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.