Uptake of CO2 through stomata inevitably leads to loss of water vapour. The relative magnitude of photosynthesis and transpiration depends on several physiological, morphological and environmental factors, such that different species in different growing conditions can have widely different C gain per unit water loss. This quantity, the ratio of the rates of net photosynthesis and transpiration (= water use efficiency, WUE), is of great ecological interest and can be measured on short or long time scales.
On short time scales (= instantaneous), WUE is often measured with infrared gas analysis (see Section 3.10). However, instantaneous WUE changes dramatically for a given leaf over short time spans, e.g. because of variable light intensity and vapour pressure deficit. This makes separating species effects and environmental effects challenging. For comparative studies, we recommend taking into account the precautions outlined in Section 3.10, and to calculate ‘instantaneous intrinsic WUE’, the ratio of net photosynthesis to stomatal conductance. This excludes the effect of differences in vapour pressure on transpiration rates. As CO2 and water vapour share the same stomatal diffusion pathway, but with diffusion of water being 1.6 times faster than that of CO2, intrinsic WUE relates to the CO2 gradient as follows:
Intrinsic WUE = A/gs = (ca – ci) / 1.6 = ca (1 – ci/ca) / 1.6,
where A is net photosynthesis, gs is stomatal conductance, ca and ci are the mole fractions of CO2 in ambient air and in the substomatal cavity, respectively.
The C-isotope approach has proved extremely useful to study WUE over longer time scales. It relies on the fact that photosynthetic enzymes discriminate against the heavier stable isotope 13C (relative to 12C) during photosynthesis, so that C in leaves is always depleted in 13C compared with that in the atmosphere. The extent of the enzyme’s discrimination against 13C depends on ci. If ci is low relative to ca, then the air inside the leaf becomes enriched in 13C, and the ability of the enzyme to discriminate declines. As a result, the plant ends up fixing a greater proportion of 13C than a plant performing photosynthesis at a higher ci. In its simplest form, for C3 plants, Δ = a + (b – a)ci/ca, where Δ is photosynthetic 13C discrimination, a = 4.4‰ and b = 27‰. Therefore, Δ allows time-integrated estimates of ci : ca and intrinsic WUE. Note that Δ is calculated from δ13C (see Section 3.12), as follows: Δ = (δ13Cair – δ13Cplant) / (1 + δ13Cplant), which highlights the requirement for assumptions or measurements of the isotope composition of the air.
Because intrinsic WUE changes rapidly, the bulk leaf 13C : 12C ratio of fixed C correlates with the ci:ca ratio for the time period during which the C comprising the leaf was fixed weighted by the photosynthetic flux. In other words, the 13C : 12C represents a longer-term measure of ci : ca, especially reflecting ci : ca during favourable periods.
What and how to collect?
For intrinsic WUE assessment, δ13C is usually determined for leaves, but can be determined on any plant part, e.g. on tree rings for a historical record. Note that, in general, there is fractionation between leaves and stems, with all non-photosynthetic organs being more enriched in 13C than are leaves. This enables differentiation between growing conditions using tree rings of different ages, and also means that leaves that grew in different years or different seasons can have different Δ, which has implications for the sampling strategy. Leaves at different positions in a tree or in a canopy can vary in Δ as a result of differences in stomatal opening and photosynthetic capacity, and also because of differences in the isotope composition of the source air. To estimate Δ, the isotope composition of the air needs to be known. In freely circulating air such as at the top of a canopy, it is generally reasonable to assume that the isotope composition of air is constant and equal to that of the lower atmosphere (δ13Cair » –8‰).
Storing and processing
Samples should be dried as soon as possible and finely ground. Grind the dried tissues thoroughly to pass through a 40-µm-mesh or finer screen. C-isotope ratio analysis requires only small samples (2–5 mg); however, it is recommended to sample and grind larger amounts of tissue to ensure representativeness.
See Section 3.12 for measuring C-isotope concentrations.
Special cases or extras
(1) Cellulose extracts Isotopes are sometimes analysed using cellulose extracts to avoid variation introduced by the slightly different isotope composition of other C compounds. In most cases, however, the Δ values of the whole tissue and those of cellulose correlate very well. Shorter-term (typically at the scale of a day) studies of Δ have sampled recent assimilates rather than structural C, either by extracting non-structural carbohydrates from snap-frozen leaves, or by sampling phloem sap.
(2) Assumptions We reiterate here that the estimation of intrinsic WUE from C-isotope composition involves several assumptions, that intrinsic WUE does not necessarily correlate well with the actual WUE (photosynthesis to transpiration ratio), with mesophyll conductance being a particular complication, and that the equation for Δ given above is a simplification of the theory. It is also important to note that, because of their different biochemistry, the equation given for Δ does not apply to C4 or CAM plants, and in these groups, C-isotope composition is not useful for estimating intrinsic WUE.
References on theory, significance and large datasets: Farquhar et al. (1989); Cernusak et al. (2009).
More on methods: Ehleringer and Osmond (2000); Seibt et al. (2008); Diefendorf et al. (2010).