Culturing Conditions and experimental design

Synechococcus strains XM24 and YX 04-1 were isolated from the coastal region and offshore water of the South China Sea, respectively (Schiksnis et al., 2024; Zheng et al., 2018). Phylogenetic analysis classified them into subclade II clade CB5 and subclade I clade II, respectively.

The cultures were grown in Aquil medium using trace metal clean artificial seawater (Sunda et al., 2005). The experiments were conducted using a matrix of two Fe concentrations (2 nM and 250 nM) and two temperatures (24℃ and 27℃) under a 12:12 dark/light cool-white fluorescent light with an intensity of ~30 μmol quanta m-2 s-1. The cultures were isolated at ~25C, and hence 24℃ and 27℃ were used in the experiments to bracket this ambient temperature. A semi-continuous approach (Yang et al., 2021) was employed to grow the cultures under each treatment condition for at least two months (12 generations or more) prior to measuring physiological responses and collecting RNA samples. The cultures were diluted every other day based on in vivo fluorescence readings. For each dilution, initial and final in vivo fluorescence were measured and growth rates were calculated to ensure cultures remained in exponential phase. Physiological parameters determined included growth rates, chlorophyll a, Fe quotas and carbon fixation rates. RNA samples were flash-frozen and stored in liquid nitrogen until extraction and sequencing.

To compare and contrast the responses of both isolates to warming and Fe limitation individually and in combination, six-treatment comparisons were conducted: 1) -Fe@27oC: Fe-limited vs Fe-replete at 27oC; 2) -Fe@24oC: Fe-limited vs Fe-replete at 24oC; 3) -Fe/warming interaction (-Fe+warming): 27oC Fe-limited vs 24oC Fe-replete; 4) 27oC@+Fe: 27oC Fe-replete vs 24oC Fe-replete; 5) 27C@-Fe: 27oC Fe-limited vs 24oC Fe-limited; 6) +Fe/warming interaction (+Fe+warming): Fe-replete at 27oC vs Fe-limited at 24oC.

Growth rates, Carbon fixation rates and elemental stoichiometry

Cell growth rates were determined by measuring in vivo fluorescence every other day, using the equation μ=ln(N/N0)/(t-t0), where N represented the final cellular in vivo fluorescence at time t, and N0 represented the initial in vivo fluorescence at time t0. Carbon fixation rates were assessed using 14C-labeled bicarbonate. Specifically, 50 mL cultures were extracted from each bottle, incubated with 14C for 3 hours, and then filtered onto GF/F membranes. Subsequently, 14C radioactivity of the filters was measured using a Beckman System 6500 liquid scintillation counter, converted to carbon fixation rates and normalized to particulate organic carbon concentration (Fu et al., 2008). To determine the particulate organic carbon and nitrogen (POC and PON), the cultures were filtered onto pre-combusted glass microfiber filters. The filters were then dried in an oven and analyzed using a Costech Elemental Analyzer that was calibrated with methionine and acetanilide (Fu et al., 2008).

Fe quota measurements

To obtain iron quota results, cell samples were filtered, digested, and analyzed by mass spectrometry following published methods (Hawco et al., 2020; Yang et al., 2021). Briefly, cultures were filtered using acid-washed 0.2 μm Supor polyethersulfone filters and rinsed with an oxalate reagent to eliminate extracellular trace metals (Kustka et al., 2003). The filters were digested with 5 ml of 50% nitric acid (HNO3) at 95°C for five days in 30 mL perfluoroalkoxy vials (Savillex). After removing the filters and drying the samples at 100°C, they were resolubilized in 200 μL of 1:1 concentrated HNO3 and hydrochloric acid (HCl), sealed and heated for approximately 2-3 hours. The samples were dried down again and then resuspended in 5 mL of 0.1 M distilled HNO3 for Fe and P concentration analysis using a Thermo Scientific Element2 inductively coupled plasma mass spectrometry (ICP-MS). The cellular Fe quota was represented by the Fe concentration normalized to phosphate concentration and POC (Kustka et al., 2003).