The chlorophylls, chlorophyll a (Chl a) and chlorophyll b (Chl b), are essential pigments for the conversion of light energy to stored chemical energy in plants and their presence and function is important from both physiological and applied perspectives (Buschmann et al., 1994; Carter, 1998; Gitelson et al., 2003; Pinar and Curran, 1996; Richardson et al., 2002). As much as 75% of the total nitrogen (N) in a plant is required for normal chloroplast formation (Kutik et al., 1995) and synthesis of components of the photosynthetic apparatus, including thylakoid membranes and photosynthetic enzymes (Evans, 1989); therefore, Chl concentration gives an indirect estimation of plant N status and photosynthetic potential (Filella et al., 1995; Moran et al., 2000). Leaf Chl concentration is often closely related to plant stress and can be used as an indicator of plant stress (Carter, 1993, 1994; Carter and Knapp, 2001; Peñuelas and Filella, 1998).
Traditionally, leaf Chl was extracted with organic solvents and measured using a spectrophotometer (Lichtenthaler, 1987; Lichtenthaler and Wellburn, 1983). Recently, alternative nondestructive optical methods based on leaf light absorbance and/or reflectance have been developed (Adams et al., 1999; Curran et al., 1990; Datt, 1999; Gamon and Surfus, 1999). These optical methods require no chemical analysis, are nondestructive, simple to use, fast, inexpensive, and can be used in field conditions (Buschmann and Nagel, 1993; Gitelson and Merzlyak, 1994; Gitelson et al., 1996a, 1996b). The most common optical methods for estimating leaf Chl concentrations are based on the use of either 1) specific Chl-related wavelengths (i.e., 550, 698, 692, or 695 nm) (Carter, 1994, 1998; Jacquemoud and Baret, 1990; Moran and Moran, 1998; Thomas and Gausman, 1977); or 2) a Chl-related wavelength in combination with a Chl-insensitive wavelength in the form of a wavelength ratio (i.e., R698/R760) or specific indices or algorithms [e.g., (R800-R445)/(R800-R680)] (Moran et al., 2000; Peñuelas et al., 1995).
Previous work has mainly focused on developing and evaluating Chl-related indices for nondestructive optical assessment of Chl (Adams et al., 1999; Blackburn, 1998; Curran et al., 1990; Datt, 1998, 1999; Gamon and Surfus, 1999; Gitelson and Merzlyak, 1994, 1996; Gitelson et al., 1996a, 1996b); however, the applicability of the proposed indices was seldom tested using a second, independent data set. Most published indices rarely have been tested using data from species other than those used in the formulation of the index (Richardson et al., 2002).
There are many reasons why reported indices or algorithms are not applicable for Chl assessment across genotypes or different studies. However, one of the main reasons is that the optimum wavelength (OW) for measuring Chl used in one study differed from it used in other studies. Differences in OW between studies are a result of variation in leaf properties among plant genotypes and phenotypes and optical characteristics of plant leaves. In many studies, the most common technique used to select the OW for developing Chl-related indices is the use of the first derivative of reflectance spectra (Curran et al., 1990; Gitelson et al., 1996a, 2003; Kochubey and Kazantsev, 2007; Richardson et al., 2002). First derivatives can be used to resolve or enhance smaller peaks that are incompletely resolved from larger peaks as a result of either the background or noise (Curran et al., 1990; Moran et al., 2000) and have been successfully used for identifying the red edge waveband for Chl assessment (Curran et al., 1990; Gitelson et al., 1996a, 2003; Kochubey and Kazantsev, 2007). However, derivative changes the original peak form and may eliminate some important peaks.
Different wavelengths have different levels of spectral (reflectance and/or transmission) sensitivity and accuracy for measuring Chl. Reflectance sensitivity explains how sensitive the reflectance is at a specific wavelength for measuring Chl, whereas r 2 is a measure of accuracy (goodness of fit) of regression response at specific wavelength to Chl concentration. Theoretically, the OW for a nondestructive Chl measurement should have the highest reflectance sensitivity and largest r 2. Although r 2 has been widely used in laboratory quantitative analysis, only few studies have been reported using r 2 for Chl-related waveband identification (Carter and Spiering, 2002; Gitelson et al., 2003; Read et al., 2002). Reflectance sensitivity has been used in some studies to identify stress-sensitive Chl-related wavelengths (Carter, 1993, 1994; Moran et al., 2000) but not used in combination with r 2 to determine OW. Our objectives were to 1) evaluate whether simple linear regression in combination with reflectance sensitivity analysis can be used to determine OW for Chl assessment using reflectance; and 2) evaluate the importance of using OW in the development of Chl-related reflectance indices.
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