Finnigan delta 5 advantage mass spectrometer

Samples of shoots and fine roots were dried (72 h at 60°C) and finely ground for subsequent analyses of C and ¹³C contents by an elemental analysis-isotope ratio mass spectrometry (EA-IRMS on a EA 1110; CE Instruments, Milan, Italy, coupled to a Finnigan MAT Delta Plus IRMS; Thermo Fisher Scientific), as well as for the determination of plant carbohydrate pools. Sucrose, glucose and fructose were extracted from aliquots of finely ground plant material with deionised water at 85°C for 30 min. After centrifugation, the supernatant was transferred to ion-exchange cartridges (OnGuard II H cation exchange and OnGuard II A anion exchange cartridges; Dionex, Thermo Scientific, Vienna, Austria) to remove ionic components. The resulting neutral fraction was then analysed by HPLC-IRMS (Dionex IC 3000 system, connected by a Finnigan LC IsoLink Interface to a Finnigan Delta V Advantage Mass Spectrometer; all Thermo Fisher Scientific) (Wild et al., 2010 (link)), on a HyperREZ XP Ca²⁺ column (Thermo Electron, Bremen, Germany) at 85°C with 0.5 ml min⁻¹ of deionised water as eluent. The starch pool in the plant material was determined after enzymatic digestion with heat stable α-amylase (Göttlicher et al., 2006 (link); Richter et al., 2009 (link)) and the resulting glucose was measured by elemental analysis-isotope ratio mass spectrometry (EA-IRMS, see above).

Leaf disks 2 cm in diameter were collected at midday during each diurnal and dried to determine specific leaf weight (SLW; g m^-2). Leaf disks 1 cm in diameter were collected at midday during each diurnal to determine chl content, chl a/b ratios, and total carotenoid content using the methods of Lichtenthaler (1987) (link) and Porra et al. (1989) (link). Near the end of the 2013 season during developmental stage R5, 2 cm leaf disks were taken from leaves at the top (uppermost 0.25m), middle (0.25–0.50 m from the top of the canopy), and bottom (0.50–0.75 m from the top of the canopy) of the canopy to determine integrated WUE using isotope analyses (Farquhar and Richards, 1984 (link)). The samples were dried and ground to a powder, after which an elemental analyzer (Elemental Combustion System 4010, Costech Analytical Technologies, Inc., Valencia, CA, USA) in parallel with an isotope ratio mass spectrometry system (Finnigan Delta V Advantage Mass Spectrometer, Thermo Fisher Scientific, Waltham, MA, USA) were used to determine δ¹³C on a per mass basis.