Skip to main content
Log in

Variability and application of the chlorophyll fluorescence emission ratio red/far-red of leaves

  • Research Article
  • Published:
Photosynthesis Research Aims and scope Submit manuscript

Abstract

Various approaches to understand and make use of the variable chlorophyll (Chl) fluorescence emission spectrum and fluorescence ratio are reviewed. The Chl fluorescence of leaves consists of two maxima in the red (near 685–690 nm), and far-red region (near 730–740 nm). The intensity and shape of the Chl fluorescence emission spectrum of leaves at room temperature are primarily dependent on the concentration of the fluorophore Chl a, and to a lower degree also on the leaf structure, the photosynthetic activity, and the leaf’s optical properties. The latter determine the penetration of excitation light into the leaf as well as the emission of Chl fluorescence from different depths of the leaf. Due to the re-absorption mainly of the red Chl fluorescence band emitted inside the leaf, the ratio between the red and the far-red Chl fluorescence maxima (near 690 and 730–740 nm, respectively), e.g., as F690/F735, decreases with increasing Chl content in a curvilinear relationship and is a good inverse indicator of the Chl content of the leaf tissue, e.g., before and after stress events. The Chl fluorescence ratio of leaves can be applied for Chl determinations in basic photosynthesis research, agriculture, horticulture, and forestry. It can be used to assess changes of the photosynthetic apparatus, developmental processes of leaves, state of health, stress events, stress tolerance, and also to detect diseases or N-deficiency of plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

Chl:

Chlorophyll

F690:

Fluorescence intensity at the red maximum of the Chl fluorescence emission spectrum of a leaf near 690 nm

F735 and F740:

Fluorescence intensity at the far-red maximum of the Chl fluorescence near 735–740 nm

PS I:

Photosystem I

PS II:

Photosystem II

References

  • Agati G (1998) Response of the in vivo chlorophyll fluorescence spectrum to environmental factors and laser excitation wavelength. Pure Appl Opt 7:797–807

    Article  CAS  Google Scholar 

  • Agati G, Fusi F, Mazzinghi P, di Paola ML (1993) A simple approach to the evaluation of the re-absorption of chlorophyll fluorescence spectra in intact leaves. J Photoch Photobio B 17:163–171

    Article  CAS  Google Scholar 

  • Agati G, Cerovic ZG, Moya I (2000) The effect of decreasing temperature up to chilling values on the in vivo F685/F735 chlorophyll fluorescence ratio in Phaseolus vulgaris and Pisum sativum: the role of the photosystem I contribution to the 735 nm fluorescence band. Photochem Photobiol 72:75–84

    Article  PubMed  CAS  Google Scholar 

  • D’Ambrosio N, Szábo K, Lichtenthaler HK (1992) Increase of the chlorophyll fluorescence ratio F690/F735 during the autumnal chlorophyll breakdown. Radiat Environ Bioph 31:51–62

    Article  CAS  Google Scholar 

  • Babani F, Lichtenthaler HK, Richter P (1996) Changes of chlorophyll fluorescence signatures during greening of etiolated barley seedlings as measured with the CCD-OMA fluorometer. J Plant Physiol 148:471–477

    CAS  Google Scholar 

  • Bartošková H, Nauš J, Výkruta M (1999) The arrangement of chloroplasts in cells influences the reabsorption of chlorophyll fluorescence emission. The effect of desiccation on the chlorophyll fluorescence of Rhizomnium punctatum leaves. Photosynth Res 62:251–260

    Article  Google Scholar 

  • Benson AA (2002) Following the path of carbon in photosynthesis: a personal story. Photosynth Res 73:29–49

    Article  PubMed  CAS  Google Scholar 

  • Benson AA, Bassham JA, Calvin M, Hall AG, Hirsch HE, Kawaguchi S, Lynch V, Tolbert NE (1952) The path of carbon in photosynthesis. XV. Ribulose and sedoheptulose. J Biol Chem 196:703–716

    PubMed  CAS  Google Scholar 

  • Bigus H-J, Voß P, Krause-Bonte J, Stransky H, Gauglitz G, Hager A (1995) Light-dependent assembly of pigment–protein complexes in etiolated leaves of Phaseolus coccineus L. monitored by seminative gel electrophoresis, fluorescence spectroscopy and pigment analysis. J Plant Physiol 147:408–418

    CAS  Google Scholar 

  • Bornman JF, Vogelmann TC, Martin G (1991) Measurement of chlorophyll fluorescence within leaves using a fibre optic microprobe. Plant Cell Environ 14:719–725

