Nah13co3

To establish the origin of phloem‐located sucrose, ¹³CO₂ feeding studies were performed in control and drought conditions. Microcentrifuge tubes containing 250 mg of NaH¹³CO₃ (99 atom % ¹³C; Sigma‐Aldrich, https://www.sigmaaldrich.com) were attached to pots that were subsequently placed in plastic zip bags (with five plants in a single pot per bag) and additionally sealed with tape. Release of ¹³CO₂ was triggered by the injection of 500 µl of saturated citric acid solution to the tubes. For each combination, samples were kept in this condition for 30 min followed by leaf excision and subsequent phloem sap collection across a time course of 0, 0.5, 1, 2, 3 and 6 h). Fractions of phloem sap were collected by moving detached leaves to fresh tubes containing water and immediately frozen in liquid nitrogen. After lyophilization and derivatization with MSTFA, samples were analysed by GC/MS as described above. The total level of sucrose was calculated by summing the intensities for all fractions, and the ratio of unlabelled to ¹³C‐labelled sucrose was determined (where sucrose molecules incorporating at least a single ¹³C were taken into account).
From this experiment we also calculated the sucrose velocity flow and presented it as the ratio of intensity observed in a particular measurement point to the time that passed from the start of the whole experiment.

The minimal medium with 30 mM NaH¹³CO₃ (Sigma-Aldrich) in a shake flask was used for the isotope labelling experiment. Strains were inoculated with OD₆₀₀ 0.01 and sampled after 72 h. 2 mL medium was transferred immediately into the 15 mL tube with 8 mL pure methanol (precooling in −80 °C fridge) and vortexed around 1 s for quenching samples. Then, the mix was centrifuged at 5000 × g for 3 min at 4 °C. Remove the supernatant and resuspend the precipitate with 3 mL of precooling methanol, 5000 × g centrifuged for 3 min, remove the supernatant, keep on ice. For hydrolysis of cellular components, 6 mL 6 N hydrochloric acid was added to quenched cells and transferred to 10 mL glass digestion tube, cells were hydrolysed for 16–20 h in metal bath at 115 °C and dried in a heating block at 85 °C, then 600 μL of ultrapure water was added, and the sample was centrifuged at the maximum speed for 1 min^{100 (link)}. Before the MS analysis, the sample was filtrated with a 0.22 µm pore size filter membrane. The isotopic labels of metabolites were determined with UPLC-MS/MS (SCIEX TRIPLETOF 6600).

The ¹³C labeling of carbamylated lysine was based on a protocol in the literature (34 (link)). Purified OXA-10 enzyme was dialyzed first overnight against degassed 25 mM sodium acetate (pH 4.5)–0.1 mM EDTA and then overnight against 50 mM sodium phosphate (pH 7.4)–0.1 mM EDTA–1 mM NaH¹³CO₃ (Sigma-Aldrich). The enzyme was then dialyzed overnight against 50 mM sodium phosphate (pH 7.4)–0.1 mM EDTA–10 mM NaH¹³CO₃, aliquoted, and frozen using liquid N₂.

Cell growth was monitored using UV-visible Spectrophotometer SU-2000 (OnLab Instruments). Maltose, glucose, xylose, xylitol, glycerol, acetate and ethanol were quantified by high performance liquid chromatography (Agilent 1200 Series HPLC system) equipped with a refractive index detector (Shimadzu, Japan) and an Bio-Rad Aminex HPX-87H organic acid analysis column (7.8 × 300 mm) which was maintained at 50 °C and used 0.05 mM sulfuric acids as mobile phase. The sample injection volume was 10 μL and the flow rate was 0.6 mL/min. Metabolites was detected by liquid chromatography-mass spectrometry/mass spectrometry system (Agilent 6460 series LC-MS/MS system) with Agilent XDC18 column (5 uM, 150 mm × 4.6 mm)36 (link). Di-n-butylammonium acetate (DBAA) and NaH¹³CO₃ were purchased from Sigma-Aldrich. Methanol was purchased from Fisher Scientific. The mobile phase was the mixture of solution A (water with 5 mM DBAA) and solution B (methanol with 5 mM DBAA) at the gradient shown in Table S6. The flow rate was 0.6 mL/min. The injection volume was 50 μL and the column temperature was 40 °C. The negative ion and selected multiple reactions monitoring (MRM) mode were used for MS detection. All experiments were conducted at least in triplicate, and the error bars in the figures denote the standard deviation from the means of independent experiments.

The rates at which carbon was fixed by the shallow and mesophotic coral colonies under their natural PAR levels were estimated using ¹³C- labelled bicarbonate according to Tremblay et al.^{4 (link)}. To take into account variation in PAR levels during the day, corals were incubated for 5 h, between 10 am and 3 pm, to cover the maximal daily irradiance⁴⁵ (Fig. S1). For each species and depth condition, 10 coral nubbins, from 10 different colonies were placed in individual beakers filled with 200 mL FSW enriched with 0.6 mM NaH¹³CO₃ (98 atom % ¹³C, #372382, Sigma-Aldrich, St-Louis, MO, USA). After this “pulse” period of 5 h, half of the corals were sampled and stored frozen at −20 °C until further analysis (T₀). The other half was transferred into 200 mL of non-enriched FSW for a chase period of 19 h (T₂₄). The determination of %¹³C enrichment, and total carbon content in the symbionts and host compartments were performed with a Delta plus Mass spectrometer coupled to a C/N analyser (Thermo Fischer Scientific, Bremen, Germany). The natural isotopic abundance of each species at each depth was determined from the corals used for the respiration measurements. Detailed calculations are described in Tremblay et al.^{4 (link)}. Data were normalized to ash-free dry weight of the organisms^{27 (link)}.

For SIP incubation, AOA subcultures were additionally supplemented with 2.5 mM NaHCO₃ (¹²C or ¹³C), and pH was adjust to 6.5 as normal cultivation using 1 M HCl. The NaH¹³CO₃ (99 atoms%) was purchased from Sigma-Aldrich (USA) Co. After one cycle of incubation, 10% of the medium was used as inoculation for a new cycle, and the rest were all used for DNA extraction. Two cycles of incubation with two replicates were performed totally. Extracted DNA (~2 μg) was added into CsCl gradients with an initial density of 1.696 g ml⁻¹. Density gradient centrifugation was performed in 4.9 ml OptiSeal polyallomer tubes (Beckman Coulter, USA) in a VTi 90 vertical rotor, subject to centrifugation at 56200 rpm for 24 h at 20 °C58 (link). Centrifuged gradients were fractionated into 24–25 equal volumes (~200 μl) as described in detail by Zhang et al.59 (link). Nucleic acids were precipitated by using PEG 6000, and then dissolved in 30 ml of TE buffer.