, Volume 245, Issue 6, pp 1215–1229 | Cite as

Lettuce flavonoids screening and phenotyping by chlorophyll fluorescence excitation ratio

  • Marek Zivcak
  • Klaudia Brückova
  • Oksana Sytar
  • Marian Brestic
  • Katarina Olsovska
  • Suleyman I. Allakhverdiev
Original Article


Main conclusion

Environmentally induced variation and the genotypic differences in flavonoid and phenolic content in lettuce can be reliably detected using the appropriate parameters derived from the records of rapid non-invasive fluorescence technique.

The chlorophyll fluorescence excitation ratio method was designed as a rapid and non-invasive tool to estimate the content of UV-absorbing phenolic compounds in plants. Using this technique, we have assessed the dynamics of accumulation of flavonoids related to developmental changes and environmental effects. Moreover, we have tested appropriateness of the method to identify the genotypic differences and fluctuations in total phenolics and flavonoid content in lettuce. Six green and two red genotypes of lettuce (Lactuca sativa L.) grown in pots were exposed to two different environments for 50 days: direct sunlight (UV-exposed) and greenhouse conditions (low UV). The indices based on the measurements of chlorophyll fluorescence after red, green and UV excitation indicated increase of the content of UV-absorbing compounds and anthocyanins in the epidermis of lettuce leaves. In similar, the biochemical analyses performed at the end of the experiment confirmed significantly higher total phenolic and flavonoid content in lettuce plants exposed to direct sun compared to greenhouse conditions and in red compared to green genotypes. As the correlation between the standard fluorescence indices and the biochemical records was negatively influenced by the presence of red genotypes, we proposed the use of a new parameter named Modified Flavonoid Index (MFI) taking into an account both absorbance changes due to flavonol and anthocyanin content, for which the correlation with flavonoid and phenolic content was relatively good. Thus, our results confirmed that the fluorescence excitation ratio method is useful for identifying the major differences in phenolic and flavonoid content in lettuce plants and it can be used for high-throughput pre-screening and phenotyping of leafy vegetables in research and breeding applications towards improvement of vegetable health effects.


Phenolic Flavonoids Chlorophyll fluorescence Lettuce UV radiation Phenotyping 



Fluorescence-based index for estimation of anthocyanin content in plant tissues




Dry weight


Fluorescence excitation ratio


Fluorescence-based index for estimation of flavonol content in plant tissues




Corrected fluorescence-based index for estimation of flavonol content


Far-red fluorescence, fluorescence with wavelength ~730 nm


Far-red fluorescence emitted after excitation by green light


Far-red fluorescence emitted after excitation by red light


Far-red fluorescence emitted after excitation by UV


Modified flavonoid index



This work was supported by the research projects VEGA-1-0923-16, APVV-15-0721,  by the EC project no. 26220220180: “Construction of the “AgroBioTech” Research Centre”, by Grants from the Russian Foundation for Basic Research, and by the Molecular and Cell Biology Programs of the Russian Academy of Sciences.


