Plant Photosynthetic Pigments: Methods and Tricks for Correct Quantification and Identification

  • Beatriz Fernández-MarínEmail author
  • José Ignacio García-Plazaola
  • Antonio Hernández
  • Raquel Esteban


Chloroplast of green photosynthetic tissues in the Viridiplantae (monophyletic group that includes green algae and terrestrial plants) is characterised by a relatively conserved composition of pigments (Esteban et al. 2015). Leaves of virtually all plant species invariably contain chlorophyll (Chl) a and Chl b, and six carotenoids. Five of them are xanthophylls (carotenoids that contain oxygen): neoxanthin (Neo), lutein (Lut), violaxanthin (Vio), antheraxanthin (Ant) and zeaxanthin (Zea). The remaining carotenoid is a carotene (no oxygen in the molecule): β-carotene (β-Car). Additionally, certain taxa contain a second carotene: α-Car, which partially substitutes β-Car in some species under low light environment (Young and Britton 1989; Esteban and García-Plazaola 2016). Some species phylogenetically unrelated also include lutein epoxide (Lx), a xanthophyll likewise related to shade acclimation (Matsubara et al. 2005; Esteban et al. 2009b). Regarding pigment concentration, in agreement with the relatively conserved composition of pigments across green photosynthetic organisms, and because the maximum Chl concentration per leaf is limited by specific and physiological constrains (Niinemets 2007), photosynthetic pigments are restricted within specific ranges of concentrations. Highly reliable ranges of pigment content for non-stressed plants that were obtained from two data-bases comprising more than 800 species can be found in (Esteban et al. 2015; Fernández-Marín et al. 2017) (summarized as reference in Table 3.1).


  1. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenolxidase in Beta vulgaris. Plant Physiol 24:1–15CrossRefPubMedPubMedCentralGoogle Scholar
  2. Britton G, Liaaen-Jensen S, Pfander H (2004) Carotenoids: handbook. Springer, BaselCrossRefGoogle Scholar
  3. Camarero JJ, Olano JM, Arroyo Alfaro SJ, Fernández-Marín B, Becerril JM, García-Plazaola JI (2012) Photoprotection mechanisms in Quercus ilex under contrasting climatic conditions. Flora 207:557–564CrossRefGoogle Scholar
  4. Cazzaniga S, Li Z, Niyogi KK, Bassi R, Dall’Osto L (2012) The Arabidopsis szl1 mutant reveals a critical role of β-carotene in photosystem I photoprotection. Plant Physiol 159:1745–1758. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Cazzaniga S, Bressan M, Carbonera D, Agostini A, Dall’Osto L (2016) Differential roles of carotenes and xanthophylls in photosystem I photoprotection. Biochemistry 55:3636–3649. CrossRefPubMedGoogle Scholar
  6. Cerovic ZG, Masdoumier G, Ben GN, Latouche G (2012) A new optical leaf-clip meter for simultaneous non-destructive assessment of leaf chlorophyll and epidermal flavonoids. Physiol Plant 146:251–260. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cherian M, Corona E (2006) Lyophilisation of biologicals. In: Briefings T (ed) Bioprocessing and biopartnering. Touch Briefings, London, pp 20–21Google Scholar
  8. Croce R (2012) Chlorophyll-binding proteins of higher plants and cyanobacteria. In: Eaton-Rye J, Tripathy B, Sharkey TD (eds) Photosynthesis: plastid biology, energy conversion and carbon assimilation, Advances in Photosynthesis and Respiration. Springer, Dordrecht, pp 127–149CrossRefGoogle Scholar
