4010 elemental analyzer

All chemicals and solvents in this project were purchased from Merck AG and Aldrich Chemical. Thomas-Hoover capillary apparatus was used to determine melting points. Infrared spectra were obtained using a Perkin Elmer Model 1420 spectrometer, and ¹H-NMR spectra were acquired by a Bruker FT-500 MHz instrument (Brucker Biosciences, USA) with TMS as the internal standard. Chloroform-D and DMSO-D₆ were used as solvents. Coupling constant (J) values were measured in hertz (Hz), and spin multiples are presented as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad). A 6410Agilent LCMS triple quadrupole mass spectrometer (LCMS) with an electrospray ionization (ESI) interface was used to perform mass spectral measurements, and there was a Costech 4010 elemental analyzer to perform the C, H, and N elemental analyses. The microanalysis values of C and H were within ± 0.4% of the theoretical values.

Dried leaves were ground into a fine powder using a 2000 Geno/Grinder (Spex Sample Prep) for 10 min at 2000 stroke/min, and 3 mg of leaf tissue was weighed and analysed for mass‐based leaf carbon (C_m) and nitrogen (N_m) concentrations using a Costech 4010 elemental analyzer (Costech Analytical Technologies, Inc). Leaf C:N ratio was calculated from C:N = C_m/N_m. Mass‐based phosphorus content was measured in triploid and tetraploid samples in 2019 with 61 triploids and 25 tetraploids at EF, and 58 triploids and 25 tetraploids at MAFES by using inductively coupled plasma optical emission spectroscopy (ICP‐OES, OPTIMA 3300 DV, Perkin–Elmer) after nitric acid digestion. Nutrient concentrations per unit area were calculated by multiplying mass‐based nutrient concentrations by LMA. Photosynthetic nitrogen and phosphorus use efficiency (A_N and A_P) were calculated as A_m divided by N_m and mass‐based phosphorus concentration (P_m), respectively.

Following photosynthesis measurements at each time point, leaf chlorophyll concentration was determined on the same leaf using a portable chlorophyll meter (SPAD 502; Minolta corporation, Ltd., Osaka, Japan). For each leaf, 6–8 measurements across the lamina were averaged. A strong correlation between SPAD readings and total (a + b) chlorophyll concentration per area are observed in both C₃ and C₄ species (Marenco et al., 2009 (link); Uddling et al., 2007 (link); Yamamoto et al., 2002 (link)), suggesting that SPAD measurements can be an accurate method to estimate chlorophyll content. Using the same leaf, we additionally determined leaf area, length and average width with a portable leaf area meter (LI‐3000C, LICOR Biosciences, Lincoln, NE, USA). To determine leaf dry mass per area (LMA), leaf disks were collected from the portion of the leaf that was enclosed in the LI‐6800 chamber and placed in an oven for at least 72 h at 65°C. LMA was calculated as leaf dry mass divided by leaf area. The oven‐dried leaf disks were ground, and mass‐based C and N concentrations (C_M and N_M) were determined from ~3 mg of fine powder sample using a Costech 4010 elemental analyzer (Costech Analytical Technologies, Inc., Valencia, CA, USA). Leaf N content per unit area (N_A) was obtained by multiplying N_M by LMA. Leaf C:N ratio was calculated as C:N = C_M / N_M.