Leaf N concentration (LNC) and leaf P concentration (LPC) are the total amounts of N and P, respectively, per unit of dry leaf mass, expressed in mg g–1 (or sometimes as %dry-leaf mass). Interspecific rankings of LNC and LPC are often correlated. Across species, LNC tends to be closely correlated with mass-based maximum photosynthetic rate and with SLA. High LNC or LPC are generally associated with high nutritional quality to the consumers in food webs. However, LNC and LPC of a given species tend to vary significantly with the N and P availability in their environments. The LNC : LPC (N : P) ratio is sometimes used as a tool to assess whether the availability of N or P is more limiting for plant growth. Actively N-fixing species, e.g. many legumes, tend to have higher LNC : LPC ratios than other plants growing at the same site.
What and how to collect?
See Section 3.1 for the leaf-collecting procedures. Initial rehydration is not necessary. Petiole or rachis are often cut off before LNC and LPC analysis, but are included in some other cases. See under Section 3.1 for discussion of when and whether to consider them. Oven dry at 60–70°C for 72 h. Leaves used for LA or SLA analysis can be also used to measure LNC and LPC, provided drying temperature has not been higher than 70°C. For replication, see under Section 3.1, but make sure that enough total leaf material per replicate is collected, according to the analytical method and equipment to be applied (~2 g dry matter per replicate for N and 5 g for P in the case of acid digestion, 0.2 g for N in the case of combustion techniques, see below under the present Section).
Storing and processing
After oven-drying the leaves, store the material air-dry and in the dark until use, to a maximum of 1 year. Grind each replicate separately. Manual grinding with mortar and pestle is an option for small numbers of samples, but is not recommended for large ones (repetitive strain injury). Effective, inexpensive mechanical grinders are available. Samples may also be ground by shaking them with steel balls in individual plastic vials on a roller mill, which is an efficient way to grind many samples at once. Avoid inter-sample contamination by cleaning the grinder or steel balls carefully between samples. Use a ball mill for small samples. Dry the ground samples again for at least 12 h before analysis.
Several techniques are available to measure LNC and LPC in ground plant material. Macro- or micro-Kjeldahl (acidic) digestion, followed by colorimetric (flow-injection) analysis (using different reagents for N and P), has been widely used. Wet acidic digestion, followed by formation of blue phosphomolybdenum complex from orthophosphate is a more precise method for measuring total P. Alternatively, you can measure P by inductively coupled plasma–optical emission spectroscopy (ICP–OES). Kjeldahl digestion for N analysis is increasingly being replaced by methods that employ a combination of combustion analysis, converting organic matter into N2 and CO2, followed by mass spectrometry or gas chromatography. These combustion techniques provide concentrations of both N and C in the leaf, and if carried out with automated N analysers, are generally less labour- and chemical-intensive than are Kjeldahl analyses. Combustion techniques also generally recover more N than do Kjeldahl analyses, because some N fractions (e.g. NO2–, NO3– and some cyclic N compounds) do not react in Kjeldahl analysis. However, we believe that all of these standard methods should give reasonably accurate LNC and LPC. We recommend running a standard reference material with known LNC and LPC along with the samples.
Special cases or extras
(1) Leafless and heterophyllous plants. See Section 3.1.
References on theory, significance and large datasets: Chapin (1980); Field and Mooney (1986); Lambers and Poorter (1992); Aerts (1996); Cornelissen et al. (1997); Grime et al. (1997); Reich et al. (1997, 2010); Aerts and Chapin (2000); Wright et al. (2004).
More on methods: Allen (1989); Anderson and Ingram (1993); Horneck and Miller (1998); Temminghoff and Houba (2004).