Simultaneous determination of ?13C and ?18O in CO2 involved in leaf gas-exchange processes by means of isotope ratio infrared spectrometry (IRIS - Delta Raytm)

The analysis of stable isotope ratios of light elements is normally used for various applications in a number of disciplines like geochemistry, physiology, ecology, paleonthology, climatology, criminology, ornithology and others. Pioneering studies by geochemists formerly highlighted peculiarity of carbon isotopes distributions in natural comparts, enclosing plant materials (Nier & Gulbransen, 1939). It is now a long lasting frame that the study of stable isotope fractionations of carbon and oxygen in plants tissues and metabolites provides insights into the photosynthetic metabolism (Wickman, 1952; Bender, 1968; O'Leary, 1981; Farquhar et al., 1982; 1983; 1984; 1989; Evans et al., 1986; Brugnoli and Farquhar, 2000). For instance, perspectives in studing WUE in natural systems are allowed by stable isotope techniques and, particularly, by analysing carbon stable isotope composition (?13C) recorded in tissues of C3 plants. A negative relationship between carbon isotope discrimination (?) and intrinsic water use efficiency has been widely tested (Farquhar et al., 1989; Brugnoli et al., 1997; Brugnoli and Farquhar, in press). The depletion of the heavy isotope 13C in plant tissues with respect to its abundance in the atmospheric CO2, is directly related to the ratio of intercellular to atmospheric CO2 molar fraction (Ci/Ca); this ratio represents the equilibrium between the availability and the requirement of CO2 at the leaf level, that is the set point for gas exchange activity (see Ehleringer, 1993). Since Ci/Ca is negatively related with WUE, a mechanistic negative relationship between ? and WUE conseques. According to this theory, carbon isotope discrimination analysis allows an assimilation weighted estimation of both Ci/Ca and intrinsic WUE integrated over different time scales, depending on which tissues or metabolites are analysed. The analysis of samples representative of the entire dry matter furnishes an evaluation of WUE integrated over the whole plant life. Istantaneous information is given by analysis on line with gas exchange measurements (Evans et al., 1986; von Caemmerer and Evans, 1991), whilst a picture of a few days is associated to the isotopic analysis of newly fixed carbon in metabolites such as leaf soluble sugars or starch (Brugnoli et al., 1988; Lauteri et al., 1993; Scartazza et al., 1998). Due to physical and climatic factors, different water resources are characterized by different isotopic signatures for both 18O/16O and D/H ratios (Craig, 1961; Dansgaard, 1964). Xylem water usually reflects the isotopic compositions of water used by plant species (Dawson, 1993, 1995; Dawson and Ehleringer, 1998; Dawson et al., 2002). Hence, stable isotopes are considered a powerful tool to investigate water relations. Especially, oxygen isotopic composition of xylem water results always in accordance with the water source used by plants and provides fundamental information in tracing the depth of root systems and the functional links between vegetation and different water sources. The broad application of stable isotopes in physiological and ecophysiological studies have led to many new insights on the processes that control primary productivity and efficiency of resource use by plants (Dawson et al., 2002). As already shown, carbon stable isotopes have provided a powerful tool for analysing constraints on photosynthesis and water-use efficiency of C3 plants (reviews by Farquhar et al., 1989; Brugnoli and Farquhar, 2000). More recently, the availability of new analytical techniques has increased the interest in using 18O/16O and D/H ratios both as tracers of the movement of water along the soil-plant-atmosphere continuum (SPAC) and as integrative indicators of microclimatic conditions and physiological processes related