  1. Agati G, Tattini M (2010) Multiple functional roles of flavonoids in photoprotection. New Phytol 186:786–793CrossRefPubMedGoogle Scholar
  2. Agati G, Pinelli P, Cortes-Ebner S, Romani A, Cartelat A, Cerovic ZG (2005) Nondestructive evaluation of anthocyanins in olive (Olea europaea) fruits by in situ chlorophyll fluorescence spectroscopy. J Agric Food Chem 53:1354–1363CrossRefPubMedGoogle Scholar
  3. Agati G, Meyer S, Matteini P et al (2007) Assessment of anthocyanins in grape (Vitis vinifera L.) berries using a non-invasive chlorophyll fluorescence method. J Agric Food Chem 55:1053–1061CrossRefPubMedGoogle Scholar
  4. Agati G, Cerovic ZG, Pinelli P, Tattini M (2011) Light-induced accumulation of ortho-dihydroxylated flavonoids as non-destructively monitored by chlorophyll fluorescence excitation techniques. Environ Exp Bot 73:3–9CrossRefGoogle Scholar
  5. Altunkaya A, Gökmen V (2009) Effect of anti-browning agents on phenolic compounds profile of fresh lettuce (L. sativa). Food Chem 117:122–126CrossRefGoogle Scholar
  6. Amarowicz R, Weidner S, Wojtowicz I, Karmać M, Kosińska A, Rybarczyk A (2010) Influence of low-temperature stress on changes in the composition of grapevine leaf phenolic compounds and their antioxidant properties. Funct Plant Sci Biotechnol 4:90–96Google Scholar
  7. Asseng S, Turner NC (2007) Modelling genotype × environment × management interactions to improve yield, water use efficiency and grain protein in wheat. In: Spiertz JHJ, Struik PC, van Laar HH (eds) Scale and complexity in plant systems research—gene–plant–crop relations. Springer, Berlin, pp 93–103CrossRefGoogle Scholar
  8. Bassman JH (2004) Ecosystem consequences of enhanced solar ultraviolet radiation: secondary plant metabolites as mediators of multiple trophic interactions in terrestrial plant communities. Photochem Photobiol 79:382–398CrossRefPubMedGoogle Scholar
  9. Behn H, Tittmann S, Walter A, Schurr U, Noga G, Ulbrich A (2010) UV-B transmittance of greenhouse covering materials affects growth and flavonoid content of lettuce seedlings. Eur J Hortic Sci 75:259–268Google Scholar
  10. Bidel LPR, Meyer S, Goulas Y, Cadot Y, Cerovic ZG (2007) Responses of epidermal phenolic compounds to light acclimation: in vivo qualitative and quantitative assessment using chlorophyll fluorescence excitation spectra in leaves of three ligneous species. J Photochem Photobiol B 88:163–179CrossRefPubMedGoogle Scholar
  11. Bidel LP, Chomicki G, Bonini F, Mondolot L, Soulé J, Coumans M, La Fisca P, Baissac Y, Petit V, Loiseau A, Cerovic ZG, Gould KS, Jay-Allemand C (2015) Dynamics of flavonol accumulation in leaf tissues under different UV-B regimes in Centella asiatica (Apiaceae). Planta 242:545–559CrossRefPubMedGoogle Scholar
  12. Bilger W, Johnsen T, Schreiber U (2001) UV-excited chlorophyll fluorescence as a tool for the assessment of UV-protection by the epidermis of plants. J Exp Bot 52:2007–2014CrossRefPubMedGoogle Scholar
  13. Brestic M, Zivcak M (2013) PSII fluorescence techniques for measurement of drought and high temperature stress signal in crop plants: protocols and applications. In: Rout GR, Das AB (eds) Molecular stress physiology of plants. Springer, India, pp 87–131CrossRefGoogle Scholar
  14. Brestic M, Zivcak M, Kalaji HM, Carpentier R, Allakhverdiev SI (2012) Photosystem II thermostability in situ: environmentally induced acclimation and genotype-specific reactions in Triticum aestivum L. Plant Physiol Biochem 57:93–105CrossRefPubMedGoogle Scholar
