Photosynthesis Research

, Volume 118, Issue 3, pp 277–295 | Cite as

Anthocyanin contribution to chlorophyll meter readings and its correction

  • Jan Hlavinka
  • Jan Nauš
  • Martina Špundová
Regular Paper


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.


Anthocyanins Chlorophyll Chlorophyll meters Correction NDVI SPAD 

List of symbols

a, b, d

Constant numbers

A535, A640

Absorbance of the sample at 535 and 640 nm, respectively




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


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

CRb, CRc

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

CTb, CTc

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


Overall specific RM absorbance at λ

DRb(λ), DRc(λ)

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


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


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


Overall specific TM absorbance at λ

DTb(λ), DTc)

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


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


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


Calculated specific RM absorbance at λ


Calculated specific TM absorbance at λ


Relative contribution of anthocyanins (b) to the SPAD signal


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


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

εRb(λ), εRc(λ)

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

εTb(λ), εTc(λ)

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


Leaf thickness


Intensity of the incident light at λ

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

Intensity of light entering the leaf at λ


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

IRe(λ), IRi(λ)

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


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


NDVI correction factor


Confidential proportionality coefficient which defines the relative SPAD units

λIR, λR

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

λ1R, λ2R, λ1T, λ2T

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


Normalized difference vegetation index


Corrected value of NDVI


Measured leaf diffusive reflectance at λ

Re(λ), Ri(λ)

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


SPAD value


Corrected SPAD value

Specific RM absorbance

Specific absorbance in the reflectance mode

Specific TM absorbance

Specific absorbance in the transmittance mode


Measured leaf diffusive transmittance at λ

xT, xR

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

Upper left index B

Abaxial side

Upper left index D

Adaxial side



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.


