1 2.1 Terminology
1.2.3 Carbon isotope discrimination as a marker of phase change
1.2.3.1
Theoretical basis of carbon isotope discrimination in plants
The CO2 in air contains a nwnber of different carbon isotopes. Two non-radioactive isotopes are 12C and l3C, the latter being the less abundant at ca. 1 . 1 % of total carbon. Plants take up and incorporate 1 2C02 preferentially to 1 3C02 as a result of their difference in molecular weight. 12C02 diffuses through plant tissue and reacts in photosynthetic reactions more rapidly than does 1 3COz (Farquhar et al., 1 982).
Therefore, a ratio of DCfllC (R) exists in plant material that is lower than the ratio of the natural abundance of these isotopes.
The value that is of interest in plant studies is the isotope fractionation, which represents the change in isotope content due to the physical, chemical, or biological processes involved in CO2 capture (O'Leary et aI., 1 992). It can be assumed that in most experiments, all plants use CO2 of the same ratio. Therefore, the actual value of R for the ambient CO2 becomes irrelevant for comparisons within experiments. The value ofR in the dry matter ofleaves is predominantly determined by three factors. These are the primary carboxylating enzyme ofthe relevant photosynthetic pathway, the ratio of intercellular to atmospheric C02 partial pressure, and the composition of source CO2, assumed in most studies to be constant at -8 %0 (Farquhar et aI., 1 982;
1 989).
O'Leary et al. ( 1 992) reported that the majority of papers on carbon isotope
discrimination gave values for isotopic composition (oI 3C) relative to the ratio of the two isotopes in the Pee Dee fossil belemnite limestone foonation (denoted RI'DB), and of value 0.0 ] 1 24.
This ratio can be used to determine values of ol3C in a sample by the equation: ODC [RsamplefRI'DW 1 ] x 1 000 ('per mil' (%0), non-dimensional units)
In using the above expression of discrimination against 1 3C in plants, it was assumed that the onC value of the source, the atmospheric CO2, was constant at about -7.8 to
-8.0 %0. However, this value can vary in practice, especially near to a source of C02 created by buming fossil fuels, when (S1 3C can reach -32.5 %0 (Gleason and Kyser,
1 984, quoted by Farquhar et aI., 1 989).
Because I 3C02 is discriminated against, 013C is always negative. To avoid confusion and for consistency of presentation, Farquhar et aI. ( 1 989) proposed discrimination be expressed by the parameter 1\, which is calculated from the difference between the isotope ratio Oa in the source substrate (air) and that in the product (plant mass)
Op,
as follows:where oa equals ca. -0.008 (-8 %0) derived from the l3C/12C ratio of air (Ra= 0.0 1 1 143) and the ratio of PDB (RpDB = 0.0 1 1 237) as follow:
13 C = 0.01 1 1 43/0.0 1 1 237 - I
Because the value ofthe denominator
(l+Op)
is always going to be close to unity in plant material, O'Leary et al. ( 1 992) argued that it can be ignored. Thediscrimination against I 3C resulting from the carbon fixation by the plant from ambient CO2 can therefore be calculated as:
Since in most carbon isotope discrimination studies (Sair is assumed to be constant at - 8%0, the main advantage of using the discrimination parameter 1\ over the carbon isotope composition parameter (SI 3C is the expression of data on a positive scale. Moreover, higher discrimination against DC is reflected in numerically greater positive values of 1\.
However, both 1\ and ol3e continue to be used to express carbon isotope
discrimination in plants. For example, Valentini et al. ( 1 994), Cordell et al. ( 1 998), Damesin et al. ( 1 997) and Waring and Silvester ( 1 994) used ODC values. On the
other hand, Fleck et al. ( 1 996), Donovan and Ehleringer ( 1 994) and Hansen ( 1 996) used the positive values of � to present results in their studies. Therefore, less negative values of carbon isotope composition (8I3C) and lower values of � refer to less discrimination against 13C02, and vice versa. Statistical differences between analyses using these two measures of carbon isotope discrimination are not likely to occur. Calculation of � is affected by the value of the divisor ( 1
+4).