  9. Dall’Osto L, Lico C, Alric J, Giuliano G, Havaux M, Bassi R (2006) Lutein is needed for efficient chlorophyll triplet quenching in the major LHCII antenna complex of higher plants and effective photoprotection in vivo under strong light. BMC Plant Biol 6:32. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Dall’Osto L, Cazzaniga S, North H, Marion-Poll A, Bassi R (2007a) The Arabidopsis aba4-1 mutant reveals a specific function for neoxanthin in protection against photooxidative stress. Plant Cell 19:1048–1064. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Dall’Osto L, Fiore A, Cazzaniga S, Giuliano G, Bassi R (2007b) Different roles of α- and β-branch xanthophylls in photosystem assembly and photoprotection. J Biol Chem 282:35056–35068. CrossRefPubMedGoogle Scholar
  12. Dall’Osto L, Cazzaniga S, Havaux M, Bassi R (2010) Enhanced photoprotection by protein-bound vs free xanthophyll pools: a comparative analysis of chlorophyll b and xanthophyll biosynthesis mutants. Mol Plant 3:576–593. CrossRefPubMedGoogle Scholar
  13. Demmig-Adams B (1998) Survey of thermal energy dissipation and pigment composition in sun and shade leaves. Plant Cell Physiol 39:474–482CrossRefGoogle Scholar
  14. Demmig-Adams B, Gilmore AM, Adams WW (1996) In vivo functions of carotenoids in higher plants. FASEB 10:403–412CrossRefGoogle Scholar
  15. Esteban R, García-Plazaola JI (2014) Involvement of a second xanthophyll cycle in non-photochemical quenching of chlorophyll fluorescence: the lutein epoxide story. In: Demmig-Adams B, Garab G, Adams WW III, Govindjee (eds) Non-photochemical quenching and energy dissipation in plants, algae and cyanobacteria. Springer, Dordrecht, pp 277–295Google Scholar
  16. Esteban R, García-Plazaola JI (2016) Nonubiquitous carotenoids in higher plants: presence, role in photosynthesis, and guidelines for identification. In: Pessarakli M (ed) Handbook of photosynthesis, 3rd edn. CRC Press, Boca Raton, pp 589–600Google Scholar
  17. Esteban R, Balaguer L, Manrique E, Rubio de Casas R, Ochoa R, Fleck I, Pintó-Marijuan M, Casals I, Morales D, Jiménez MS, Lorenzo R, Artetxe U, Becerril JM, García-Plazaola JI (2009a) Alternative methods for sampling and preservation of photosynthetic pigments and tocopherols in plant material from remote locations. Photosynth Res 101:77–88. CrossRefPubMedGoogle Scholar
  18. Esteban R, Becerril JM, García-Plazaola JI (2009b) Lutein epoxide cycle, more than just a forest tale. Plant Signal Behav 4:342–344. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Esteban R, Olano JM, Castresana J, Fernández-Marín B, Hernández A, Becerril JM, García-Plazaola JI (2009c) Distribution and evolutionary trends of photoprotective isoprenoids (xanthophylls and tocopherols) within the plant kingdom. Physiol Plant 135:379–389. CrossRefPubMedGoogle Scholar
  20. Esteban R, Matsubara S, Jiménez MS, Morales D, Brito P, Lorenzo R, Fernández-Marín B, Becerril JM, García-Plazaola JI (2010) Operation and regulation of the lutein epoxide cycle in seedlings of Ocotea foetens. Funct Plant Biol 37:859–869. CrossRefGoogle Scholar
  21. Esteban R, Barrutia O, Artetxe U, Fernández-Marín B, Hernández A, García-Plazaola JI (2015) Internal and external factors affecting photosynthetic pigment composition in plants: a meta-analytical approach. New Phytol 206:268–280. CrossRefPubMedGoogle Scholar
  22. Fernández-Marín B, Balaguer L, Esteban R, Becerril JM, García-Plazaola JI (2009) Dark induction of the photoprotective xanthophyll cycle in response to dehydration. J Plant Physiol 166:1734–1744. CrossRefPubMedGoogle Scholar
