Protocol

AUTHOR

This article is modified from Perez Harguindeguy et al. (2013). The “New handbook for standardised measurement of plant functional traits worldwide” is a product of and is hosted by Nucleo Diversus (with additional Spanish translation). For more on this trait and on its context as part of the entire trait handbook visit its primary site Nucleo DiverSus at http://www.nucleodiversus.org/?lang=en

Contributing Editor

Gowland, K.M.

OVERVIEW

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 Light saturated photosynthetic rate). 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 Light saturated photosynthetic rate, 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/g_s = (c_a – c_i) / 1.6 = c_a (1 – c_i/c_a) / 1.6,

where A is net photosynthesis, g_s is stomatal conductance, c_a and c_i 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 c_i. If c_i is low relative to c_a, 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 c_i. In its simplest form, for C₃ plants, = a + (b – a)c_i/c_a, where is photosynthetic ¹³C discrimination, a = 4.4‰ and b = 27‰. Therefore, allows time-integrated estimates of c_i : c_a and intrinsic WUE. Note that is calculated from ¹³C (see Photosynthetic pathway), as follows: = ( ¹³C_air – ¹³C_plant) / (1 + ¹³C_plant), 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 c_i:c_a 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 c_i : c_a, especially reflecting c_i : c_a during favourable periods.

UNITS, TERMS, DEFINITIONS

A – net photosynthesis

C- Carbon

c_a – mole fractions of CO₂ in ambient air

c_i – mole fractions of CO₂ in the substomatal cavity

CO₂ – carbon dioxide

g_s – stomatal conductance

WUE – Water use efficiency

Procedure

What and how to collect

For intrinsic WUE assessment, ¹³C is usually determined for leaves, but can be determined on any plant part, e.g. on tree rings for a historical record. 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 , which has implications for the sampling strategy. Leaves at different positions in a tree or in a canopy can vary in as a result of differences in stomatal opening and photosynthetic capacity. Note that, in general, there is fractionation between leaves and stems, with all non-photosynthetic organs being more enriched in ¹³C than are leaves. To estimate , 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 ( ¹³C_air »-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.

Measuring

See Photosynthetic pathway for measuring C-isotope concentrations.

NOTES AND TROUBLESHOOTING TIPS

(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 values of the whole tissue and those of cellulose correlate very well. Shorter-term (typically at the scale of a day) studies of 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 given above is a simplification of the theory. It is also important to note that, because of their different biochemistry, the equation given for does not apply to C₄ or CAM plants, and in these groups, C-isotope composition is not useful for estimating intrinsic WUE.

LITERATURE REFERENCES

References on theory, significance and large datasets:

Cernusak LA, Tcherkez G, Keitel C, Cornwell WK, Santiago LS, Knohl A, Barbour MM, Williams DG, Reich PB, Ellsworth DS, Dawson TE, Griffiths HG, Farquhar GD, Wright IJ (2009) Why are nonphotosynthetic tissues generally 13C enriched compared with leaves in C3 plants Review and synthesis of current hypotheses. Functional Plant Biology 36, 199-213. doi:10.1071/FP08216

Farquhar GD, Ehleringer JR, Hubick KT (1989) Carbon isotope discrimination and photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 40, 503-537. doi:10.1146/annurev.pp.40.060189.002443

More on methods:

Diefendorf AF, Mueller KE, Wing SL, Koch PL, Freeman KH (2010) Global patterns in leaf 13C discrimination and implications for studies of past and future climate. Proceedings of the National Academy of Sciences, USA 107, 5738-5743. doi:10.1073/pnas.0910513107

Ehleringer JR, Osmond CB (2000) Stable isotopes. In Plant physiological ecology: field methods and instrumentation. Eds RW Pearcy, J Ehleringer, HA Mooney, PW Rundel, pp. 281-300. Kluwer Academic Publishers: Dordrecht, The Netherlands.

Seibt U, Rajabi A, Griffiths H, Berry JA (2008) Carbon isotopes and water use efficiency: sense and sensitivity. Oecologia 155, 441-454. doi:10.1007/s00442-007-0932-7