However, this typically ranges in value from ca. 0.98 to 0.97 for C3 plants (8l3C being ca. -20%0). Thus, the relative differences between 813C and � values can reach a maximum of approximately one percent. The subtraction of the value for air813C of -8%0 changes the absolute differences between 8 and �, but has minimal effect on statistical analysis of these data.Detailed reviews of the physical and chemical basis for carbon isotope
discrimination in plants have been published by several authors (e.g. Craig, 1 953; Bender, 1 97 1 ; O'Leary, 1 98 1 ; Farquhar et aI., 1 982; Farquhar et aI., 1 989; O'Leary et aI., 1 992). These show that the majority of discrimination against 1 3C02 in C3 plants is accounted for by carbon fixation by Rubisco, at a value of ca. -20 %0. However, the intercellular partial pressure of CO2 also significantly affects discrimination during diffusion through stomata and membranes (Lin et aI.,
Ehleringer, 1 997). As the C02 taken up progresses through the leaf, the gradient in CO2 concentration and transition into the liquid phase further affects discrimination, although this is less significant than that due to diffusion (O'Leary, 1 98 1 ; Farquhar et aI., 1 982). Theoretical values of discrimination were calculated at -4.4%0 for diffusion of CO2 through the stomatal pore (Craig, 1 953), and at -2.9%0 for diffusion through the boundary layer to the stomata (Farquhar, 1 983).
Carbon isotope discrimination in plant material has been used extensively in studies of photosynthetic pathways (Farquhar et aI., 1 989), and gas exchange and water use at the single leaf level (Robinson et aI., 1 993), and for communities of leaves (Vitousek et aI., 1 990; Donovan and Ehleringer, 1 99 1 , 1 994, 1 998; Gutierrez and Meinzer, 1 994; Stewart et aI., 1 995). Typically, in C3 plants carbon isotope composition (813C) varies between - 1 8 and -35%0 due to environmental or genetically-based influences. In CAM plants, the range of values of 81 3C was
reported to be - 1 3 to -25%0 (Medina and Troughton, 1 974, quoted by O'Leary, 1 98 1 ). C4 plants discriminate less against 1 3C due to leaf anatomical and biochemical factors, with ol 3C ranging from - 1 0 to - 1 2%0 (O'Leary, 1 98 1 ; Farquhar, 1 982).
In C3 plants, the level of discrimination is dependent largely on the ratio of
intercellular to atmospheric partial pressure of CO2 (P/Pa) prevailing in the leaf when carbon is assimilated (O'Leary, 1 98 1 ; Farquhar et aI., 1 982; Gutierrez and Meinzer, 1 994). As leaf conductance decreases, the lower P/Pa and discrimination become (O'Leary, 1 98 1 ; Wang et aI., 1 997). In turn, discrimination is related to the ratio of assimilation (A) to stomatal conductance to water vapour (g), and to intrinsic water use efficiency (WUE) (Farquhar et aI., 1 989). The relationship between
discrimination, A and g was expressed by Geber and Dawson ( 1 990) and Gutierrez and Meinzer ( 1 994) as instantaneous WUE AlE, where E is transpiration, which is positively related to g. Similarly, leaf anatomy and/or morphology can affect
discrimination through their effects on P/Pa and the availability of CO2 at the site of carboxylation (O'Leary, 1 98 1 ; Farquhar et aI., 1 982; 1 989).
1.2.3.2
Factors responsible for variation of carbon isotope
discrimination in leaves
The importance of considering external factors as well as intrinsic factors, such as leaf dimension and crown structure that in turn influence boundary layer
conductance, have been discussed with respect to the interpretation of physiological functions of individual leaves and their ecological significance (Farquhar et aI.,
1 989). A number of authors have addressed the differences in discrimination within a species caused by morphological, anatomical and physiological differences between leaves, or differences between positions in a crown ( Koerner and Diemer,
1 987; Geber and Dawson, 1 990; Vitousek et aI., 1 990; Bostrack, 1 993; Gutierrez and Meinzer, 1 994; Waring and Silvester, 1 994; Wang et aI., 1 997). In general, observed differences in discrimination were attributed to specific
microenvironmental factors, such as temperature, position within the canopy, water and light availability, and salinity (Farquhar ( 1 982; Farquhar et aI., 1 989). Some of
these studies examined differences in discrimination along environmental gradients, e.g. that within the tree crown (Waring and Silvester, 1 994; Wang et aI., 1 997), an elevation gradient associated with leaf thickness in Metrosideros poZvmorpha (Vitousek et aI., 1 990), and a rainfall gradient (Stewart et aI., 1 995).
