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 to water use by plants. Leaf water is generally enriched in the heavier isotopes with respect to xylem water because of transpiration while xylem water reflects the isotopic signature of soil water taken up by plants. The extent of the enrichment of leaf water relative to soil water depends on both atmospheric conditions and stomatal regulation of water loss. Particularly, leaf isotope enrichment is affected by leaf and air vapour pressures, which are dependent on leaf and air temperatures, the fraction of evaporating water to source water and their mixing to compose the bulk leaf water, the isotopic composition of atmospheric water vapour and the stomatal and boundary layer resistances to water vapour diffusion. Farquhar and Lloyd (1993) have modelled the leaf water enrichment during transpiration after Craig and Gordon (1965) model of evaporative enrichment from a free water body (e.g. lake). It should be noted here that the isotope fractionation occurs in a precise pool of the leaf water called evaporating water; this is the fraction of leaf water at the sites of transpiration. In contrast, bulk leaf water is all of the water pools present in the leaf. The enrichment of leaf water that occurs during transpiration above the source (input) water value (?18Oe) equals: ?18Oe = ?* + ?k + (?18Ov - ?k) ea/ei, where ?* is the equilibrium fractionation between liquid and vapour, ?k is the kinetic fractionation factor when vapour diffuses through stomata and leaf boundary layer to the atmosphere, ?18Ov is the isotopic enrichment of atmospheric water vapour relative to source water, ea/ei is the ratio of ambient to intercellular vapour pressures (? relative humidity). The variation in the oxygen isotope compositions of soil, xylem and leaf water and vapour surrounding the leaf has the potential to be a relevant signal of plant-environment interactions and adaptive processes. It is also worth noting that the oxygen isotopic composition of evaporative water is at a certain extent reflected on that of the CO2 molecule. Indeed, the molecule is isotopically labelled when entering the water solution of the mesophyll cell from the intercellular air spaces, a process facilitated by the enzymatic activity of carbonic anhydrase (Cernusak et al., 2004). Important insights on the above mentioned processes are typically gained by coupling a leaf gas-exchange system and an isotope ratio mass spectrometer (IRMS). Air samples ingoing and outgoing the leaf cuvette are respectively collected or diverted to the IRMS. The discrimination occurring during the leaf gas exchange and affecting the 13C and 18O abundances in the CO2 molecules can be then calculated. In recent years, new instruments have been developed, based on absorption of electromagnetic radiation. Such tools allow to measure isotope ratios of different atomic species in real time. Furthermore, their reduced size and weight allow to perform experiments in field conditions. Main objective of this work was to test the reliability of results obtained by coupling an isotope ratio infrared spectrometer (IRIS; Delta RayTM, Thermofisher) with a laboratory gas-exchange open system. To achieve the purpose, leaves of several plant species differing in life form (herbaceous and trees) and photosynthetic metabolism (C3, C4 and CAM) were subjected to a range of controlled environmental conditions in the leaf cuvette by manipulating humidity, temperature and CO2 concentration. Particular attention in C3 plants was spent in estimating the mesophyll conductance to CO2 transfer to the carboxylation sites into the chloroplasts, by adopting the on-line discrimination technique (Evans, 1986).