    Article  CAS  Google Scholar 

  • Buschmann C (1981) The characterization of the developing photosynthetic apparatus in greening barley leaves by means of (slow) fluorescence kinetic measurements. In: Akoyunoglou G (ed) Photosynthesis. Balaban International Science Services, Philadelphia, pp 417–426

    Google Scholar 

  • Buschmann C, Lichtenthaler HK (1988a) Complete fluorescence emission spectra determined during the induction kinetic using a diode-array detector. In: Lichtenthaler HK (ed) Applications of chlorophyll fluorescence. Kluwer Academic Publishers, Dordrecht, pp 77–84

    Google Scholar 

  • Buschmann C, Lichtenthaler HK (1988b) Reflectance and chlorophyll fluorescence signatures of leaves. In: Lichtenthaler HK (ed) Applications of chlorophyll fluorescence. Kluwer Academic Publishers, Dordrecht, pp 325–332

    Google Scholar 

  • Buschmann C, Lichtenthaler HK (1998) Principles and characteristics of multi-colour fluorescence imaging of plants. J Plant Physiol 152:297–314

    CAS  Google Scholar 

  • Buschmann C, Lichtenthaler HK (1999) Contribution of chlorophyll fluorescence to the reflectance of leaves in stressed plants as determined with the VIRAF-spectrometer. Z Naturforsch 54c:849–855

    Google Scholar 

  • Buschmann C, Nagel E (1993) In vivo spectroscopy and internal optics of leaves as basis for remote sensing of vegetation. Int J Remote Sens 14:711–722

    Article  Google Scholar 

  • Buschmann C, Schrey H (1981) Fluorescence induction kinetics of green and etiolated leaves by recording the complete in vivo emission spectra. Photosynth Res 1:233–241

    Article  Google Scholar 

  • Buschmann C, Nagel E, Szabó K, Kocsányi L (1994) Spectrometer for fast measurements of in vivo reflection, absorption and fluorescence in the visible and near infrared. Remote Sens Environ 48:18–24

    Article  Google Scholar 

  • Cerovic ZG, Samson G, Morales F, Tremblay N, Moya I (1999) Ultraviolet-induced fluorescence for plant monitoring: present state and prospects. Agronomie 19:543–578

    Google Scholar 

  • Chappelle EW, Wood FM, McMurtrey YE, Newcomb WW (1984) Laser induced fluorescence of green plants. 1: A technique for remote detection of plant stress and species differentiation. Appl Optics 23:134–138

    CAS  Google Scholar 

  • Cordón GB, Lagorio MG (2006) Re-absorption of chlorophyll fluorescence in leaves revisited. A comparison of correction models. Photochem Photobio Sci 5:735–740

    Article  CAS  Google Scholar 

  • Csintalan Z, Tubá Z, Lichtenthaler HK (1998) Changes in laser-induced chlorophyll fluorescence ratio F690/F735 in the poikilochlorophyllous desiccation tolerant plant Xerophyta scabrida during desiccation. J Plant Physiol 151:540–544

    Google Scholar 

  • Daley PF, Raschke K, Ball JT, Berry JA (1989) Topography of photosynthetic activity of leaves obtained from video imaging of chlorophyll fluorescence. Plant Physiol 90:1233–1238

    PubMed  CAS  Google Scholar 

  • Edner H, Johansson J, Svanberg S, Lichtenthaler HK, Lang M, Stober F, Schindler C, Björn L-O (1995) Remote multi-colour fluorescence imaging of selected broad-leaf plants. EARSeL Adv Remote Sens 3:2–14

    Google Scholar 

  • Falkowski PG, Koblížek M, Gorbunov M, Kolber Z (2004) Development and application of variable chlorophyll fluorescence techniques in marine ecosystems. In: Papageorgiou GC, Govindjee (eds) Chlorophyll a fluorescence: a signature of photosynthesis. Springer, Dordrecht, pp 757–778

  • Gilroy S (1997) Fluorescence microscopy of living plant cells. Annu Rev Plant Phys 48:165–190

    Article  CAS  Google Scholar 

  • Gitelson AA, Buschmann C, Lichtenthaler HK (1998) Leaf chlorophyll fluorescence corrected for re-absorption by means of absorption and reflectance measurements. J Plant Physiol 152:283–296

    CAS  Google Scholar 

  • Gitelson AA, Buschmann C, Lichtenthaler HK (1999) The chlorophyll fluorescence ratio F735/F700 as an accurate measure of the chlorophyll content in plants. Remote Sens Environ 69:296–302

    Article  Google Scholar 

  • Goodwin RH (1953) Fluorescent substances in plants. Annu Rev Plant Phys 4:283–304

