*4.2. Cell Density*

Cell density was monitored during the experiments to obtain growth curves. Briefly, 2 mL subsamples from each flask were collected and fixed with Lugol's iodine solution (1.5% *v*/*v*). One milliliter of this solution was used to fill a Sedgewick Rafter counting cell chamber and cell counts were performed by using a Zeiss Axioskop 2 Plus light microscope (Carl Zeiss, Göttingen, Germany).

### *4.3. RNA Extraction, Reverse Transcription and Best Reference Genes Assessment*

Diatom samples (50 mL) were collected at di fferent times from dawn by centrifugation at 3200 rcf for 30 min at 4 ◦C. The final pellets were resuspended in 800 μL TRIZOL, frozen in liquid nitrogen and stored at −80 ◦C until use. The total RNA was extracted according to Barra et al., 2013 [41] and subjected to DNase treatment using DNase I recombinant, RNase-free (Roche, Basel, Switzerland), according to the manufacturer's protocol. RNeasy MinElute Cleanup Kit (Qiagen, Venlo, The Netherlands) was used to purify and concentrate the total RNA, finally eluted in 20 μL RNase-free water. RNA samples were quantified by assessing the absorbance at 260 nm (ND-1000 Spectrophotometer; NanoDrop Technologies, Wilmington, DE, USA.) and then checked for integrity by agarose gel electrophoresis. Possible gDNA contamination was checked by PCR on RNA samples and agarose gel electrophoresis. From each RNA sample 1 μg was retrotranscribed in complementary DNA (cDNA) using the iScriptTM cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) and the T100 Thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA), following the manufacturer's instructions. In order to analyze the expression levels of target genes, five putative reference genes were analyzed by RT-qPCR to find the most stable genes in our conditions. The selected genes were histone 4 (*H4*), α- tubulin (*TUB A*), elongation factor 1 α (*EF1*α), glyceraldehyde-3-phosphate dehydrogenase (*GAPDH*) and actin (*ACT*). For the amplification of putative reference genes, specific primers reported in the literature were used [72]. The best reference gene for each condition was identified by crossing results obtained with two di fferent algorithms: BestKeeper [73] and NormFinder [74]. In particular, *H4* was used as a reference gene in the following conditions: Sin600, Square300 and darkness; *GADPH* was used for Square10; *ACT* for Square600 and *TUB A* for Sin10 and Sin150.

### *4.4. Reverse Transcription-Quantitative PCR (RT-qPCR) Experiments*

For *ovoA*, the expression levels of the transcript SmOvoA\_2388 containing all the canonical domains of metazoan OvoAs [23] were examined. For *nos* genes, the expression of two di fferent transcripts previously reported in *S. marinoi* [44] was analyzed. We named the two *nos* genes: *nos1* and *nos2* (for details see Materials and Methods 4.8.). Specific primers were designed using Primer3 program V. 4.1.0 (primer3.ut.ee) considering the putative sequences reported in the *S. marinoi* transcriptome (MMETSP1039, http://datacommons.cyverse.org/browse/iplant/home/shared/imicrobe/camera/camera\_ mmetsp\_ncgr). RT-qPCR was performed in a MicroAmp Optical 384-Well reaction plate (Applied Biosystems, Foster City, CA, USA) with optical adhesive covers in a Viia7 Real Time PCR System (Applied Biosystem, Foster City, CA, USA). The oligos used to amplify the target genes were reported in Table S2. Serial dilutions of cDNA and the obtained cycle (Ct) mean values were used to generate the standard curves in order to calculate primer reaction e fficiency (E= <sup>10</sup>−1/slope) and correlation factor R2 (Table S2). The RT-qPCR reaction was carried out in 10 μL for each sample, including 5 μL of SYBR Green Master Mix (Roche), 1 μL of cDNA template (1:25 template dilution) and 0.7 pmol/μ<sup>L</sup> of each primer. The procedure used to obtain the RT-qPCR thermal profile was: 95 ◦C for 20 s, 40 cycles of 95 ◦C for 1 s and 60 ◦C for 20 s. The melting curve of each amplicon was revealed by the program from 60 to 95 ◦C, reading every 0.5 ◦C. The gene-specific amplification and the absence of primer-dimers were confirmed by the presence of single peaks for all genes. qPCR was carried out in triplicate (technical replicates) on cDNA, deriving from three independent cultures (biological replicates) and each assay included three no template negative controls for each primer pair.

### *4.5. Nitric Oxide (NO) Determination*

NO levels were measured by monitoring the formation of nitrite, the oxidation product of NO, through the Griess assay [75]. At di fferent times from dawn, diatom samples (50 mL) were collected by centrifugation at 2600 rcf for 15 min at 4 ◦C (Eppendorf 5810 R, Eppendorf AG, Hamburg, Germany). The pellets were washed in phosphate bu ffer (KH2PO4 50 mM pH 7.5, 0.5 M NaCl) and centrifuged again under the same conditions. The final pellets were weighed, frozen in liquid nitrogen and kept at −80 ◦C until use. Samples were homogenized in 1 mL of phosphate buffer, sonicated two times at 30% amplitude for 1 min with a one-minute-break between the two sonication cycles. The samples were centrifuged at 13,000 rcf for 15 min at 4 ◦C and the supernatants were analyzed for nitrite content. Aliquots (300 μL) were incubated at room temperature (25 ◦C) with nitrate reductase (1 U/mL) and the enzyme cofactors: flavin adenine dinucleotide (100 μM) and nicotinamide adenine dinucleotide phosphate hydrogen (0.6 mM). After 2 h, samples were treated for 10 min in the dark with 300 μL of 1% (*w*/*v*) sulphanilamide in 5% H3PO4 and then with 300 μL of 0.1% (*w*/*v*) N-(1-naphthy)-ethylenediamine dihydrochloride for additional 10 min. The absorbance at 540 nm was measured in 1 mL glass cuvettes and the molar concentration of nitrite in the sample was calculated by interpolation from a standard curve generated using known concentrations of sodium nitrite (0–5 μM). Nitrite content in each sample was determined in triplicate (technical replicates) on samples deriving from three independent cultures (biological replicates).

### *4.6. Reactive Oxygen Species (ROS) Determination*

ROS levels were measured in vivo using a fluorescent ROS-sensitive dye, 2,7-dichlorofluorescein diacetate (H2DCF-DA; Sigma-Aldrich, Saint Louis, MO, USA). At different times from dawn, diatom samples (15 mL) were incubated for 30 min in the dark with H2DCF-DA (20 μM final concentration). The cells were then collected by centrifugation, as described above, homogenized in phosphate buffer (0.5 mL), and sonicated as described above. The samples were centrifuged at 13,000 rcf for 15 min at 4 ◦C and the supernatants were analyzed for ROS content. Aliquots (5 μL) of samples were diluted in 100 μL of MilliQ water in a 96 multiwell plate and the fluorescence was measured using excitation and emission wavelengths of 485 and 530 nm, respectively. The molar concentration of ROS in the sample was calculated from a standard curve generated using known concentrations of 2,7-dichlorofluorescein (H2DCF; 0–1 μM). ROS content in each sample was determined in triplicate (technical replicates) on samples deriving from three independent cultures (biological replicates).
