A nondestructive method to estimate the chlorophyll content of Arabidopsis seedlings

Abstract

Background: Chlorophyll content decreases in plants under stress conditions, therefore it is used commonly as an indicator of plant health. Arabidopsis thaliana offers a convenient and fast way to test physiological phenotypes of mutations and treatments. However, chlorophyll measurements with conventional solvent extraction are not applicable to Arabidopsis leaves due to their small size, especially when grown on culture dishes.

Results: We provide a nondestructive method for chlorophyll measurement whereby the red, green and blue (RGB) values of a color leaf image is used to estimate the chlorophyll content from Arabidopsis leaves. The method accommodates different profiles of digital cameras by incorporating the ColorChecker chart to make the digital negative profiles, to adjust the white balance, and to calibrate the exposure rate differences caused by the environment so that this method is applicable in any environment. We chose an exponential function model to estimate chlorophyll content from the RGB values, and fitted the model parameters with physical measurements of chlorophyll contents. As proof of utility, this method was used to estimate chlorophyll content of G protein mutants grown on different sugar to nitrogen ratios.

Conclusion: This method is a simple, fast, inexpensive, and nondestructive estimation of chlorophyll content of Arabidopsis seedlings. This method lead to the discovery that G proteins are important in sensing the C/N balance to control chlorophyll content in Arabidopsis.

Keywords: Arabidopsis thaliana; C/N sensing; Chlorophyll content; ColorChecker chart; Heterotrimeric G protein complex; Stress assay.

Fig. 1

Examples of images needed in converting RGB value to chlorophyll content. a The picture of the X-rite ColorChecker classic chart. b The original picture of the Arabidopsis seedlings grown on the plates under 4% glucose and 2 mM nitrogen for 12 days. c The dialogue box of the plugin in ImageJ. d The grey scale pictures represent the chlorophyll content, which is calculated by the equation estimate the chlorophyll content

Fig. 2

Correlation of the chlorophyll content estimated by RGB value and chemical extraction. a, b Comparison of extracted chlorophyll calculated by the Lichthenther (a) and Arnon (b) methods to chlorophyll content estimated by RGB value of Arabidopsis seedlings grown in agar plates under different C/N treatment (n = 234, four independent experiment). c, d Chlorophyll content estimated by RGB value and chemical extraction in soil-grown-plant using the defaulted coefficients (c) versus the refitted coefficients (d), respectively (n = 15)

Fig. 3

Growth of the wild type Arabidopsis in response to C/N ratios. The image of the plate-grown plants under different C/N ratios, including six different glucose concentration (0, 1, 2, 4, 5, and 6%) and five nitrogen (0.1, 0.3, 0.5, 2 and 6 mM KNO₃) concentrations

Fig. 4

The regulatory pathway of G protein signaling in the C/N sensing evaluated by RGB value. a Proportion of green leaves of the G protein mutants in response to nitrogen under 6% glucose. To distinguish from green and not green leaves, a threshold of was established (>15 ng/mm² = green) Experiments were repeated twice with 48 seedlings each. The curves were created through global curve fitting (sigmoid equation with four parameters) with SigmaPlot 12.5. b The chlorophyll content estimated by RGB value under moderate C/N stress (4% glucose and 6 mM nitrate, n = 40). For the box plot, solid line indicates the median and the dotted line indicates the mean value. Different lowercase letters indicate the significant differences among six genotypes (p < 0.05)