    Article  Google Scholar 

  • Günther KP, Dahn H-G, Lüdeker W (1994) Remote sensing vegetation status by laser-induced fluorescence. Remote Sens Environ 47:10–17

    Article  Google Scholar 

  • Hák R, Lichtenthaler HK, Rinderle U (1990) Decrease of the chlorophyll fluorescence ratio F690/F730 during greening and development of leaves. Radiat Environ Bioph 29:329–336

    Article  Google Scholar 

  • Hák R, Rinderle-Zimmer U, Lichtenthaler HK, Nátr L (1993) Chlorophyll a fluorescence signatures of nitrogen deficient barley leaves. Photosynthetica 28:151–159

    Google Scholar 

  • Heber U, Lange OL, Shuvalov VA (2006) Conservation and dissipation of light energy as complementary processes: homoiohydric and poikilohydric autotrophs. J Exp Bot 57:1211–1223

    Article  PubMed  CAS  Google Scholar 

  • Hoge FE, Swift RN, Yungel JK (1983) Feasibility of airborne detection of laser-induced fluorescence emissions from green terrestrial plants. Appl Optics 22:2991–3000

    CAS  Google Scholar 

  • Holzwarth AR, Wendler J, Haehnel W (1985) Time-resolved picosecond fluorescence spectra of the antenna chlorophylls in Chlorella vulgaris. Resolution of photosystem I fluorescence. Biochim Biophys Acta 807:155–167

    Article  CAS  Google Scholar 

  • Itoh S, Sugiura K (2004) Fluorescence of photosystem I. In: Papageorgiou GC, Govindjee (eds) Chlorophyll a fluorescence: a signature of photosynthesis. Springer, Dordrecht, pp 231–250

    Google Scholar 

  • Koizumi M, Takahashi K, Mineuchi K, Nakamura T, Kano H (1998) Light gradients and the transverse distribution of chlorophyll fluorescence in mangrove and Camellia leaves. Ann Bot 81:527–533

    Article  CAS  Google Scholar 

  • Lang M, Stober F, Lichtenthaler HK (1991) Fluorescence emission spectra of plant leaves and plant constituents. Radiat Environ Bioph 30:333–347

    Article  CAS  Google Scholar 

  • Lichtenthaler HK (1987) Chlorophyll fluorescence signatures of leaves during the autumnal chlorophyll breakdown. J Plant Physiol 131:101–110

    CAS  Google Scholar 

  • Lichtenthaler HK (1992) The Kautsky effect: 60 years of chlorophyll fluorescence induction kinetics. Photosynthetica 27:45–55

    CAS  Google Scholar 

  • Lichtenthaler HK, Babani F (2004) Light adaptation and senescence of the photosynthetic apparatus. Changes in pigment composition, chlorophyll fluorescence parameters and photosynthetic activity. In: Papageorgiou GC, Govindjee (eds) Chlorophyll a fluorescence: a signature of photosynthesis. Springer, Dordrecht, pp 713–736

    Google Scholar 

  • Lichtenthaler HK, Miehé JA (1997) Fluorescence imaging as a diagnostic tool for plant stress. Trends Plant Sci 2:316–320

    Article  Google Scholar 

  • Lichtenthaler HK, Rinderle U (1988a) The role of chlorophyll fluorescence in the detection of stress conditions in plants. CRC Cr Rev Anal Chem 19:S29–S85

    Google Scholar 

  • Lichtenthaler HK, Rinderle (1988b) Chlorophyll fluorescence spectra of leaves as induced by blue light and red laser light. In: Proceedings of the 4th international colloquium on spectral signatures of objects in remote sensing, Aussois, ESA Publications Division, Noordwijk, pp 251–254

  • Lichtenthaler HK, Schweiger J (1998) Cell wall bound ferulic acid, the major substance of the blue-green fluorescence emission of plants. J Plant Physiol 152:272–282

    CAS  Google Scholar 

  • Lichtenthaler HK, Prenzel U, Kuhn G (1982a) Carotenoid composition of chlorophyll-carotenoid-proteins from radish chloroplasts. Z Naturforsch 37c:10–12

    CAS  Google Scholar 

  • Lichtenthaler HK, Kuhn G, Prenzel U, Buschmann C, Meier D (1982b) Adaptation of chloroplast-ultrastructure and of chlorophyll-protein levels to high-light and low-light growth conditions. Z Naturforsch 37c:464–475

    CAS  Google Scholar 

  • Lichtenthaler HK, Hák R, Rinderle U (1990) The chlorophyll fluorescence ratio F690/F730 in leaves of different chlorophyll content. Photosynth Res 25:295–298

