Evaluating acetate in the transpiration stream as a
substrate for cell wall O-acetylation
In order to evaluate potential mechanisms involving rapid changes in
cell wall O -acetylation in response to drought stress,
experiments investigating the long-distance transport of doubly13C-labeled13C2-acetate in the transpiration
stream of detached branches and a whole intact tree were carried out. To
evaluate leaf cell wall O -acetylation responses to13C2-acetate in the transpiration
stream, cell wall preparations (AIR) were isolated from canopy leaves,
and saponified with deuterated sodium hydroxide (NaOD) to quantitatively
hydrolyze the esters. The resulting solution was analyzed for acetate
isotopologues including monoisotopic acetate
(12C2-acetate) and acetate with one
(13C-1-acetate, 13C-2-acetate), and
two-13C-atoms
(13C2-acetate), by one-dimensional1H-NMR (Figure 11 ).
Acetate released upon saponification of the AIR (from 10 mg dried AIR/mL
0.4M NaOD) ranged in concentration between 212-333 nmol/mg AIR (dry
wt.), corresponding to 2.12 mM and 3.33 mM. The concentration of acetate
in the method blank was 0.006 mM and, to quantify any additional free
acetate that may have been present, incubations of AIR in only
D2O were also carried out. Free acetate was only
quantifiable in two detached branch leaf samples at (1.2 and 1.4 nmol/mg
AIR (dry wt.), or 0.012 mM and 0.014 mM. In the spectra of the other AIR
D2O incubations, signals from lysine, which were not
observed in the saponified AIR, prevented detection and quantitation of
acetate. In the two cases where it was quantifiable, the highest amount
of free acetate in the AIR amounted to less than 0.7 % of the total
concentration of acetate observed after saponification with 0.4 M NaOD.
The isotopologue distributions were determined from the experimental
spectra (Figure 11 ) by integrating the peak areas corresponding
to each isotopologue and dividing by the sum of the integrated areas of
all acetate peaks. The results are summarized in Table 1 as the
fraction of isotopologue divided by its expected fraction at natural
abundance wherein a value of 1 indicates no change.
In leaf cell AIR, there was an
increase in the 13C2-acetate
isotopologue by a factor of 125 +/- 31 above its expected fraction at
natural abundance along with concomitant decreases in the fractions
corresponding to the remaining isotopologues. For example, no
significant changes or slight decreases were detected in the relative
abundances of mono-labeled 13C-1-acetate and13C-2-acetate isotopologues. An increase in the
fraction of 13C2-acetate isotopologue
by a factor of 48 +/- 7 was also observed in two of the three canopy
leaf samples collected following one week of 10 mM13C2-acetate solution continuously
injected into the xylem of an intact potted tree. Although branch
emissions of 13C2-AA were not observed
during the whole tree 13C2-acetate
xylem injections, leaf and branch level emissions of13C2-AA could be observed in some of
the experiments (e.g. supplementary Figure S8 ), confirming the
delivery of the labeled acetate to the leaves.