  1. Albert NW, Lewis DH, Zhang H, Irving LJ, Jameson PE, Davies KM (2009) Light-induced vegetative anthocyanin pigmentation in petunia. J Exp Bot 60:2191–2202PubMedCrossRefGoogle Scholar
  2. Atlassi Pak V, Nabipour M, Meskarbashee M (2009) Effect of salt stress on chlorophyll content, fluorescence, Na+ and K+ ions content in rape plants (Brassica napus L.). Asian J Agric Res 3:28–37CrossRefGoogle Scholar
  3. Cerovic ZG, Masdoumier G, Ghozlen NB, Latouche G (2012) A new optical leaf-clip meter for simultaneous non-destructive assessment of leaf chlorophyll and epidermal flavonoids. Physiol Plantarum 146:251–260CrossRefGoogle Scholar
  4. Chalker-Scott L (1999) Environmental significance of anthocyanins in plant stress responses. Photochem Photobiol 70:1–9CrossRefGoogle Scholar
  5. Close DC, Beadle CL (2003) The ecophysiology of foliar anthocyanin. Bot Rev 69:149–161CrossRefGoogle Scholar
  6. Coste S, Baraloto C, Leroy C, Marcon É, Renaud A, Richardson AD, Roggy J-C, Schimann H, Uddling J, Hérault B (2010) Assessing foliar chlorophyll contents with the SPAD-502DL chlorophyll meter: a calibration test with thirteen tree species of tropical rainforest in French Guiana. Ann For Sci 67:607CrossRefGoogle Scholar
  7. Feild TS, Lee DW, Holbrook NM (2001) Why leaves turn red in autumn. The role of anthocyanins in senescing leaves of red-osier dogwood. Plant Physiol 127:566–574PubMedCrossRefGoogle Scholar
  8. Gamon JA, Surfus JS (1999) Assessing leaf pigment content and activity with a reflectometer. New Phytol 143:105–117CrossRefGoogle Scholar
  9. Gamon JA, Peñuelas J, Field CB (1992) A narrow-waveband spectral index that tracks diurnal changes in photosynthetic efficiency. Remote Sens Environ 41:35–44CrossRefGoogle Scholar
  10. Gauche C, Malagoli ED, Luiz MTB (2010) Effect of pH on the copigmentation of anthocyanins from Cabernet Sauvignon grape extracts with organic acids. Sci Agric 67:41–46CrossRefGoogle Scholar
  11. Gitelson AA, Merzlyak MN, Chivkunova OB (2001) Optical properties and nondestructive estimation of anthocyanin content in plant leaves. Photochem Photobiol 74:38–45PubMedCrossRefGoogle Scholar
  12. Gitelson AA, Peng Y, Masek JG, Rundquist DC, Verma S, Suyker A, Baker JM, Hatfield JL, Meyers T (2012) Remote estimation of crop gross primary production with Landsat data. Remote Sens Environ 121:404–414CrossRefGoogle Scholar
  13. Giusti MM, Wrolstad RE (2001) Characterization and measurement of anthocyanins by UV-visible spectroscopy. Curr Protoc Food Analyt Chem F1.2.1–F1.2.13Google Scholar
  14. Guo J, Wang M-H (2010) Ultraviolet A-specific induction of anthocyanin biosynthesis and PAL expression in tomato (Solanum lycopersicum L.). Plant Growth Regul 62:1–8CrossRefGoogle Scholar
  15. Hoch WA, Zeldin EL, McCown BH (2001) Physiological significance of anthocyanins during autumnal leaf senescence. Tree Physiol 21:1–8PubMedCrossRefGoogle Scholar
  16. Hörtensteiner S (2006) Chlorophyll degradation during senescence. Annu Rev Plant Biol 57:55–77PubMedCrossRefGoogle Scholar
  17. Hughes NM, Smith WK (2007) Attenuation of incident light in Galax urceolata (Diapensiaceae): concerted influence of adaxial and abaxial anthocyanic layers on photoprotection. Am J Bot 94:784–790PubMedCrossRefGoogle Scholar
  18. Jifon JL, Syvertsen JP, Whaley E (2005) Growth environment and leaf anatomy affect nondestructive estimates of chlorophyll and nitrogen in Citrus sp. leaves. J Amer Soc Hort Sci 130:152–158Google Scholar
  19. Junka N, Kanlayanarat S, Buanong M, Wongs-Aree C (2012) Characterisation of floral anthocyanins and their antioxidant activity in Vanda hybrid (V. teres x V. hookeriana). J Food Agric Environ 10:221–226Google Scholar
  20. Karageorgou P, Manetas Y (2006) The importance of being red when young: anthocyanins and the protection of young leaves of Quercus coccifera from insect herbivory and excess light. Tree Physiol 26:613–621PubMedCrossRefGoogle Scholar
  21. Kytridis V-P, Manetas Y (2006) Mesophyll versus epidermal anthocyanins as potential in vivo antioxidants: evidence linking the putative antioxidant role to the proximity of oxy-radical source. J Exp Bot 57:2203–2210PubMedCrossRefGoogle Scholar