    Article  CAS  Google Scholar 

  • Lichtenthaler HK, Stober F, Lang M (1992) The nature of the different laser-induced fluorescence signatures of plants. EARSeL Adv Remote Sens 1:20–32

    Google Scholar 

  • Lichtenthaler HK, Lang M, Sowinska M, Heisel F, Miehé JA (1996) Detection of vegetation stress via a new high resolution fluorescence imaging system. J Plant Physiol 148:599–612

    CAS  Google Scholar 

  • Lichtenthaler HK, Lang M, Sowinska M, Summ P, Heisel F, Miehé JA (1997) Uptake of the herbicide diuron (DCMU) as visualized by the fluorescence imaging technique. Bot Acta 110:158–163

    CAS  Google Scholar 

  • Lombard F, Strasser RJ (1984) Evidence for spill over changes during state 1-state 2 transition in green leaves. In: Sybesma C (ed) Photosynthesis, vol 3. Dr W Junk Publishers, The Hague, pp 4.271–4.274

    Google Scholar 

  • Louis J, Cerovic Z, Moya I (2006) Quantitative study of fluorescence excitation and emission spectra of bean leaves. J Photochem Photobiol B 84:65–71

    Article  CAS  Google Scholar 

  • McFarlane JC, Watson RD, Theisen AF, Jackson RD, Ehrler WL, Pinter PJ, Idso SB, Reginato RJ (1980) Plant stress detection by remote measurement of fluorescence. Appl Optics 19:3287–3289

    Article  Google Scholar 

  • Moya I, Cerovic ZG (2004) Remote sensing of chlorophyll fluorescence: Instrumentation and analysis. In: Papageorgiou GC, Govindjee (eds) Chlorophyll a fluorescence: a signature of photosynthesis. Springer, Dordrecht, pp 429–445

    Google Scholar 

  • Murata N, Nishimura M, Takamiya A (1966) Fluorescence of chlorophyll in photosynthetic systems. 3. Emission and action spectra of fluorescence – three emission bands of chlorophyll a and the energy transfer between two pigment systems. Biochim Biophys Acta 126:234–243

    Article  PubMed  CAS  Google Scholar 

  • Nedbal L, Whitmarsh J (2004) Chlorophyll fluorescence imaging of leaves and fruits. In: Papageorgiou GC, Govindjee (eds) Chlorophyll a fluorescence: a signature of photosynthesis. Springer, Dordrecht, pp 389–407

    Google Scholar 

  • Oxborough K (2004) Using chlorophyll fluorescence imaging to monitor photosynthetic performances. In: Papageorgiou GC, Govindjee (eds) Chlorophyll a fluorescence: a signature of photosynthesis. Springer, Dordrecht, pp 409–428

    Google Scholar 

  • Papageorgiou G, Govindjee (eds) (2004) Chlorophyll a fluorescence – a signature of photosynthesis. Springer, Dordrecht

  • Peterson RB, Oja V, Laisk A (2001) Chlorophyll fluorescence at 680 and 730 nm and leaf photosynthesis. Photosynth Res 70:185–196

    Article  PubMed  CAS  Google Scholar 

  • Pfündel E (1998) Estimating the contribution of photosystem I to total leaf chlorophyll fluorescence. Photosynth Res 56:185–195

    Article  Google Scholar 

  • Ramos ME, Lagorio MG (2004) True fluorescence spectra of leaves. Photochem Photobio Sci 3:1063–1066

    Article  CAS  Google Scholar 

  • Rinderle U, Lichtenthaler HK (1988) The chlorophyll fluorescence ratio F690/F735 as a possible stress indicator. In: Lichtenthaler HK (ed) Applications of chlorophyll fluorescence. Kluwer Academic Publishers, Dordrecht, pp 189–196

    Google Scholar 

  • Rolfe SA, Scholes JD (1995) Quantitative imaging of chlorophyll fluorescence. New Phytol 131:69–79

    Article  Google Scholar 

  • Rosema A, Cecchi G, Pantani L, Radicatti B, Romuli M, Mazzinghi P, van Kooten O, Kliffen C (1992) Monitoring photosynthetic activity and ozone stress by laser induced fluorescence in trees. Int J Remote Sens 13:737–751

    Article  Google Scholar 

  • Šestak Z, Šiffel P (1997) Leaf-age difference in chlorophyll fluorescence. Photosynthetica 33:347–369

    Google Scholar 

  • Shibata K, Benson AA, Calvin M (1954) The absorption spectra of suspensions of living micro-organisms. Biochim Biophys Acta 15:461–470