  15. Burchard P, Bilger W, Weissenböck G (2000) Contribution of hydroxycinnamates and flavonoids to epidermal shielding of UV-A and UV-B radiation in developing rye primary leaves as assessed by ultraviolet-induced chlorophyll fluorescence measurements. Plant Cell Environ 23:1373–1380CrossRefGoogle Scholar
  16. Cerovic ZG, Ounis A, Cartelat A, Latouche G, Goulas Y, Meyer S, Moya I (2002) The use of chlorophyll fluorescence excitation spectra for the non-destructive in situ assessment of UV-absorbing compounds in leaves. Plant Cell Environ 25:1663–1676CrossRefGoogle Scholar
  17. Cerovic ZG, Ghozlen NB, Milhade C, Obert M, Debuisson S, Moigne ML (2015) Nondestructive diagnostic test for nitrogen nutrition of grapevine (Vitis vinifera L.) based on dualex leaf-clip measurements in the field. J Agric Food Chem 63:3669–3680CrossRefPubMedGoogle Scholar
  18. Chandrasekara A, Shahidi F (2010) Content of insoluble bound phenolics in millets and their contribution to antioxidant capacity. J Agric Food Chem 58:6706–6714CrossRefPubMedGoogle Scholar
  19. Cook NC, Samman S (1996) Flavonoids—chemistry, metabolism, cardioprotective effects, and dietary sources. J Nutr Biochem 7:66–76CrossRefGoogle Scholar
  20. Crozier A, Lean MEJ, McDonald MS, Black C (1997) Quantitative analysis of the flavonoid content of commercial tomatoes, onions, lettuce and celery. J Agric Food Chem 45:590–595CrossRefGoogle Scholar
  21. Dumpert K, Knacker T (1985) A comparison of the effects of enhanced UV-B radiation on crop plants exposed to greenhouse and field conditions. Biochemy Physiol Pflanzen 180:599–612CrossRefGoogle Scholar
  22. Furbank RT, Tester M (2011) Phenomics–technologies to relieve the phenotyping bottleneck. Trends Plant Sci 16:635–644CrossRefPubMedGoogle Scholar
  23. Garcia-Macias P, Ordidge M, Vysini E, Waroonphan S, Battey NH, Gordon MH, Hadley P, John P, Lovegrove JA, Wagstaffe A (2007) Changes in the flavonoid and phenolic acid contents and antioxidant activity of red leaf lettuce (Lollo Rosso) due to cultivation under plastic films varying in ultraviolet transparency. J Agric Food Chem 55:10168–10172CrossRefPubMedGoogle Scholar
  24. Ghozlen NB, Cerovic ZG, Germain C, Toutain S, Latouche G (2010) Non-destructive optical monitoring of grape maturation by proximal sensing. Sensors 10:10040–10068CrossRefPubMedPubMedCentralGoogle Scholar
  25. Gould KS, McKelvie J, Markham KR (2002) Do anthocyanins function as antioxidants in leaves? Imaging of H2O2 in red and green leaves after mechanical injury. Plant Cell Environ 25:1261–1269CrossRefGoogle Scholar
  26. Harbinson J, Prinzenburg AE, Kruijer W, Aarts MGM (2012) High throughput screening with chlorophyll fluorescence imaging and its use in crop improvement. Curr Opin Biotechnol 23:221–226CrossRefPubMedGoogle Scholar
  27. Havaux M, Kloppstech K (2001) The protective functions of carotenoid and flavonoid pigments against excess visible radiation at chilling temperature investigated in Arabidopsis npq and tt mutants. Planta 213:953–966CrossRefGoogle Scholar
  28. Jordan BR (2002) Review: molecular response of plant cells to UV-B stress. Funct Plant Biol 29:909–916CrossRefGoogle Scholar
  29. Kalaji MH, Jajoo A, Oukarroum A, Brestic M, Zivcak M, Samborska AI, Cetner DM, Lukasik I, Goltsev V, Ladle JR (2016) Chlorophyll a fluorescence as a tool to monitor physiological status of plants under abiotic stress conditions. Acta Physiol Plant 38:102CrossRefGoogle Scholar
  30. Kalaji HM, Schansker G, Ladle RJ et al (2017) Frequently asked questions about in vivo chlorophyll fluorescence: the sequel. Photosynth Res. doi: 10.1007/s11120-016-0318-y Google Scholar
  31. 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(5):613–621CrossRefPubMedGoogle Scholar
