Tracing D2O in xylem tissues
To examine and track the dynamics of water uptake, transport and storage
in trees of the two species, we used a marker technique with
isotopically labelled water similar to that described by
James et al. (2003) and
Meinzer et al. (2006) We injected
deuterium oxide (99.9 atom% D2O, Cambridge Isotope
Laboratories, Inc.) into the xylem transpiration stream after having
drilled four holes at equidistant points (90° apart) around the trunk
circumference at 130 cm height. The dosage was kept constant at ≈0.7 g
D2O per cm of sapwood circumference. This assay was
performed once during a dry season (spring 2013) and once after a winter
rain (winter 2014) to examine the dynamics of water transport and
storage in trees. Deuterium injections in three pine trees per plot of
pure and mixed stands (total = 24 injected trees) (δD = 366‰; i.e. 5.6
times enriched compared to natural abundance of deuterium) occurred in
spring 2013 (March 28 between 11:00 h and 13:30 h). Also, in winter 2014
(February 21 between 10:00 h and 14:00 h), injections of δD = 503‰ (17.1
times enriched compared to natural abundance of deuterium) deuterium was
applied in each of four pine and oak trees per plot in pure and mixed
stands (total = 48 injected trees). The presence of D2O
tracer in stem water was determined by periodic collection of twigs
oriented N, S, W, E in the tree crown. Twigs (3 cm long) were
immediately sealed in borosilicate (VWR®) and then wrapped with Parafilm
to prevent evaporation. Prior to D2O injections stem
samples were collected to establish the baseline for hydrogen isotope
ratios. After the injection of D2O (day 0), twigs (total
720 samples) and wood cores (total 216 samples) were collected at noon
on the following 10 days (for water extraction method and stable isotope
analysis and calculations see below).