Assessing the rate and timing of leaf wax regeneration in Fraxinus americana using stable hydrogen isotope labeling.

Rationale: Understanding leaf wax regeneration and recycling is crucial for plant physiology and paleoclimate studies. However, our recent isotope labeling experiments on a grass species (Phleum pratense) yielded different conclusions from published data on a tree species (Populus trichocarpa), with the former showing rapid regeneration and the latter little regeneration in mature leaves. It is therefore important to determine if the discrepancies in published results were due to differing dynamics of leaf wax regeneration and/or caveats in experimental methods.

Methods: Leaves from a native New England tree species (Fraxinus americana) were collected at 1 to 3 h intervals over a 2-day experimental period, and, subsequently, the leaf wax δ(2) H isotopic ratios were measured using gas chromatography/isotope ratio mass spectrometry.

Results: It was necessary to irrigate the tree using water with significantly higher δ(2) H values than that used for the grass in order to obtain readily measurable isotopic responses over diurnal cycles. In addition, diurnal leaf wax regeneration in Fraxinus americana was delayed by 1-4 h relative to Phleum pratense, suggesting that the latter produced leaf waxes from more recently photosynthesized substrates.

Conclusions: The isotopic inertia in Fraxinus americana was due to lower leaf wax regeneration rates than in Phleum pratense by one to two orders of magnitude. The difference in the timing of leaf wax biosynthesis might partially account for the observed leaf wax hydrogen isotopic difference between trees and grasses.