This article is modified from Perez-Harguindeguy et al (2013). “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
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 specific leaf area (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 SLA 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 SLA 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 SLA, 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).
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.
Notes and troubleshooting tips
(1) Leafless and heterophyllous plants See SLA
References on theory, significance and large datasets:
Aerts R (1996) Nutrient resorption from senescing leaves of perennials are there general patterns Journal of Ecology 84, 597-608. doi:10.2307/2261481
Aerts R, Chapin S III (1999) The mineral nitrition of wild plants revisited: a re-evaluation of processes and patterns. Advances in Ecological Research 30, 1-67. doi:10.1016/S0065-2504(08)60016-1
Chapin FS III (1980) The mineral nutrition of wild plants. Annual Review of Ecology and Systematics 11, 233-260. doi:10.1146/annurev.es.11.110180.001313
Cornelissen JHC, Werger MJA, Castro-Díez P, Van Rheenen JWA, Rowland AP(1997) Foliar nutrients in relation to growth, allocation and leaf traits in seedlings of a wide range of woody plant species and types. Oecologia 111, 460-469. doi:10.1007/s004420050259
Field C, Mooney HA (1986) The photosynthesis-nitrogen relationship in wild plants. In On the economy of plant form and function. Ed. TJ Givnish, pp. 25-55. Cambridge University Press: Cambridge, UK
Grime JP, Thompson K, Hunt R, Hodgson JG, Cornelissen JHC, Rorison IH, Hendry GAF, Ashenden TW, Askew AP, Band SR, Booth RE, Bossard CC, Campbell BD, Cooper JEL, Davison AW, Gupta PL, Hall W, Hand DW, Hannah MA, Hillier SH, Hodkinson DJ, Jalili A, Liu Z, Mackey JML, Matthews N, MowforthMA, NealAM,Reader RJ, Reiling K, Ross-Fraser W, Spencer RE, Sutton F, Tasker DE, Thorpe PC, Whitehouse J (1997) Integrated screening validates primary axes of specialisation in plants. Oikos 79, 259-281. doi:10.2307/3546011
Lambers H, Poorter H (1992) Inherent variation in growth rate between higher plants: a search for physiological causes and ecological consequences. Advances in Ecological Research 23, 187-261. doi:10.1016/S0065-2504(08)60148-8
Reich PB, Walters MB, Ellsworth DS (1997) From tropics to tundra: global convergence in plant functioning. Proceedings of the National Academy of Sciences, USA 94, 13730-13734. doi:10.1073/pnas.94.25.13730
Reich PB, Oleksyn J, Wright IJ, Niklas KJ, Hedin L, Elser J (2010) Evidence of a general 2/3-power law of scaling leaf nitrogen to phosphorus among major plant groups and biomes. Proceedings. Biological Sciences 277, 877-883. doi:10.1098/rspb.2009.1818
Wright IJ, Reich PB, Westoby M, Ackerly DD, Baruch Z, Bongers F, Cavender-Bares J, Chapin T, Cornelissen JHC, Diemer M, Flexas J, Garnier E, Groom PK, Gulias J, Hikosaka K, Lamont BB, Lee T, Lee W, Lusk C, Midgley JJ, NavasML, Niinemets U, Oleksyn J, Osada N, Poorter H, Poot P, Prior L, Pyankov VI, Roumet C, Thomas SC, Tjoelker MG, Veneklaas EJ, Villar R (2004) The worldwide leaf economics spectrum. Nature 428, 821-827. doi:10.1038/nature02403
More on methods:
Allen SE (1989) Chemical analysis of ecological material. 2nd edn. Blackwell: Oxford, UK.
Anderson JM, Ingram JSI (1993) Tropical soil biology and fertility: a handbook of methods. 2nd edn. CAB International: Wallingford, UK
Horneck DA, Miller RO (1998) Determination of total nitrogen in plant tissue. In Handbook and reference methods for plant analysis. Ed. YP Kalra, pp. 75-84. CRC Press: New York
Temminghoff EEJM, Houba VJG (2004) Plant analyses procedures. Kluwer Academic Publishers: Dodrecht, The Netherlands.