  23. Fernández-Marín B, Míguez F, Becerril J, García-Plazaola J (2011a) Activation of violaxanthin cycle in darkness is a common response to different abiotic stresses: a case study in Pelvetia canaliculata. BMC Plant Biol 11:181. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Fernández-Marín B, Míguez F, Becerril JM, García-Plazaola JI (2011b) Dehydration-mediated activation of the xanthophyll cycle in darkness: is it related to desiccation tolerance? Planta 234:579–588. CrossRefPubMedGoogle Scholar
  25. Fernández-Marín B, Kranner I, Sebastián MS, Artetxe U, Laza JM, Vilas JL, Pritchard HW, Nadajaran J, Míguez F, Becerril JM, García-Plazaola JI (2013) Evidence for the absence of enzymatic reactions in the glassy state. A case study of xanthophyll cycle pigments in the desiccation-tolerant moss Syntrichia ruralis. J Exp Bot 64:3033–3043. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Fernández-Marín B, Artetxe U, Barrutia O, Esteban R, Hernández A, García-Plazaola JI (2015) Opening Pandora’s box: cause and impact of errors on plant pigment studies. Front Plant Sci 6:148. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Fernández-Marín B, Hernández A, García-Plazaola JI, Esteban R, Míguez F, Artetxe U, Gómez-Sagasti MT (2017) Photoprotective strategies of Mediterranean plants in relation to morphological traits and natural environmental pressure: a meta-analytical approach. Font Plant Sci 19:1051. CrossRefGoogle Scholar
  28. Fernàndez-Martínez J, Joffre R, Zacchini M, Fernández-Marín B, García-Plazaola JI, Fleck I (2017) Near-infrared reflectance spectroscopy allows rapid and simultaneous evaluation of chloroplast pigments and antioxidants, carbon isotope discrimination and nitrogen content in Populus spp. leaves. For Ecol Manag 399:227–234. CrossRefGoogle Scholar
  29. Fukushima A, Kusano M, Nakamichi N, Kobayashi M, Hayashi N, Sakakibara H, Mizuno T, Saito K (2009) Impact of clock-associated Arabidopsis pseudo-response regulators in metabolic coordination. Proc Natl Acad Sci 106:8791–8791. CrossRefGoogle Scholar
  30. Gamon JA, Field CB, Fredeen AL, Thayer S (2001) Assessing photosynthetic downregulation in sunflower stands with an optically-based model. Photosynth Res 67:113–125. CrossRefPubMedGoogle Scholar
  31. García-Plazaola JI, Becerril JM (1999) A rapid high-performance liquid chromatography method to measure lipophilic antioxidants in stressed plants: simultaneous determination of carotenoids and tocopherols. Phytochem Anal 10:307–313CrossRefGoogle Scholar
  32. García-Plazaola JI, Becerril JM (2001) Seasonal changes in photosynthetic pigments and antioxidants in beech (Fagus sylvatica) in a Mediterranean climate: implications for tree decline diagnosis. Aust J Plant Physiol 28:225–232. CrossRefGoogle Scholar
  33. García-Plazaola JI, Fernández-Marín B, Ferrio JP, Alday JG, Hoch G, Landais D, Milcu A, Tissue DT, Voltas J, Gessler A, Roy J, Resco de Dios V (2017) Endogenous circadian rhythms in pigment composition induce changes in photochemical efficiency in plant canopies. Plant Cell Environ 40:1153–1162. CrossRefPubMedGoogle Scholar
  34. Gilmore AM, Yamamoto HY (1991) Resolution of lutein and zeaxanthin using a non-endcapped, lightly carbon-loaded C18 high-performance liquid chromatographic column. J Chromatogr A 543:137–145. CrossRefGoogle Scholar
  35. Gruszecki WI, Strzalka K (2005) Carotenoids as modulators of lipid membrane physical properties. Biochim Biophys Acta Mol basis Dis 1740:108–115. CrossRefGoogle Scholar
  36. Havaux M (1998) Carotenoids as membrane stabilizers in chloroplasts. Trends Plant Sci 3:147–151. CrossRefGoogle Scholar