  22. Lamb DW, Steyn-Ross M, Schaare P, Hanna MM, Silvester W, Steyn-Ross A (2002) Estimating leaf nitrogen concentration in ryegrass (Lolium spp.) pasture using the chlorophyll red-edge: theoretical modelling and experimental observations. Int J Remote Sens 23:3619–3648CrossRefGoogle Scholar
  23. Leyva A, Jarillo JA, Salinas J, Martinez-Zapater JM (1995) Low temperature induces the accumulation of phenylalanine ammonia-lyase and chalcone synthase mRNAs of Arabidopsis thaliana in a light-dependent manner. Plant Physiol 108:39–46PubMedGoogle Scholar
  24. Lichtenthaler HK (1987) Chlorophyll and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148:350–382CrossRefGoogle Scholar
  25. Loh FCW, Grabosky JC, Bassuk NL (2002) Using the SPAD 502 meter to assess chlorophyll and nitrogen content of benjamin fig and cottonwood leaves. Hort Technol 12:682–686Google Scholar
  26. Manetas Y, Grammatikopoulos G, Kyparissis A (1998) The use of the portable, non-destructive, SPAD-502DL (Minolta) chlorophyll meter with leaves of varying trichome density and anthocyanin content. J Plant Physiol 53:513–516CrossRefGoogle Scholar
  27. Markwell J, Osterman JC, Mitchell JL (1995) Calibration of the Minolta SPAD-502DL leaf chlorophyll meter. Photosynth Res 46:467–472CrossRefGoogle Scholar
  28. McClendon JH, Fukshansky L (1990) On the interpretation of absorption-spectra of leaves. 1. Introduction and the correction of leaf spectra for surface reflection. Photochem Photobiol 51:203–210CrossRefGoogle Scholar
  29. Mendez M, Jones DG, Manetas Y (1999) Enhanced UV-B radiation under field conditions increases anthocyanin and reduces the risk of photoinhibition but does not affect growth in the carnivorous plant Pinguicula vulgaris. New Phytol 144:275–282CrossRefGoogle Scholar
  30. Merzlyak MN, Chivkunova OB, Solovchenko AE, Naqvi KR (2008) Light absorption by anthocyanins in juvenile, stressed, and senescing leaves. J Exp Bot 59:3903–3911PubMedCrossRefGoogle Scholar
  31. Nauš J, Rolencová M, Hlaváčková V (2008) Is chloroplast movement in tobacco plants influenced systemically after local illumination or burning stress? J Integr Plant Biol 50:1292–1299PubMedCrossRefGoogle Scholar
  32. Nauš J, Prokopová J, Řebíček J, Špundová M (2010) SPAD chlorophyll meter reading can be pronouncedly affected by chloroplast movement. Photosynth Res 105:265–271PubMedCrossRefGoogle Scholar
  33. Pietrini F, Iannelli MA, Massacci A (2002) Anthocyanin accumulation in the illuminated surface of maize leaves enhances protection from photo-inhibitory risks at low temperature, without further limitation to photosynthesis. Plant Cell Environ 25:1251–1259CrossRefGoogle Scholar
  34. Qin C, Li Y, Niu W, Ding Y, Zhang R, Shang X (2010) Analysis and characterisation of anthocyanins in mulberry fruit. Czech J Food Sci 28:117–126Google Scholar
  35. Sheoran IS, Dumonceaux T, Datla R, Sawhney VK (2006) Anthocyanin accumulation in the hypocotyl of an ABA-over producing male-sterile tomato (Lycopersicon esculentum) mutant. Physiol Plant 127:681–689CrossRefGoogle Scholar
  36. Sims DA, Gamon JA (2002) Relationships between leaf pigment content and spectral reflectance across a wide range of species, leaf structures and developmental stages. Remote Sens Environ 81:337–354CrossRefGoogle Scholar
  37. Solovchenko AE, Chivkunova OB (2011) Physiological role of anthocyanin accumulation in common hazel juvenile leaves. Russ J Plant Physiol 58:674–680CrossRefGoogle Scholar
  38. Tanaka Y, Sasaki N, Ohmiya A (2008) Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J 54:733–749PubMedCrossRefGoogle Scholar
  39. Terashima I, Saeki T (1983) Light environment within a leaf I. optical properties of paradermal sections of Camellia leaves with special reference to differences in the optical properties of palisade and spongy tissues. Plant Cell Physiol 24:1493–1501Google Scholar
  40. Uddling J, Gelang-Alfredsson J, Piikki K, Pleijel H (2007) Evaluating the relationship between leaf chlorophyll concentration and SPAD-502DL chlorophyll meter readings. Photosynth Res 91:37–46PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.Department of Biophysics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of SciencePalacký University, OlomoucOlomoucCzech Republic

Personalised recommendations