Curve-fit analysis of chlorophyll fluorescence spectra: Application to nutrient stress detection in sunflower

Abstract

The in vivo laser-induced chlorophyll fluorescence (LICF) spectra of healthy and nutrient-deficient sunflower plants were measured on a Jobin Yvon monochromator with HeNe laser excitation. To correctly determine the peak center and to evaluate the relative contributions of the bands in the total fluorescence spectrum, the steady state LICF spectra were analyzed with a nonlinear iterative procedure using Gaussian, Lorentzian, Pearson, Voigt, and exponential Gaussian spectral functions. It was observed that curve fitting performed by using two Gaussian peaks centered at 690 and 730 nm usually fits well to the chlorophyll fluorescence spectra. After curve fitting, the mean peak centers of the red and far-red chlorophyll bands of control plants were observed at 688.2 and 725.4 nm, respectively. A blue shift of as much as 9 nm in the peak position of the far-red band was observed with nutrient stress, whereas the shift in position of the red band was only of the order of a few nanometers. Further, the width at half maximum of the far-red band was found to increase by as much as 20 nm with nutrient stress. Curve fitting could thus separate out the red and far-red fluorescence spectra from a pair of normally distributed curves centered at 690 and 730 nm wavelengths, thereby differentiating the effects due to reabsorption from those due directly to changes in photosynthetic electron transport. The F690F730 fluorescence intensity ratio obtained from curve-fitted parameters was found to be more sensitive to plant stress than were fluorescence values alone. Results indicate that a curve-fitting analysis of LICF spectra using Gaussian spectral functions is a very useful and sensitive method of evaluating spectral features from a statistical point of view and for accurate determination of contributions from constituent bands in the whole leaf fluorescence spectrum.