Vitousek et al. ( 1 990), Geeske et al. ( 1 994) and Cordell et al. ( 1 998) found that pubescent leaves of Metrosideros polymorpha exhibited lower discrimination against 13C02 than did glabrous leaves of the same species. They attributed this difference to effects of altitude and water availability on the ratio of leaf mass to leaf area. Leaf mass/area ratio and leaf pubescence increased with altitude from 70 to 2350 m, pubescence accounting for up to 3 5 % of the leaf mass. Vitousek et al. ( 1 990) concluded that lower discrimination resulted from increased internal resistance to CO2 diffusion in thicker leaves found at higher elevations.
Genetically determined morphological differences, such as size of leaves, internodes, flowers and seeds, were linked to the rate of photosynthesis, leaf conductance, water use efficiency (WUE) and leaf carbon isotope discrimination in the annual plant Polygonum arenas/um (Geber and Dawson, 1 990). Small-leaved families tended to have higher gas exchange, lower long-tenn WUE and higher carbon isotope
discrimination. Discrimination was positively correlated with both assimilation rate and leaf conductance.
Wang et al. ( 1 997) compared the physiology of two morphological types of Populus euphratica leaves, lanceolate (LL) and broad-oval (BOL), which were associated with particular positions within the tree canopy. The BOL leaves had lower stomatal conductance, resulting in lower transpiration rates, lower CO2 concentration in intercellular spaces, and lesser discrimination against DC02.
At the whole canopy level, Gutierrez and Meinzer ( 1 994) examined the effect of seasonal changes in environmental conditions and leaf position within the developing canopy upon discrimination in leaves of coffee (Co/lea) hedgerows. Differences in discrimination between leaves from different positions were ascribed to the effect of shading within the canopy.
Similarly, Waring and Silvester ( 1 994) found that variation in discrimination within the crowns of Pinus radiata trees, which varied by as much as 6%0, could be best explained by the combined effects of shading (about 2%0) and relative branch hydraulic conductivity on stomatal conductance.
Damesin et al. ( 1 997) compared discrimination in leaves of evergreen (Quercus ilex) and deciduous (Q. pubescens) oaks. They also attributed the differences in
discrimination to the effect of environment and differences in leaf mass/area ratio, particularly in the evergreen Q. ilex. Later, Damesin et al. ( 1 998) examined the long term changes in discrimination in developing leaves of the two oak species, and found inter-specific differences were linked to the seasonal plant water potentiaL In a two year comprehensive study of Pin us contorta, Populus tremuloides, Acer negundo and A. grandidentatum. Buchmann et aL ( 1 997) found a positive correlation between leaf area index (LAI) and discrimination in leaves of the deciduous trees, but not in the evergreen species (P. contorta).
An effect of soil water utilisation on carbon isotope discrimination has frequently been reported in the literature. For example, studying the effect of water utilisation among several species of conifer at two altitudes in the Italian Alps ( 1 000 m and
1 500), Valentini et a1. ( 1 994) reported that in the predominantly deep rooting Larix decidua (8L 1C -29.0%0), and in the surface water utilising P. sylvestris (81 3e = - 25.9%0), discrimination was principally affected by water availability.
Apart from other results, Aitken et aL ( 1 995) found that trees of Pseudotsuga
menziesii growing m a COIIDnon garden experiment discriminated more against l3e if grown without water stress, while stressed trees discriminated less and had a higher WUE. The average values for discrimination also appeared to be related to mean annual precipitation. Assessing the relative effects of annual precipitation and soil moisture availability, Stewart et a1. ( 1 995) measured discrimination in the leaves of 348 species from 1 2 plant communities along a 900 km-long rainfall gradient. The authors concluded that the average carbon isotope discrimination signature gave a strong indication of soil moisture availability. They also believed that because of its ability to integrate the effects of long-tenn conditions, carbon isotope discrimination provides a more meaningful measure of soil water availability than rainfall .