Non-destructive measurement of chlorophyll b: AAAAA ratios and identification of photosynthetic pathways in grasses by reflectance spectroscopy

Equations for non-destructive determination of chlorophyll b:a ratios in grasses were developed from reflectance spectra of intact leaves of barley (Hordeum vulgare L.) and two barley mutants: clorina f2, which lacks chlorophyll b and clorina f104, which has a low chlorophyll b content. These plants enabled separation of effects of chlorophyll composition on reflectance spectra due to differential light absorption by chlorophylls a and b and to measure the effects of chlorophyll b on the contribution of fluorescence emitted by chlorophyll a to the reflectance spectra. Indices developed from these data were then tested on growth chamber-grown leaves from six C₃ and 17C₄ grass species (7 NAD-ME and 10 NADP-ME subtypes). We used the chlorophyll b:a ratio because the data were less skewed than the chlorophyll a:b ratio. The best index for determination of the chlorophyll b:a ratio utilised wavelengths affected by chlorophyll absorbance: [R₆₂₆ 0.5 (R₆₀₃+R ₆₄₇)]/[R₅₅₂ R₆₂₆]. The chlorophyll b:a ratio was significantly lower in the C₄ than C₃ grasses, but was not sufficient in itself to separate these two functional groups. However, because of differences in fluorescence characteristics, C₃ and C ₄ species could be distinguished by an index based on wavelengths affected by chlorophyll fluorescence: [R_{696 to 709}/R _{545 to 567}].