    Article  PubMed  CAS  Google Scholar 

  • Smorenburg K, Bazalgette Courrèges-Lacoste G, Berger M, Buschmann C, Court A, Del Bello U, Langsdorf G, Lichtenthaler HK, Sioris C, Stoll M-P, Visser H (2002) Remote sensing of solar induced fluorescence of vegetation. Proc SPIE 4542:178–190

    Article  Google Scholar 

  • Sowinska M, Cunin B, Deruyver A, Heisel F, Miehé JA, Langsdorf G, Lichtenthaler HK (1999) Near-field measurements of vegetation by laser-induced fluorescence imaging. Proc SPIE 3868:120–131

    Article  Google Scholar 

  • Stober F, Lichtenthaler HK (1992) Changes of the laser-induced blue, green and red fluorescence signatures during greening of etiolated leaves of wheat. J Plant Physiol 140:673–680

    CAS  Google Scholar 

  • Strasser RJ, Butler WL (1977) Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants. Biochim Biophys Acta 462:307–313

    Article  PubMed  CAS  Google Scholar 

  • Strasser RJ, Schwarz B, Bucher JB (1987) Simultane Messung der Chlorophyll-Fluoreszenz-Kinetik bei verschiedenen Wellenlängen als rasches Verfahren zur Frühdiagnose von Immissionsbelastungen an Waldbäumen: Ozoneinwirkungen auf Buchen und Pappeln. Eur J Forest Pathol 17:149–157

    Article  CAS  Google Scholar 

  • Szabó K, Lichtenthaler HK, Kocsányi L, Richter P (1992) A CCD-OMA device for the measurement of complete chlorophyll fluorescence emission spectra of leaves during the fluorescence induction kinetics. Radiat Environ Bioph 31:153–160

    Article  Google Scholar 

  • Takács Z, Lichtenthaler HK, Tubá Z (2000) Fluorescence emission spectra of desiccation-tolerant cryptogamic plants during a rehydration-desiccation cycle. J Plant Physiol 156:375–379

    Google Scholar 

  • Takahashi K, Mineuchi K, Nakamura T, Koizumi M, Kano H (1994) A system for imaging transverse distribution of scattered light and chlorophyll fluorescence in intact rice leaves. Plant Cell Environ 17:105–110

    Article  Google Scholar 

  • Terjung F (1998) Reabsorption of chlorophyll fluorescence and its effects on the spectral distribution and the picosecond decay of higher plant leaves. Z Naturforsch 53c:924–926

    Google Scholar 

  • Thornber JP (1975) Chlorophyll-proteins: light-harvesting and reaction center components of plants. Annu Rev Plant Phys 26:127–158

    Article  CAS  Google Scholar 

  • Valeur C (2002) Molecular fluorescence – principles and applications. Wiley-VCH, Weinheim

    Google Scholar 

  • Virgin HI (1954) The distortion of fluorescence spectra in leaves by light scattering and its reduction by infiltration. Physiol Plant 7:560–570

    Article  Google Scholar 

  • Vogelmann TC (1993) Plant tissue optics. Annu Rev Plant Phys 44:231–251

    Article  Google Scholar 

  • Vogelmann TC, Evans JR (2002) Profiles of light absorption and chlorophyll within spinach leaves from chlorophyll fluorescence. Plant Cell Environ 25:1313–1323

    Article  Google Scholar 

  • Wollman F-A (2001) State transitions reveal the dynamics and flexibility of the photosynthetic apparatus. EMBO J 20:3623–3630

    Article  PubMed  CAS  Google Scholar 

  • Zarco-Tejada PJ, Miller JR, Mohammed GH, Noland TL (2000) Chlorophyll fluorescence effects on vegetation apparent reflectance – I. Leaf-level measurements and model simulation. Remote Sens Environ 74:582–595

    Article  Google Scholar 

Download references

Acknowledgements

Financial support of the European Community provided through the Human Potential Program under contract HPRN-CT-2002-00254 within the European Research Training Network STRESSIMAGING and of the European Space Agency (ESA) within the ‘FLEX (Fluorescence Explorer) Instrument Feasibility Study on the Utilization of Fluorescence Measurements in Remote Sensing of Vegetation’ is gratefully acknowledged. I wish to thank Ms Gabrielle Johnson for English language assistance.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Claus Buschmann.

Additional information

This manuscript is dedicated to Andy Benson on the occasion of his 90th birthday.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Buschmann, C. Variability and application of the chlorophyll fluorescence emission ratio red/far-red of leaves. Photosynth Res 92, 261–271 (2007). https://doi.org/10.1007/s11120-007-9187-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11120-007-9187-8

Keywords

Navigation