom model used in this study, as alternative source of antioxidant molecules. This study also shows the relevance of light driven-modulation on the intracellular concentrations of these molecules.
Current results highlight a substantial infradiel variability in the cellular concentrations of antioxidants. The increase in antioxidants observed at midday confirms the role of light in controlling antioxidant synthesis; antioxidants counteract the detrimental effect of the ROS which are produced as consequence of light exposure, as already observed in higher plants [
65,
66]. In the absence of light or under an extremely low sinusoidal light, infradiel variations of protective or antioxidant responses disappear, highlighting a direct light stimulus control, excluding an internal circadian clock, of these variations. A circadian clock synchronized with predictable daily environmental cyclic variations generally represents an evolutionary adaptation able to increase the fitness of the organism [
67]. Instead, under the highly fluctuating light environment naturally experienced by diatoms, which frequently move along the water column, the presence of a rigid scheme ruling cell physiology could be a disadvantage. A better strategy might consist of promptly modifying the metabolism following the external stimuli, resulting in a great plasticity, which is a known feature attributed to diatoms.
In contrast with what was reported under sinusoidal light distribution, square wave distribution does not induce cyclical infradiel variability. The sinusoidal high light distribution, although slowing microalgal growth, is well tolerated, thanks to the activation and functioning of the antioxidant-photoprotective network. By contrast, square wave distribution with high light intensity strongly affects cell performance impairing the normal functioning of the defense processes network.
Light climate changes experienced by cells induce an uncoupling of the regulative responses (photoprotection vs. AsA, phenol and flavonoid contents) compared to the synergy of these photoresponses observed under pre-acclimation light (Sin 150). Sinusoidal high light exposition leads ABTS scavenging activity to be related to phenolic content as well as Dt and β-car while a non-significant role of flavonoids or AsA content is observed. Parallel responses of Dt and phenolic compounds’ concentrations have been already reported [
24], along with no significant relationship between Dt and NPQ ([
24,
37,
47]; this study) confirming an alternative role of this pigment, which is likely to have an additional antioxidant function. Under sinusoidal light distribution, with either moderate or high intensity, significant contribution of Dt in ROS scavenging activity is detected. Intriguingly, the relationship between Dt and ABTS is always accompanied by the significant correlation between β-car and ABTS (except when cells enter the stationary phase) that might reveal a similar role of these two pigments in ROS scavenging. The discrepancy between NPQ and Dt is related to an earlier NPQ response compared to Dt as observed under Sin 600, with the highest NPQ recorded after 2 h and subsequently decreasing. This uncoupling between NPQ and Dt confirms the role of NPQ as first defense strategy against light-related stress and that of Dt as a less quick ROS quencher [
47].
In Quad 600 the significant role of flavonoids into the ABTS scavenging activity, by contrast to the other phenolic compounds, agree with the fact that flavonoids are known to have strong antioxidant activity [
68,
69,
70], together with a relevant role in photoprotection [
58] that relies on their enhanced concentration in chloroplasts, sites of light-driven ROS production [
71,
72].
The peculiar response of AsA under Quad 600, with a decrease recorded after 10 min of light exposure, might be due to its fast consumption to counteract the oxidative process induced by abrupt and strong high light exposure.
By contrast in lower light square wave distribution (Quad 300) AsA seems to control ABTS scavenging activity since they are both significantly correlated.
Under low light conditions, different bioactive compounds families with respect to the light climate modulate ABTS scavenging activity.
Under prolonged darkness the increased concentration of Dt is induced by the chlororespiration-dependent trans-thylakoid ΔpH [
39,
73,
74], and significantly linked to ABTS scavenging activity. Under very low light conditions (Sin 10), ABTS only significantly relies on phenolic content, as it was also observed—together with Dt—in Sin 600. This very low light intensity does not determine any increase in Dt, probably because of the absence of chlororespiratory pathway development as observed in prolonged darkness.
By contrast, the continuous low light causes a strong impairment of the normal cell functioning inducing high cell mortality. Under this condition, such as under Quad 600 ABTS scavenging activity is only related to flavonoids content.
Not only light distribution and/or intensity, but also culture age changes dramatically the photoresponses of the cells. All the antioxidant molecules as well as Dt increase during cell senescence. The accumulation AsA has been already observed in the senescent diatom
S. marinoi [
64]. The infradiel variations observed during the active growth phase were disrupted during the stationary phase, vouching for the drastic changes to which the cells were subjected [
75]. Conversely to exponentially grown cells, NPQ remains high at midday together with the antioxidant capacity and molecule concentration. The integrated defense strategy development suggests the high level of ROS produced in senescent cultures. In higher plants, the early event of cell senescence is the inactivation of the enzyme Rubisco [
76,
77] not paralleled by a loss of the thylakoid proteins, which happens at a later time [
77]. Therefore, the potential exposure to increasing light induces the development of the first defense mechanism represented by NPQ and, subsequently, the antioxidant network is involved in the scavenging of the ROS, which are produced by the accumulation of electrons from the photosynthetic process.