The pH of green leaf tissue and of senesced leaves (leaf litter), measured by grinding up the tissue and extracting it with distilled water, varies substantially among species. The variation is at least partly intrinsic (presumably genetic) because this pH can differ greatly among different species growing in the same soil (and also during the day in CAM plants, see below under Special cases or extras in the present Section) and is robust to differences in soil chemistry (including pH). Ground leaf-tissue pH integrates the effects of many compounds and processes in the leaf that affect its exchange capacity for H+ ions. However, some substances are particularly strong determinants of leaf-tissue pH. High concentrations of metal cations (calcium, magnesium, potassium) will give high pH, whereas high concentrations of organic acids and of C-rich secondary metabolites (chemical-defence compounds) such as tannins, will tend to give a lower pH. The latter may explain why leaf-tissue pH tends to be correlated (negatively) with important biogeochemical traits such as C concentration or C : N ratio, and (positively) with SLA (Section 3.1). Green leaf-tissue pH correlates positively with digestibility, making it an important predictor of palatability to herbivores, which may actually ‘taste’ acidity. Differences in leaf-tissue pH among species tend to persist during senescence, making leaf-litter pH a worthwhile trait also. It can be a reasonable proxy for litter decomposability, because substrate pH can be important to decomposers. Shifts in species composition, among species that differ in leaf-litter pH, can drive changes in the pH of the litter layer and of the soil’s organic horizon. For example, planting pines on high-pH soils dominated by bacteria can, via the leaf litter, lead to more acidic soil organic matter within decades, and a decomposer community dominated by fungi. Whether green leaves or senesced leaves, or both, should be measured depends on the question to be addressed; however, green leaves should be used if in doubt and if decomposition is not the main issue.
The cells of any plant tissue consist of three or more membrane-delimited compartments that can, and usually do, have different internal pH values. Therefore, the pH of the type of crude, water-extracted homogenate or ground material used for this method, although it can be useful as an ecological trait, must not be mistaken for the actual pH of the leaf as a whole or any part thereof. It will, at best, be a weighted average of the pH values of the various compartments of the leaf, possibly modified by reactions that occur between components of different compartments, including vacuoles, when they are mixed together. If leaves are oven-dried before grinding, loss of compartmentalisation under conditions favouring pH-modifying reactions will have occurred long before the tissue is ground. Still, the pH measured this way has been shown to also represent the pH of green leaves. Because, in leaf litter, metabolic processes have already stopped, unlike in living leaves, litter pH values should reflect the actual situation in the litter at the time of sampling.
What and how to collect
For green leaves, see Section 3.1 for the collecting and storing procedure before processing. If leaves are small, make sure enough of them are collected so as to get enough material for the analysis. Initial leaf rehydration is not necessary.
Processing and storing
As a default option, any petiole or rachis should be removed before pH analysis; however, see Special cases or extras in Section 3.1. Fresh green leaves can simply be ground or chopped as noted below. Leaves collected for leaf SLA analysis can instead be used, after being oven-dried to obtain their biomass (see Section 3.1), and subsequently stored air-dry, because pH values obtained from oven-dried leaves are largely comparable to those from fresh leaves. Air-dried leaves or leaf litter are ground as for leaf N analysis (see Section 3.6) and stored air-dry until the analysis.
Add distilled or de-mineralised water to the ground leaf sample, to give an 8 : 1 volume ratio of water to leaf sample. Shake the samples in a laboratory rotary shaker for 1 h, then centrifuge until there is a clear separation of the sediment and the supernatant. The supernatant can then be measured for pH by using any of a wide range of laboratory pH meters, as long as calibration is adequate (using buffer solutions of pH 4 and pH 7). If samples are small in volume, we recommend adding 1.2 mL of water to 0.15 mL of ground leaf material in a 2.5-mL Eppendorf tube, then following the above procedure. A thin SenTix 41 electrode connected to an Inolab level 2 pH meter (both WTW, Weilheim, Germany) fits nicely inside such Eppendorf tubes.
Special cases or extras
(1) Additional measurements on fresh leaves. Although measurements on dried, ground leaves tend to match those on fresh leaves fairly well, for various specific purposes, it may be of interest to (additionally) measure fresh leaves, the cell contents of which have remained intact until shortly before measurement. For instance, this may be useful to follow diurnal changes in leaf pH to indicate possible CAM (see Section 3.12). Related to this, in comparisons of CAM plants with other plants for leaf pH, leaves of CAM plants are best collected in the afternoon, i.e. well after nocturnal acid accumulation. Grinding fresh leaves does not always work well because solids may stick to the surfaces or to the balls in a ball mill, making cleaning between samples laborious. Instead, we recommend chopping the leaf sample into ~1-mm-diameter pieces with a razor blade or an automatic chopper that gives comparable fragmentation. This should be carried out immediately before shaking and centrifuging.
References on theory, significance and large datasets: Zinke (1962); Marschner (2012); Finzi et al. (1998); Cornelissen et al. (2006, 2011); Freschet et al. (2010).
More on methods: Cornelissen et al. (2006, 2011).