Delta 5 isotope ratio mass spectrometer

Samples (40–70 mL) for Particulate Organic Carbon (POC) concentration and stable isotopic composition were processed as described in Remize et al. [33 (link)]. The POC concentrations of all samples were measured using a CE Elantech NC2100 (ThermoScientific, Lakewood, NJ, USA) according to the protocol of the United States Environmental Protection Agency [67 ] with acetanilide (99.9% purity, C₈H₁₉NO CASRN 103-84-4) as a standard. The POC concentrations are given in mmol L⁻¹. The bulk carbon isotopic composition (¹³C-POC) was analyzed by continuous flow on an Elemental Analyzer (EA, Flash 2000; Thermo Scientific, Bremen, Germany) coupled with a Delta V+ isotope ratio mass spectrometer (Thermo Scientific, Bremen, Germany). Calibration was performed with international standards and the in-house standards described in Table 1.

Blood, muscle, insect, and plant samples were freeze-dried for 24 to 48 h, homogenised into a powder, and weighed into tin capsules for stable isotope analysis. Freeze-dried POM samples were removed from filters and placed into tin capsules for stable isotope analysis. Samples were flash combusted using a Costech ECS4010 Elemental Analyzer and analysed for δ¹³C and δ¹⁵N values via a coupled to a Thermo Scientific XP or Delta V Isotope Ratio Mass Spectrometer. Stable isotope ratios are expressed in δ notation in per mil units (‰), according to the following equation:
δX = [(R_sample/R_standard) - 1] x 1000
Where X is ¹³C or ¹⁵N and R is the corresponding ratio ¹³C /¹²C or ¹⁵N /¹⁴N. The R_standard values are referenced to the Vienna PeeDee Belemnite (VPDB) for δ¹³C and atmospheric N₂ for δ¹⁵N.
Raw δ values were normalized on a two-point scale using glutamic acid reference materials with low and high values [i.e., USGS40 (δ¹³C = −26.4‰, δ¹⁵N = −4.5‰) and USGS41 (δ¹³C = 37.6‰, δ¹⁵N = 47.6‰)]. The analytical precision, based on standard deviations of repeated reference materials were 0.1‰and 0.2‰for δ¹³C and δ¹⁵N, respectively.

For each measurement, the amount of 220 μg ± 10% of chemically untreated pollen material was weighed directly into silver capsules using a high-precision scale (Mettler Toledo AX 26 Delta Range). δ¹³C and δ¹⁸O were determined using a DELTA V isotope ratio mass spectrometer (IRMS; Thermo Fisher Scientific^™, Bremen) at the dendrochronological laboratory, section 4.3, GFZ Potsdam, Germany. To exclude potential water contamination from air humidity, all samples were vacuum dried at 100 °C for at least 12 hours in a Thermo Scientific Heraeus VT 6060 P prior to measurement. The pollen material was reduced to CO for simultaneous IRMS analysis of carbon and oxygen isotope ratios in a High Temperature Conversion Elemental Analyzer (TC/EA; 1400 °C; Thermo Fisher Scientific^™, Bremen) coupled to the IRMS. All isotope ratios are expressed relative to VPDB for δ¹³C and VSMOW for δ¹⁸O. Isotope data were compared against international and lab-internal reference material (IAEA-CH3, IAEA-CH6 and IAEA 601 and 602) using two reference standards with widespread isotopic compositions for a single-point normalisation [42 ]. Most of the 809 individual pollen samples were weighed and measured with two or three repetitions. In total, we conducted 2132 measurements of stable isotopes. The pollen-isotope dataset is deposited at Pangea Database (https://doi.org/10.1594/PANGAEA.910977).

A portion of the studied rock sample was initially crushed with a stainless steel mortar and pestle and sieved to collect 1–2 mm chips. These chips were then heated at 60 °C under vacuum to remove surficial water. Approximately 0.23 g of these chips were placed into a hydraulic rock crusher with a continuous He stream similar to that of Potter and Longstaffe (2007)^{94 (link)} and the crusher activated several times until the CH₄ signal approached that of the blank. The gas released by crushing was focused on a Porapak Q filled quartz capillary trap held at liquid nitrogen temperature. Gases were released from the trap by moving it out of the liquid nitrogen and into a 150 °C heating block.
The released gases were separated on a HP 6890 gas chromatograph fitted with an Agilent Poraplot Q column (50 m, 0.32 mm wide bore, 10 μm film) temperature programmed from −30 to 80 °C. The column effluent was fed into an oxidation oven containing NiO, CuO and Pt catalysts where the reduced gases were converted to CO₂. Following the oxidation oven, the gases entered a Thermo Fischer Delta V isotope ratio mass spectrometer (IRMS). Data reduction was performed by comparing an in house CH₄ isotope standard to Indiana University Biogeochemical Laboratory CH₄ standards #1, #2, #5, and #7.

From each plant, an aliquot of the leaves, roots and soil was sampled at the start of the experiment (0 h), after 8 h and at the end of the incubation period (after 24 h). Samples were dried for 24 h at 60 °C and finely ground using a vibrating disc mill (RS200, Retsch, Germany). Stable isotope ratios (¹³C/¹²C) were determined using an Elemental Analyzer (EA) Flash 2000 HT (Thermo Fisher Scientific, Bremen, Germany), which was coupled with a Delta V isotope ratio mass spectrometer (IRMS) via a ConFlo IV interface (Thermo Fisher Scientific, Bremen, Germany). Stable carbon isotope values (δ¹³C) were expressed as per mil (‰) relative to the international standard.δ¹³C values were normalized to the international scale Vienna Pee Dee Belemnite (VPDB) by analyses of the international standards USGS40 and USGS41 (L-glutamic acid) within the sequence [39 (link)]. The precision, defined as the standard deviation (± 1σ) of the laboratory control standard along the run, was smaller than ± 0.1‰

Approximately 50 mg of mature leaf samples at the heading stage of Yugu1 and sistl2 were harvested. The samples were dried off at 80°C for 24 h. Pulverized samples were then disposed in accordance with a method described in a previous study (Cousins et al., 2008 (link)). The carbon isotopes were measured by using a Thermo Fisher Delta V isotope ratio mass spectrometer and calculated from a previously described equation (Stutz et al., 2014 (link)).
To verify how a low CO₂ environment impacts SiSTL2 and photosynthesis-associated genes, 4-week-old plants of the WT and sistl2 were grown in a CO₂ level control box. The CO₂ level was set to 40 ppm with 16 h of light and 8 h of dark at 28°C. To avoid the influence due to the daily fluctuation of the genes, samples are harvested from the same parts of the leaves at the same time in each day (10 o’clock a.m.).