The main issues are the variability of the leaf responses within the crown/canopy and the ecological scale of the investigation (assessment of the response of the whole tree/plant, or of a target population of leaves). PF-6463922 A complete representation of a plant should take into account the different levels, age, and position of leaves. This would be the approach of choice but would require a large number of samples, and this would be difficult to realize in large-scale sampling. Thus, normally only one or a few leaf positions (e.g., sun leaves in the upper part of the crown, south exposed leaves, flag leaves, or fully developed leaves) are considered, depending

on the purpose of the survey. The number of leaves to be sampled depends on the internal variability of the parameters of interest. The following selleck formula can be used for this calculation: $$ n \, = \, Z_\alpha ^2 s^2 / \, B^2 $$where n is the sample size; Z α is the standard normal coefficient (= 1.96 for a 95 % confidence level); s is the SD; B is the desired precision level expressed as percent of the mean value (Elzinga et al. 2001; Gottardini

et al. 2014). A recent study of boreal forests (Pollastrini et al. 2014) found that, in the higher external part of a crown of Betula pendula, the CV among different leaves was very low for F V/F M (1.6 %), and increased for the parameters related to the step J (1 − V J, CV = 7 %) and the step I (ΔV IP = 1 − V I, CV = 14 %). We mention here that this type of studies demonstrated that the IP phase, linked to the PSI

content (Oukarroum et al. 2009; Ceppi et al. 2012), is quite sensitive to different types of stress; e.g., it decreased in response to ozone (Bussotti et al. 2011b) and nitrogen deprivation (Nikiforou and Manetas 2011), while it increased in response to high light conditions (Desotgiu et al. 2012). In order to sample as many leaves as possible during a single day, sampling must be performed during the whole day and cannot be limited to specific hours. As a consequence, leaves are sampled under different conditions of short-term light acclimation and different extents of photoinhibition. To reduce the associated variability, Nintedanib (BIBF 1120) it is necessary to allow the regulatory mechanisms induced by the ambient light to relax and to allow the leaves to recover from photoinhibition, which means a sufficient period of at least 4–5 h of dark acclimation at a constant temperature must be made before measurement. In addition, to avoid the onset of leaf senescence or the induction of other stress factors that can change the physiological state of the leaf during sampling and dark acclimation of the leaves, all fieldwork must be performed as fast as possible. Managing a large number of samples in a short time, e.g., 1,000 samples in one day, requires fast instruments/experimental protocols.