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In photosynthesis, antenna pigments in leaf chloroplasts absorb solar radiation, and through resonance transfer the resulting excitation is channeled to the reaction centre pigments, which release electrons and set in motion the photochemical process. The chlorophylls, Chla and Chlb, are the most important of these pigments, and are thus virtually essential for the oxygenic conversion of light energy to the stored chemical energy that powers the biosphere. From a physiological perspective, leaf Chl content (for example, how it varies both between and within species) is therefore a parameter of significant interest in its own right. However, from an applied perspective, leaf pigmentation is important to both land managers and ecophysiologists. There are several reasons for this. First, the amount of solar radiation absorbed by a leaf is largely a function of the foliar concentrations of photosynthetic pigments, and therefore low concentrations of chlorophyll can directly limit photosynthetic potential and hence primary production (Curran et al., 1990; Filella et al., 1995). Second, much of leaf nitrogen is incorporated in chlorophyll, so quantifying Chl content gives an indirect measure of nutrient status (Filella et al., 1995; Moran et al., 2000). Third, pigmentation can be directly related to stress physiology, as concentrations of carotenoids increase and chlorophylls generally decrease under stress and during senescence (Peñuelas & Filella, 1998). Fourth, the relative concentrations of pigments are known to change with abiotic factors such as light (e.g. sun leaves have a higher Chla : Chlb ratio; Larcher, 1995) and so quantifying these proportions can provide important information about relationships between plants and their environment.
The amount of chlorophyll in a leaf is normally expressed in terms of either concentration (i.e. µg Chl g−1 tissue) or content (i.e. µg Chl cm−2 tissue); preference for one over the other may depend on the researcher’s objectives. Sometimes, Chl concentration or content is expressed in terms of moles per amount of leaf mass or area, since photon flux and carbon assimilation rates are usually expressed in similar units, and this permits better understanding of physiological processes. The molecular weights of Chla and Chlb are 892 and 906, respectively.
Traditionally, wet chemical methods have required Chl extraction in a solvent, followed by the spectrophotometric determination of absorbance by the chlorophyll solution, and conversion from absorbance to concentration using standard published equations (e.g. those of Arnon (1949) and modifications thereof). Although long considered the standard method for Chl determination, extraction requires destructive sampling (which precludes, for example, developmental studies of single leaves) and is relatively time consuming.
More recently, nondestructive optical methods, based on the absorbance and/or reflectance of light by the intact leaf, have been developed. Optical methods generally yield a ‘chlorophyll index’ value that expresses relative chlorophyll content but not absolute Chl content per unit leaf area, or concentration per gram of leaf tissue. These newer methods are nondestructive, very quick, and now possible in the field (Markwell et al., 1995; Gamon & Surfus, 1999). In this paper, we evaluate both nondestructive absorbance and reflectance methods.
Hand-held Chl absorbance meters, of which several are commercially available, measure absorbance by the leaf of two different wavelengths of light: c. 660 nm (red) and c. 940 nm (near-infrared). The red light is strongly absorbed by Chl; the near-infrared light is a ‘reference wavelength’ that is used to adjust for differences in leaf structure. The theoretical principles on which these meters are based are described in detail by Markwell et al. (1995).
Portable reflectometers, which essentially allow the application of remote sensing technology at the leaf or branch level, record reflectance at many closely spaced wavelengths across an entire spectrum. This spectrum typically spans ultraviolet, visible, and near-infrared wavelengths. Mathematical indices have been developed which reduce complex spectra to a single value; these indices, which are based on knowledge of the reflectance properties of the biochemical components in leaves, can be targeted to estimate (among other things) pigment content. More complete reviews of some of the practical and theoretical considerations of reflectance spectroscopy are given by Curran et al. (1990), Adams et al. (1999), Datt (1999), and Gamon & Surfus (1999).
Compared to hand-held Chl absorbance meters, which yield just a single index value, one of the benefits of reflectance spectroscopy is the wealth of information that can be obtained from each leaf scan. A typical spectrum, containing 256 or more data points, can be transformed in an almost infinite number of ways. However, a key problem for the researcher is to choose an appropriate transformation index from among the vast array of those available. The application of reflectance spectroscopy to the estimation of leaf Chl content has recently received considerable attention in the literature, and many of these papers have presented new indices which are well-correlated with Chl (Curran et al., 1990; Gitelson & Merzlyak, 1994, 1996; Gitelson et al., 1996; Blackburn, 1998; Datt, 1998, 1999; Adams et al., 1999; Gamon & Surfus, 1999). The researcher’s choice of index is not made any easier by the fact that some of these authors have failed to test the applicability of the proposed index by using a second, independent, data set. In some cases, the results of such tests are not presented in a way that allows meaningful comparison of the indices across different studies. Finally, these indices have rarely been tested using data from species other than those used in the formulation of the index. Notwithstanding the fact that reflectance spectroscopy can provide considerably more data than hand-held Chl absorbance meters, there is no evidence that more is actually better. Although two studies have tested hand-held Chl absorbance meters (Monje & Bugbee, 1992; Markwell et al., 1995), we are not aware of any studies that attempted a comparative test of absorbance meters and different reflectance indices, to determine which noninvasive method produces the index best correlated with leaf Chl.
Thus, the purpose of this study is to compare the performance of two commercially available hand-held Chl absorbance meters with that of several reflectance indices for leaf-level Chl (other indices may be more appropriate for remote sensing applications at the canopy or stand level; e.g. Datt, 1999; Gamon & Surfus, 1999). As our standard against which the noninvasive methods would be judged, we measured leaf Chl using standard extraction techniques.