  32. Kaushik P, Andújar I, Vilanova S, Plazas M, Gramazio P, Herraiz FJ, Brar NS, Prohens J (2015) Breeding vegetables with increased content in bioactive phenolic acids. Molecules 20:18464–18481CrossRefPubMedGoogle Scholar
  33. Kolb CA, Pfündel E (2005) Origins of non-linear and dissimilar relationships between epidermal UV absorbance and UV absorbance of extracted phenolics in leaves of grapevine and barley. Plant Cell Environ 25:580–590CrossRefGoogle Scholar
  34. Kolb C, Käser M, Kopecký J, Zotz G, Riederer M, Pfündel E (2001) Effects of natural intensities of visible and UV radiation on epidermal UV-screening and photosynthesis in grape leaves (Vitis vinifera cv. Silvaner). Plant Physiol 127:863–875CrossRefPubMedPubMedCentralGoogle Scholar
  35. Krizek DT, Britz SJ, Mirecki RM (1998) Inhibitory effects of ambient levels of solar UV-A and UV-B radiation on growth of cv. New Red Fire lettuce. Physiol Plantarum 103:1–7CrossRefGoogle Scholar
  36. Król A, Amarowicz R, Weidner S (2014) Changes in the composition of phenolic compounds and antioxidant properties of grapevine roots and leaves (Vitis vinifera L.) under continuous of long-term drought stress. Acta Physiol Plant 36:1491–1499CrossRefGoogle Scholar
  37. Kumar S, Pandey AK (2013) Chemistry and biological activities of flavonoids: an overview. Sci World J 2013:1–16Google Scholar
  38. Lachman J, Proněk D, Hejtmánková A, Dudjak J, Pivec V, Faitová K (2003) Total polyphenol and main flavonoid antioxidants in different onion (Allium cepa L.) varieties. Sci Hortic 30:142–147Google Scholar
  39. Latouche G, Bellow S, Poutaraud A, Meyer S, Cerovic ZG (2013) Influence of constitutive phenolic compounds on the response of grapevine (Vitis vinifera L.) leaves to infection by Plasmopara viticola. Planta 237:351–361CrossRefPubMedGoogle Scholar
  40. Lejealle S, Evain S, Cerovic ZG (2010) Multiplex: a new diagnostic tool for management of nitrogen fertilization of turfgrass. In: 10th International conference on precision agriculture, vol 15, DenverGoogle Scholar
  41. Lindoo SJ, Caldwell MM (1978) UV-B radiation induced inhibition of leaf expansion and promotion of anthocyanidin production. Plant Physiol 61:278–282CrossRefPubMedPubMedCentralGoogle Scholar
  42. Llorach R, Martínez-Sánchez A, Tomás-Barberán FA, Gil MI, Ferreres F (2008) Characterisation of polyphenols and antioxidant properties of five lettuce varieties and escarole. Food Chem 108:1028–1038CrossRefPubMedGoogle Scholar
  43. Lovdal T, Olsen KM, Slimestad R, Verheul M, Lillo C (2010) Synergetic effects of nitrogen depletion, temperature, and light on the content of phenolic compounds and gene expression in leaves of tomato. Phytochemistry 71:605–613CrossRefPubMedGoogle Scholar
  44. Mabry TJ, Markham KR, Thomas MB (1970) The systematic identification of flavonoids. Springer, BerlinCrossRefGoogle Scholar
  45. 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
  46. Meyer S, Louis J, Moise N et al (2009) Developmental changes in spatial distribution of in vivo fluorescence and epidermal UV absorbance over Quercus petraea leaves. Ann Bot Lond 104:621–633CrossRefGoogle Scholar
  47. Morales LO, Tegelberg R, Brosche M, Keinanen M, Linfors A, Aphalo PJ (2010) Effects of solar UV-A and UV-B radiation on gene expression and phenolic accumulation in Betula pendula leaves. Tree Phisyol 30:923–934CrossRefGoogle Scholar
  48. Morales LO, Tegelberg R, Brosché M, Lindfors A, Siipola S, Aphalo PJ (2011) Temporal variation in epidermal flavonoids due to altered solar UV radiation is moderated by the leaf position in Betula pendula. Physiol Plant 143:261–270CrossRefPubMedGoogle Scholar
