Carbon stable isotope ratio of phloem sugars in mature pine trees throughout the growing season: comparison of two extraction methods.

The study presents a comparison of two phloem sugar extraction methods. The amount of phloem sugar extracted and the carbon isotope composition (delta(13)C) of the total extracts and of the main phloem compounds separated by high-performance liquid chromatography (sucrose, glucose, fructose and pinitol) are compared. These two phloem sap extraction methods are exudation in distilled water and a new method using centrifugation, which avoids the addition of any solvent. We applied both extraction methods on phloem discs sampled from 38-year-old Pinus pinaster trees in south-western France throughout the period from June 2007 to December 2008 on different time-scales: hourly, daily and monthly. We found that the centrifugation method systematically extracted ca. 50% less compounds from the phloem discs than the exudation method. In addition, the two extraction methods provided similar delta(13)C values of the total extracts, but the values obtained by the exudation method were 0.6 per thousand more negative than those calculated from the mass balance using the individual constituents. Over the growing season, both extraction methods exhibited lower total sugar content and more (13)C-enriched phloem sap in summer compared with winter values. These findings suggest that both extraction methods can be applied to study the carbon isotope composition of phloem sap, and the centrifugation method has the advantage that no solvent has to be added. The exudation method, however, is more appropriate for the quantification of the amounts of phloem sugars.