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Anthocyanin contribution to chlorophyll meter readings and its correction

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Abstract

Leaf chlorophyll content is an important physiological parameter which can serve as an indicator of nutritional status, plant stress or senescence. Signals proportional to the chlorophyll content can be measured non-destructively with instruments detecting leaf transmittance (e.g., SPAD-502) or reflectance (e.g., showing normalized differential vegetation index, NDVI) in red and near infrared spectral regions. The measurements are based on the assumption that only chlorophylls absorb in the examined red regions. However, there is a question whether accumulation of other pigments (e.g., anthocyanins) could in some cases affect the chlorophyll meter readings. To answer this question, we cultivated tomato plants (Solanum lycopersicum L.) for a long time under low light conditions and then exposed them for several weeks (4 h a day) to high sunlight containing the UV-A spectral region. The senescent leaves of these plants evolved a high relative content of anthocyanins and visually revealed a distinct blue color. The SPAD and NDVI data were collected and the spectra of diffusive transmittance and reflectance of the leaves were measured using an integration sphere. The content of anthocyanins and chlorophylls was measured analytically. Our results show that SPAD and NDVI measurement can be significantly affected by the accumulated anthocyanins in the leaves with relatively high anthocyanin content. To describe theoretically this effect of anthocyanins, concepts of a specific absorbance and a leaf spectral polarity were developed. Corrective procedures of the chlorophyll meter readings for the anthocyanin contribution are suggested both for the transmittance and reflectance mode.

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Abbreviations

a, b, d :

Constant numbers

A 535, A 640 :

Absorbance of the sample at 535 and 640 nm, respectively

ACN:

Anthocyanins

C ACN :

Anthocyanin content in leaf disk [nmol cm−2] estimated analytically

C CHL :

Chlorophyll a+b content in leaf disk [nmol cm−2] estimated analytically

C Rb, C Rc :

Mean theoretical anthocyanin (b) or chlorophyll (c) molar concentration [M] along the reflection pathway, respectively

C Tb, C Tc :

Mean theoretical anthocyanin (b) or chlorophyll (c) molar concentration [M] along the transmission pathway, respectively

D R(λ):

Overall specific RM absorbance at λ

D Rb(λ), D Rc(λ):

Specific RM absorbance of anthocyanin (b) or chlorophyll (c) at λ, respectively

D Rg(λ):

Mean normalized specific RM absorbance of a leaf without anthocyanins at λ

D Rr(λ):

Specific RM absorbance caused by reflection, scatter, refraction, and diffraction at λ

D T(λ):

Overall specific TM absorbance at λ

D Tb(λ), D Tc (λ):

Specific TM absorbance of anthocyanins (b) or chlorophyll (c) at λ, respectively

D Tg(λ):

Mean normalized specific TM absorbance of a leaf without anthocyanins at λ

D Tr(λ):

Specific TM absorbance caused by reflection, scatter, refraction, and diffraction at λ

D sR(λ):

Calculated specific RM absorbance at λ

D sT(λ):

Calculated specific TM absorbance at λ

Δ b(SPAD):

Relative contribution of anthocyanins (b) to the SPAD signal

Δ Rb(λ 1R):

Relative contribution of anthocyanins (b) to the specific RM absorbance at λ 1R

Δ Tb(λ 1T):

Relative contribution of anthocyanins (b) to the specific TM absorbance at λ 1T

ε Rb(λ), ε Rc(λ):

Mean molar absorption coefficients of anthocyanins (b) or chlorophyll (c) along the reflection (R) pathway at λ, respectively

ε Tb(λ), ε Tc(λ):

Mean molar absorption coefficients of anthocyanins (b) or chlorophyll (c) along the transverse (T) pathway at λ, respectively

h :

Leaf thickness

I 0(λ):

Intensity of the incident light at λ

\( I^{\prime}_{ 0} (\lambda ) \) :

Intensity of light entering the leaf at λ

I R(λ):

Intensity of light reflected (R) from the leaf at λ

I Re(λ), I Ri(λ):

Intensity of light reflected (R) from the leaf surface at λ (external reflection) or from internal leaf structures at λ (internal reflection), respectively

I T(λ):

Intensity of light transmitted (T) through the leaf at λ

K :

NDVI correction factor

l :

Confidential proportionality coefficient which defines the relative SPAD units

λ IR, λ R :

Wavelength in the infrared (IR) and red (R) region

λ 1R, λ 2R, λ 1T, λ 2T :

Detection wavelengths for the reflectance (R) and transmittance (T) mode

NDVI :

Normalized difference vegetation index

NDVI C :

Corrected value of NDVI

R m(λ):

Measured leaf diffusive reflectance at λ

R e(λ), R i(λ):

External (surface) or internal reflectance of the leaf at λ, respectively

SPAD :

SPAD value

SPAD C :

Corrected SPAD value

Specific RM absorbance:

Specific absorbance in the reflectance mode

Specific TM absorbance:

Specific absorbance in the transmittance mode

T m(λ):

Measured leaf diffusive transmittance at λ

x T, x R :

A beam trajectory in the transmittance (T) or reflectance (R) mode, respectively

Upper left index B:

Abaxial side

Upper left index D:

Adaxial side

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Acknowledgements

We would like to thank Eliška Ježilová for her help with measurements. This work was supported by the Ministry of Youth and Education of the Czech Republic (MSM6198959215); by the Czech Science Foundation (GD522/08/H003); and by the Grant no. ED0007/01/01 Centre of the Region Haná for Biotechnological and Agricultural Research.

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  1. Department of Biophysics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Šlechtitelů 11, 78371, Olomouc, Czech Republic

    Jan Hlavinka, Jan Nauš & Martina Špundová

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Correspondence to Martina Špundová.

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Hlavinka, J., Nauš, J. & Špundová, M. Anthocyanin contribution to chlorophyll meter readings and its correction. Photosynth Res 118, 277–295 (2013). https://doi.org/10.1007/s11120-013-9934-y

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