Delta 5

Plants were grown under sterile and long-day conditions for 19 days on N-free MS medium, 0.5 mM L-Gln, 1% (w/v) agar and 0.5% (w/v) sucrose, buffered to pH 5.8 using 7.7 mM MES. The seedlings were dried at 60 °C and subsequently weighed. A minimum of four replicates were used for biomass determination. Afterwards, seedlings were homogenized for determination of total N content. Partially, samples were pooled to obtain a minimum of three biological replicates. The analysis was performed using an Elemental Analyzer—Isotope Ratio Mass Spectrometer (EA-IRMS) (EA: Flash EA 2000, IRMS: Delta V, both from Thermo Fisher Scientific) (Werner et al. 1999 (link)). Data presented are mean values ± SE (P < 0.05, one-way ANOVA with an additional Dunnett’s test, n = 3). Black stars indicate the comparison between lht1, aap5 and lht1 aap5 with the control Col-0, as their genetical background is Col-0. T1:3 and T4:4 were compared with lht1 aap5, as their genetical background is lht1 aap5 (gray stars).

Fluid samples for subsequent laboratory analysis were collected from the depressurized line (Fig. 1b). To determine the carbon isotope composition of the dissolved inorganic carbon, 1 to 2 ml of water were injected immediately after sampling in 12 ml septum capped borosilicate vials containing 150  μl of 85% phosphoric acid and previously flushed with high purity He to convert the dissolved inorganic carbon completely to CO₂. The generated CO₂ was analyzed on a Gasbench II coupled to a Delta V mass spectrometer (Thermo Fisher Scientific, Bremen). Solutions of Na-bicarbonate of known carbon isotope composition were used for standardization.
Three samples for stable isotope analysis were taken and analyzed per sampling date and their mean and standard deviation is used as value and error, respectively.
The ionic concentrations were measured with a Dionex DX-120 Ion Chromatograph (Thermo Fischer). For the anions, the concentrations in fluoride, chloride, nitrite, bromide, nitrate, phosphate and sulphate were quantified. Concentrations in lithium, sodium, ammonium, potassium, magnesium, calcium as well as strontium was measured for the cations.

The δ¹³C values of finely ground homogenous powder from the pooled dried leaves of whole plants were measured in an isotope ratio mass spectrometer (Delta V; Thermo Fisher Scientific) in the Stable Isotope Laboratory of the Smithsonian Tropical Research Institute. The abundance of ¹³C in each sample was calculated relative to the abundance of ¹³C in standard CO₂ that had been calibrated against Pee Dee belemnite (Belemnitella americana). Relative abundance was determined using the relationship:
The δ¹³C values of two C₄ plant species, Saccharum spontaneum and Portulaca oleracea, grown in each glasshouse and outside in the open air were used to correct for differences in the δ¹³C value of the source CO₂ (Cernusak et al., 2011 (link)). The CO₂ purchased for CO₂ enrichment was from a natural CO₂ spring and a correction of 2‰ was applied.

Diploptene δ¹³C values were determined using gas chromatography–isotope ratio mass spectrometry (GC–IRMS) with a ThermoFisher Delta V. A fused silica capillary column (60 m × 0.32 mm) coated with CP-Sil-5 (film thickness 0.10 μm) was used with the same GC temperature programme as above. The δ¹³C values are reported relative to the Vienna Pee Dee Belemnite (VPDB) standard, and the analytical error, determined by using co-injected standards, is ±0.5‰.

The δ¹³C analysis of carbonates was performed using a gas stable isotope mass spectrometer (Thermo Scientific Delta V – GasBench II). Before measurement, plant residues and microbiological shells were picked out under a magnifying glass. After that, each sample was sieved less than 200 mesh. The treated sediment samples were loaded into glass vials and placed in an aluminum heating block of GasBench II. Then the samples (∼0.1 g) were purged with helium for 7 min followed by a treatment with supersaturated phosphoric acid (98%) for 2 h; the reaction temperature was 72°C. Ultimately the CO₂ generated by the reaction was carried into the Delta V for detection using helium as the carrier gas. NBS-18 was used as the international reference material, while GBW04405 was used as the national reference material. The δ values are reported in per mil relative to the Vienna Pee Dee Belemnite, with an error less than 0.1‰.

The δ¹³C value of sugar fraction samples
was measured using an elemental analyzer (Flash EA 1112, Thermo Scientific,
Bremen, Germany), coupled with IRMS (DELTA V, Thermo Scientific) through
a ConFlo IV dilutor device (Thermo Finningan, Bremen, Germany).