  49. Müller V, Albert A, Winkler JB, Lankes C, Noga G, Hunsche M (2013) Ecologically relevant UV-B dose combined with high PAR intensity distinctly affect plant growth and accumulation of secondary metabolites in leaves of Centella asiatica L. Urban. J Photochem Photobiol B Biol 127:161–169CrossRefGoogle Scholar
  50. Murata N, Takahashi S, Nishiyama Y, Allakhverdiev SI (2007) Photoinhibition of photosystem II under environmental stress. Biochim Biophys Acta 1767:414–421CrossRefPubMedGoogle Scholar
  51. Nicolle C, Carnat A, Fraisse D, Lamaison JL, Rock E, Michel H, Amouroux P, Remesy C (2004) Characterisation and variation of antioxidant micronutrients in lettuce (Lactuca sativa folium). J Sci Food Agric 84:2061–2069CrossRefGoogle Scholar
  52. Nishiyama Y, Yamamoto H, Allakhverdiev SI, Inaba M, Yokota A, Murata N (2001) Oxidative stress inhibits the repair of photodamage to the photosynthetic machinery. EMBO J 20:5587–5594CrossRefPubMedPubMedCentralGoogle Scholar
  53. Nishiyama Y, Allakhverdiev SI, Murata N (2006) A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II. Biochim Biophys Acta 1757:742–749CrossRefPubMedGoogle Scholar
  54. Ozgen S, Sekerci S (2011) Effect of leaf position on the distribution of phytochemicals and antioxidant capacity among green and red lettuce cultivars. Spanish J Agric Res 9:801–809CrossRefGoogle Scholar
  55. Parr AJ, Bolwell GP (2000) Phenols in the plant and in man. The potential for possible nutritional enhancement of the diet by modifying the phenols content in profile. J Sci Food Agric 80:985–1012CrossRefGoogle Scholar
  56. Pfündel EE, Ghozlen NB, Meyer S, Cerovic ZG (2007) Investigating UV screening in leaves by two different types of portable UV fluorimeters reveals in vivo screening by anthocyanins and carotenoids. Photosynth Res 93:205–221CrossRefPubMedGoogle Scholar
  57. 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
  58. Romani A, Pimnelli P, Galardi C, Sani G, Cimato A, Heimler D (2002) Polyphenols in greenhouse and open-air-grown lettuce. Food Chem 79:337–342CrossRefGoogle Scholar
  59. Scalbert A, Williamson G (2000) Dietary intake and bioavailability of polyphenols. J Nutr 130:2073–2085Google Scholar
  60. Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic 16:144–158Google Scholar
  61. Sivankalyani V, Feygenberg O, Diskin S, Wright B, Alkan N (2016) Increased anthocyanin and flavonoids in mango fruit peel are associated with cold and pathogen resistance. Postharvest Biol Technol 111:132–139CrossRefGoogle Scholar
  62. Skaar I, Adaku C, Jordheim M, Byamukama R, Kiremire B, Andersen ØM (2014) Purple anthocyanin colouration on lower (abaxial) leaf surface of Hemigraphis colorata (Acanthaceae). Phytochemistry 105:141–146CrossRefPubMedPubMedCentralGoogle Scholar
  63. Suthaparan A, Stensvand A, Solhaug KA, Torre S, Mortensen LM, Gadoury DM, Seem RC, Gislerød HR (2012) Suppression of powdery mildew (Podosphaera pannosa) in greenhouse roses by brief exposure to supplemental UV-B radiation. Plant Dis 96:1653–1660CrossRefGoogle Scholar
  64. Sytar O, Brestic M, Rai M, Shao HB (2012) Plant phenolic compounds for food, pharmaceutical and cosmetics production. J Med Plants Res 6:2526–2539Google Scholar
  65. Sytar O, Kosyan A, Taran N, Smetanska I (2014) Antocyanins as marker for selection of buckwheat plants with high rutin content. Gesunde Pflanz 66:165–169CrossRefGoogle Scholar
  66. Sytar O, Bruckova K, Hunkova E, Zivcak M, Konate K, Brestic M (2015) The application of multiplex fluorimetric sensor for the analysis of flavonoids content in the medicinal herbs family Asteraceae, Lamiaceae, Rosaceae. Biol Res 48:1–9CrossRefGoogle Scholar
