Stable isotope composition of organic compounds transported in the phloem of European beech--evaluation of different methods of phloem sap collection and assessment of gradients in carbon isotope composition during leaf-to-stem transport.

The analysis of stable isotope composition (delta13C, delta15N, delta18O) of phloem-transported organic matter is a useful tool for assessing short-term carbon and water balance of trees. A major constraint of the general application of this method to trees at natural field sites is that the collection of phloem sap with the "phloem bleeding" technique is restricted to particular species and plant parts. To overcome this restriction, we compared the contents (amino compounds and sugars) and isotope signatures (delta13C, delta15N, delta18O) of phloem sap directly obtained from incisions in the bark (bleeding technique) with phloem exudates where bark pieces were incubated in aqueous solutions (phloem exudation technique with and without chelating agents [EDTA, polyphosphate] in the initial sampling solution, which prevent blocking of sieve tubes). A comparable spectrum of amino compounds and sugars was detected using the different techniques. O, C, or N compounds in the initial sampling solution originating from the chelating agents always decreased precision of determination of the respective isotopic signatures, as indicated by higher standard deviation, and/or led to a significant difference of mean delta as compared to the phloem bleeding technique. Hence, depending on the element from which the ratio of heavy to light isotope is determined, compounds lacking C, N, and/or O should be used as chelating agents in the exudation solution. In applying the different techniques, delta13C of organic compounds transported in the phloem of the twig (exudation technique with polyphosphate as chelating agent) were compared with those in the phloem of the main stem (phloem bleeding technique) in order to assess possible differences in carbon isotope composition of phloem carbohydrates along the tree axis. In July, organic compounds in the stem phloem were significantly enriched in 13C by > 1.3 per thousand as compared to the twig phloem, whereas this effect was not observed in September. Correlation analysis between delta13C and stomatal conductance (Gs) revealed the gradient from the twigs to the stem observed in July may be attributed to temporal differences rather than to spatial differences in carbon isotope composition of sugars. As various authors have produced conflicting results regarding the enrichment/depletion of 13C in organic compounds in the leaf-to-stem transition, the different techniques presented in this paper can be used to provide further insight into fractionation processes associated with transport of C compounds from leaves to branches and down the main stem.