  37. Havaux M, Dall’Osto L, Cuiné S, Giuliano G, Bassi R (2004) The effect of zeaxanthin as the only xanthophyll on the structure and function of the photosynthetic apparatus in Arabidopsis thaliana. J Biol Chem 279:13878–13888. CrossRefPubMedGoogle Scholar
  38. Jeffrey SW, Humphrey GF (1975) New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem und Physiol der Pflanz 167:191–194. CrossRefGoogle Scholar
  39. Johnson MP, Goral TK, Duffy CD, Brain AP, Mullineaux CW, Ruban AV (2011) Photoprotective energy dissipation involves the reorganization of photosystem II light-harvesting complexes in the grana membranes of spinach chloroplasts. Plant Cell 23:1468–1479. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Junker LV, Ensminger I (2016a) Relationship between leaf optical properties, chlorophyll fluorescence and pigment changes in senescing Acer saccharum leaves. Tree Physiol 36:694–711. CrossRefPubMedGoogle Scholar
  41. Junker LV, Ensminger I (2016b) Fast detection of leaf pigments and isoprenoids for ecophysiological studies, plant phenotyping and validating remote-sensing of vegetation. Physiol Plant 158:369–381. CrossRefPubMedGoogle Scholar
  42. Kromdijk J, Glowacka K, Leonelli L, Gabilly ST, Iwai M, Niyogi KK, Long SP (2016) Improving photosynthesis and crop productivity by accelerating recovery from photoprotection. Science 354(6314):857–861CrossRefPubMedGoogle Scholar
  43. Küpper H, Seibert S, Parameswaran A (2007) Fast, sensitive, and inexpensive alternative to analytical pigment HPLC: quantification of chlorophylls and carotenoids in crude extracts by fitting with Gauss peak spectra. Anal Chem 79:7611–7627. CrossRefPubMedGoogle Scholar
  44. Lichtenthaler HK, Buschmann C (2001) Chlorophylls and carotenoids: measurement and characterization by UV-VIS spectroscopy. In: Current protocols in food analytical chemistry. Wiley, HobokenGoogle Scholar
  45. Lichtenthaler HK, Wellburn AR (1983) Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem Soc Trans 11:591–592CrossRefGoogle Scholar
  46. Lokstein H, Tian L, Polle JEW, DellaPenna D (2002) Xanthophyll biosynthetic mutants of Arabidopsis thaliana: altered nonphotochemical quenching of chlorophyll fluorescence is due to changes in photosystem II antenna size and stability. Biochim Biophys Acta Bioenerg 1553:309–319. CrossRefGoogle Scholar
  47. Lu S, Lu X, Zhao W, Liu Y, Wang Z, Omasa K (2015) Comparing vegetation indices for remote chlorophyll measurement of white poplar and Chinese elm leaves with different adaxial and abaxial surfaces. J Exp Bot 66:5625–5637. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Matsubara S, Naumann M, Martin R, Nichol C, Rascher U, Morosinotto T, Bassi R, Osmond B (2005) Slowly reversible de-epoxidation of lutein-epoxide in deep shade leaves of a tropical tree legume may “lock-in” lutein-based photoprotection during acclimation to strong light. J Exp Bot 56:461–468. CrossRefPubMedGoogle Scholar
  49. Maurer MM, Mein JR, Chaudhuri SK, Constant HL (2014) An improved UHPLC-UV method for separation and quantification of carotenoids in vegetable crops. Food Chem 165:475–482. CrossRefPubMedGoogle Scholar
  50. Míguez F, Fernández-Marín B, Becerril JM, García-Plazaola JI (2017) Diversity of winter photoinhibitory responses: a case study in co-occurring lichens, mosses, herbs and woody plants from subalpine environments. Physiol Plant 160:282–296CrossRefPubMedGoogle Scholar
  51. Moradzadeh M, Sadeghnia HR, Tabarraei A, Sahebkar A (2017) Anti-tumor effects of crocetin and related molecular targets. J Cell Physiol 233:2170–2182. CrossRefPubMedGoogle Scholar
