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.
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
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
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
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
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
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
Benson AA (2002) Following the path of carbon in photosynthesis: a personal story. Photosynth Res 73:29–49
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
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
Bornman JF, Vogelmann TC, Martin G (1991) Measurement of chlorophyll fluorescence within leaves using a fibre optic microprobe. Plant Cell Environ 14:719–725
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
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
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
Buschmann C, Lichtenthaler HK (1998) Principles and characteristics of multi-colour fluorescence imaging of plants. J Plant Physiol 152:297–314
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
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
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
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
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
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
Cordón GB, Lagorio MG (2006) Re-absorption of chlorophyll fluorescence in leaves revisited. A comparison of correction models. Photochem Photobio Sci 5:735–740
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
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
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
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
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
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
Goodwin RH (1953) Fluorescent substances in plants. Annu Rev Plant Phys 4:283–304
Günther KP, Dahn H-G, Lüdeker W (1994) Remote sensing vegetation status by laser-induced fluorescence. Remote Sens Environ 47:10–17
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
Hák R, Rinderle-Zimmer U, Lichtenthaler HK, Nátr L (1993) Chlorophyll a fluorescence signatures of nitrogen deficient barley leaves. Photosynthetica 28:151–159
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
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
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
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
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
Lang M, Stober F, Lichtenthaler HK (1991) Fluorescence emission spectra of plant leaves and plant constituents. Radiat Environ Bioph 30:333–347
Lichtenthaler HK (1987) Chlorophyll fluorescence signatures of leaves during the autumnal chlorophyll breakdown. J Plant Physiol 131:101–110
Lichtenthaler HK (1992) The Kautsky effect: 60 years of chlorophyll fluorescence induction kinetics. Photosynthetica 27:45–55
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
Lichtenthaler HK, Miehé JA (1997) Fluorescence imaging as a diagnostic tool for plant stress. Trends Plant Sci 2:316–320
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
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
Lichtenthaler HK, Prenzel U, Kuhn G (1982a) Carotenoid composition of chlorophyll-carotenoid-proteins from radish chloroplasts. Z Naturforsch 37c:10–12
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
Lichtenthaler HK, Hák R, Rinderle U (1990) The chlorophyll fluorescence ratio F690/F730 in leaves of different chlorophyll content. Photosynth Res 25:295–298
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
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
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
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
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
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
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
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
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
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
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
Pfündel E (1998) Estimating the contribution of photosystem I to total leaf chlorophyll fluorescence. Photosynth Res 56:185–195
Ramos ME, Lagorio MG (2004) True fluorescence spectra of leaves. Photochem Photobio Sci 3:1063–1066
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
Rolfe SA, Scholes JD (1995) Quantitative imaging of chlorophyll fluorescence. New Phytol 131:69–79
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
Šestak Z, Šiffel P (1997) Leaf-age difference in chlorophyll fluorescence. Photosynthetica 33:347–369
Shibata K, Benson AA, Calvin M (1954) The absorption spectra of suspensions of living micro-organisms. Biochim Biophys Acta 15:461–470
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
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
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
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
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
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
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
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
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
Thornber JP (1975) Chlorophyll-proteins: light-harvesting and reaction center components of plants. Annu Rev Plant Phys 26:127–158
Valeur C (2002) Molecular fluorescence – principles and applications. Wiley-VCH, Weinheim
Virgin HI (1954) The distortion of fluorescence spectra in leaves by light scattering and its reduction by infiltration. Physiol Plant 7:560–570
Vogelmann TC (1993) Plant tissue optics. Annu Rev Plant Phys 44:231–251
Vogelmann TC, Evans JR (2002) Profiles of light absorption and chlorophyll within spinach leaves from chlorophyll fluorescence. Plant Cell Environ 25:1313–1323
Wollman F-A (2001) State transitions reveal the dynamics and flexibility of the photosynthetic apparatus. EMBO J 20:3623–3630
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
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
Corresponding author
Additional information
This manuscript is dedicated to Andy Benson on the occasion of his 90th birthday.
Rights 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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11120-007-9187-8