  67. Sytar O, Zivcak M, Brestic M (2016) Noninvasive methods to support metabolomic studies targeted at plant phenolics for food and medicinal use. In: Hakeem KR, Tombuloglu H, Tombuloğlu G (eds) Plant omics: trends and applications. Springer, Switzerland, pp 406–432Google Scholar
  68. Teramura AH, Murali NS (1986) Intraspecific differences in growth and yield of soybean exposed to ultraviolet-B radiation under greenhouse and field conditions. Environ Exp Bot 26:89–95CrossRefGoogle Scholar
  69. Tevini M, Braun J, Fieser G (1991) The protective function of the epidermal layer of rye seedlings against ultraviolet-B radiation. Photochem Photobiol 53:329–333CrossRefGoogle Scholar
  70. Tremblay N, Wang Z, Bélec C (2007) Evaluation of the Dualex for the assessment of corn nitrogen status. J Plant Nutr 30:1355–1369CrossRefGoogle Scholar
  71. Tsormpatsidis E, Henbest RGC, Battey NH, Hadley P (2010) The influence of ultraviolet radiation on growth, photosynthesis and phenolic levels of green and red lettuce: potential for exploiting effects of ultraviolet radiation in a production system. Ann Appl Biol 156:357–366CrossRefGoogle Scholar
  72. Tuccio L, Remorini D, Pinelli P, Fierini E, Tonutti P, Scalabrelli G, Agati G (2011) Rapid and non-destructive method to assess in the vineyard grape berry anthocyanins under different seasonal and water conditions. Aust J Grape Wine Res 17:181–189CrossRefGoogle Scholar
  73. Weidner S, Frączek E, Amarowicz R, Abe S (2001) Alternations in phenolic acids content in developing rye grains in normal environment and during enforced dehydration. Acta Physiol Plant 23:475–482CrossRefGoogle Scholar
  74. Weidner S, Karamać M, Amarowicz R, Szypulska E, Golgowska A (2007) Changes in composition of phenolic compounds and antioxidant properties of Vitis amurensis seeds germinated under osmotic stress. Acta Physiol Plant 29:238–290CrossRefGoogle Scholar
  75. Wróbel M, Karmać M, Amarowicz R, Frączek E, Weidner S (2005) Metabolism of phenolic compounds in Vitis riparia seeds during stratification and during germination under optimal and low temperature stress conditions. Acta Physiol Plant 27:313–320CrossRefGoogle Scholar
  76. Zhang WJ, Björn LO (2009) The effect of ultraviolet radiation on the accumulation of medicinal compounds in plants. Fitoterapia 80:207–218CrossRefPubMedGoogle Scholar
  77. Zivcak M, Olsovska K, Slamka P, Galambosova J, Rataj V, Shao HB, Brestic M (2014) Application of chlorophyll fluorescence performance indices to assess the wheat photosynthetic functions influenced by nitrogen deficiency. Plant Soil Environ 60:210–215Google Scholar

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© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Department of Plant PhysiologySlovak Agricultural UniversityNitraSlovak Republic
  2. 2.Agrobiotech Research CenterSlovak Agricultural UniversityNitraSlovak Republic
  3. 3.Educational and Scientific Centre, Institute of Biology and MedicineTaras Shevchenko National University of KyivKyivUkraine
  4. 4.Institute of Plant PhysiologyRussian Academy of SciencesMoscowRussia
  5. 5.Institute of Basic Biological ProblemsRussian Academy of SciencesPushchino, Moscow RegionRussia
  6. 6.Department of Plant Physiology, Faculty of BiologyM.V. Lomonosov Moscow State UniversityMoscowRussia
  7. 7.Institute of Molecular Biology and Biotechnology, Azerbaijan National Academy of SciencesBakuAzerbaijan

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