2.3 | Measurements of
δ13Cp
We measured the δ13C of the solutes in the fluid
moving through the phloem (δ13Cp, ‰).
Phloem samples were collected at breast height on 15 tree trunks in each
plot with a cork-corer 9 mm in diameter. The samples were collected on
18 October 2011 and 11 November 2011 and then every 14 days from 26
April to 25 September, 2012. In the field, bark and wood were carefully
removed and a disc, which included the active phloem, was dropped into a
6 mL vial containing 2 mL of exudation solution (15 mM polyphosphate
buffer: sodium hexametaphosphate, Sigma, München, Germany). The solution
was chosen to minimise the blockage of cut phloem cells without adding
carbon to the exudate solution. The exudation lasted for 5 hours
(Gessler, Rennenberg, & Keitel, 2004) and the exudate was then stored
in a freezer until it was freeze-dried. Because the phloem solute
concentration is much higher than in adjacent tissues, the exudate was
dominated by phloem sap. The solutes were redissolved in 150 µL and the
resulting solution was pipetted into a tin capsule and dried at 60 °C
for 12 hours. The samples were then loaded into an elemental analyser
(NA 2500; CE Instruments, Milan, Italy) coupled to an isotope ratio mass
spectrometer (Delta Plus; Finnigan MAT GmbH, Bremen, Germany) for
δ13C analysis. The analysis were performed at the SLU
stable isotope laboratory (SSIL, Umeå, Sweden,
www.slu.se/en/departments/forest-ecology-management/ssil).
Isotopic results were expressed in ‰ relative to VPDB (Vienna Pee Dee
Belemnite).