  52. Niinemets Ü (2007) Photosynthesis and resource distribution through plant canopies. In: Eaton-Rye J, Tripathy B, Sharkey T (eds) Photosynthesis: plastid biology, energy conversion and carbon assimilation, Advances in Photosynthesis and Respiration. Blackwell Publishing Ltd, Dordrecht, pp 1052–1071Google Scholar
  53. Parry C, Blonquist JM, Bugbee B (2014) In situ measurement of leaf chlorophyll concentration: analysis of the optical/absolute relationship. Plant Cell Environ 37:2508–2520. CrossRefPubMedGoogle Scholar
  54. Peguero-Pina JJ, Gil-Pelegrín E, Morales F (2013) Three pools of zeaxanthin in Quercus coccifera leaves during light transitions with different roles in rapidly reversible photoprotective energy dissipation and photoprotection. J Exp Bot 64:1649–1661. CrossRefPubMedPubMedCentralGoogle Scholar
  55. Pintó-Marijuan M, Joffre R, Casals I, De Agazio M, Zacchini M, García-Plazaola JI, Esteban R, Aranda X, Guàrdia M, Fleck I (2013) Antioxidant and photoprotective responses to elevated CO2 and heat stress during holm oak regeneration by resprouting, evaluated with NIRS (near-infrared reflectance spectroscopy). Plant Biol 15:5–17. CrossRefPubMedGoogle Scholar
  56. Polívka T, Frank HA (2010) Light harvesting by carotenoids. Acc Chem Res 43:1125–1134CrossRefPubMedPubMedCentralGoogle Scholar
  57. Porra RJ (2002) The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth Res 73:149–156. CrossRefPubMedGoogle Scholar
  58. Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta Bioenerg 975:384–394. CrossRefGoogle Scholar
  59. Shrestha S, Brueck H, Asch F (2012) Chlorophyll index, photochemical reflectance index and chlorophyll fluorescence measurements of rice leaves supplied with different N levels. J Photochem Photobiol B Biol 113:7–13. CrossRefGoogle Scholar
  60. 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 81:337–354. CrossRefGoogle Scholar
  61. Smith JHC, Benitez A (1955) Chlorophylls: analysis in plant materials. In: Modern methods of plant analysis/Moderne Methoden der Pflanzenanalyselant analysis. Springer, Berlin/Heidelberg, pp 142–196CrossRefGoogle Scholar
  62. Tausz M, Wonisch A, Grill D, Morales D, Jiménez MS (2003) Measuring antioxidants in tree species in the natural environment: from sampling to data evaluation. J Exp Bot 54:1505–1510. CrossRefPubMedGoogle Scholar
  63. Wang K, Tu W, Liu C, Rao Y, Gao Z, Yang C (2017) 9-cis-Neoxanthin in light harvesting complexes of photosystem II regulates the binding of violaxanthin and xanthophyll cycle. Plant Physiol 174:86–96. CrossRefPubMedPubMedCentralGoogle Scholar
  64. Wellburn AR (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144:307–313. CrossRefGoogle Scholar
  65. Young AJ, Britton G (1989) The distribution of α-carotene in the photosynthetic pigment-protein complexes of higher plants. Plant Sci 64:179–183. CrossRefGoogle Scholar
  66. Young A, Phillip D, Savill J (1996) Methods for carotenoid analysis. In: Pessaraki M (ed) Handbook of photosynthesis. Marcel Dekker Inc, New York, pp 597–622Google Scholar
  67. Ziegler R, Egle K (1965) Zur quantitativen Analyse der Chloroplastenpigmente. I. Kritische. berprüfung der spektralphotometrischen Chlorophyll-Bestimmung. Beitr Biol Pflanz 41:11–37Google Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Beatriz Fernández-Marín
    • 1
    Email author
  • José Ignacio García-Plazaola
    • 1
  • Antonio Hernández
    • 1
  • Raquel Esteban
    • 1
  1. 1.Department of Plant Biology and EcologyUniversity of the Basque Country (UPV/EHU)BilbaoSpain

Personalised recommendations