Determination of plant carbon and isotopes

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).