Differentiation between two strains of microalga Parachlorella kessleri using modern spectroscopic method
© Saleh et al.; licensee Springer 2014
Received: 14 April 2014
Accepted: 23 June 2014
Published: 12 July 2014
The differentiation between wild type of Parachlorella kessleri and its mutant strains PC Mut2, PC Mut4 by using the Multi-functional Plant Efficiency Analyzer (М-РЕА-2) was studied. Mutant algal cells of P. kessleri have been obtained by UV-C during 3 and 10 min respectively.
Light-induced kinetics of prompt fluorescence (OJIP transients), delayed fluorescence and modulated reflection at 820 nm (redox transitions of P700 in PSI) showed disturbance of electron transport flow in photosystem II (PSII) and an increase fraction of non-reducing centers of secondary quinone acceptors of electron (QB). In addition, the amplitudes of the fast and slow peak in the kinetics of the delayed light emission and non-photochemical fluorescence quenching ( NPQ) were significantly reduced in mutant cells, indicating low level of the membrane energization of photosynthetic membranes. Changes of photosynthetic reactions of mutants may lead to an increase of the carotenoids content, which protect cells against the light stress.
It is suggested to use parameters of induction curves of prompt and delayed fluorescence to characterize mutant algal cells in biotechnological studies.
KeywordsParachlorella kessleri Chlorophyll a fluorescence Delayed fluorescence JIP-test Photosynthesis
The solar radiation sustains life on Earth. However significant increase of UV-C radiation may lead to inhibition of many biological processes. The major cellular targets of UV-C are various biomolecules, which directly absorb this radiation or are indirectly affected by various UV-C induced photochemical reactions. The biological and, ultimately, ecological consequences are numerous (Charles ). It is known that UV-C induces some physiological effects such as declining photosynthetic rates, which can be related not only to damaged biomolecules, but also to ultrastructural changes in organelles or membranes (Holzinger and Lütz ). Typical alterations include swollen mitochondrial cristae, disrupted thylakoids or detached phycobilisomes in chloroplasts, bent-shaped dictyosomes, and damaged plasmalemma. An intact ultrastructure of the algal cell is a prerequisite for the optimum functioning of all physiological processes (Charles ). Parachlorella kessleri was described as a new genus, derived from the true spherical Chlorella. The wild strain SAG 211-11 h was formerly referred to as Chlorella vulgaris but later reassigned as Chlorella kessleri (Fott and Nováková ). Today this species is referred to as P. kessleri (Krienitz et al. ).
Fluorescence methods are used in biotechnological experiments for monitoring photosynthesis processes, which give detailed information of primary defects of cell metabolism, mainly at the membrane level (Schreiber ; Antal et al. ; Matorin et al. ; Schansker et al. ). The basis of fluorescence methods lies in the ability of chlorophyll located in photosynthesis membranes to serve as a natural detector for the algae cells, emitting quanta fluorescence. Importantly, such methods allow to gain information of alga state in real time. Measurement of the ratio between the fluorescence intensity under the photosynthesis saturating illumination (F M ) and under low light intensity, which induces no changes in the state of the photosynthetic apparatus (F O ) makes it possible to determine the maximum efficiency of the PSII processes, which is equal to F V /FM= (F M - F O )/F M . The F V /F M ratio presents dimensionless characteristics of the efficiency of photosynthesis, which is independent of the species of organisms. Measurements of fluorescence induction curves are carried out during several seconds by using PAM or PEA instrument. Measurements of fluorescence induction curves with high resolution (starting from 20 μs) have been recently used in studies of photosynthetic reactions in higher plants and algae cultures (Schreiber ; Strasser et al. ; Matorin and Rubin ). The M-PEA-2 instrument allows to measure separate reactions in PSI and PSII simultaneously (Strasser et al. ; Oukarroum et al. ; Bulychev et al. ). Moreover, it monitors induction changes of delayed fluorescence, which indicate the level of the membrane energization (Goltsev et al. ).
In the present paper we investigated processes in PSI and PSII of wild type of P. kessleri and two mutant strains by using a newly designed Multi-functional Plant Efficiency Analyzer (M-PEA-2, Hansatech). This instrument gives a comprehensive picture of primary photosynthetic events by simultaneous recording the prompt and delayed fluorescence kinetics and reflectance at 820 nm at high time resolution. Here we introduce results of the first attempt of assessment of photosynthetic characteristics of the mutant strains of algal cells by using M-PEA-2.
Strains and growth conditions
The strain of Parachlorella kessleri microalga was obtained from Al-Farabi Kazakh National University, Biotechnology Department culture collection. P. kessleri wild type strain was grown in tris-acetate-phosphate (TAP) medium (pH 6–7) (Gorman ), in 250 ml Erlenmeyer flask at 28°C under continuous illumination of photosynthetic photon flux density (PPFD) 120 μmol photons m−2 s−1 and constant shaking.
UV irradiation and mutagenesis
According to Harris (), 5 ml of the liquid culture with a density of 1×106/ml algal cells were placed in 9 cm Petri dish, where the culture formed a thin layer, covering the bottom. The dish was placed on shaker at 20 rpm and exposed to UV-C lamp (254 nm and 40 erg/mm2) at distance 15 cm during 3 and 10 min respectively. After UV irradiation, the cells were inoculated in solid TAP medium and incubated in darkness for 24 h to prevent photoreactivation. After the 24 h, some dishes were incubated photoautotrophically (photon flux density 120 μmol photons m−2 s−1), others were incubated heterotrophically (in dark) during 15 days.
Selection of the mutant strains
After the incubation period, two mutant strains were selected based on their phenotypic characteristics, using colonies whose color and size differed from the wild type colonies. These mutant strains, PC Mut2 and PC Mut4, were obtained from P. kessleri after irradiation time of 3 and 10 min respectively. These mutants were transferred from the solid to liquid medium and kept under phototrophic growth conditions (photon flux density 120 μmol photons m−2 s−1).
Analysis of chlorophyll a and b content
Spectrophotometry method was used according to Merchant et al. (). The calculation of the concentration of the pigments was determined by the optical density of pigment solutions at appropriate wavelength.
Chlorophyll fluorescence and absorbance measurements
Light-induced kinetics of prompt Chl fluorescence (PF), delayed Chl fluorescence (DF) and modulated reflectance at 820 nm (MR) were recorded by using Multi-functional Plant Efficiency Analyzer (M-PEA-2, Hansatech Instrument Ltd., King’s Lynn, Norfolk, UK). Prior to the measurements, the cells were concentrated on a membrane filter. Then a thin layer of cells was fixed by a special clip on the surface of the measuring chamber and incubated in the darkness for 10 min. It has previously been verified in algal suspension by using Aqua-Pen fluorometer (Photon System Instruments, Czech Republic) that concentration algal cells on the filter surface doesn’t affect the physiological state of cells (Matorin et al. ).
The design of the measuring chamber, timing protocol of a single measurement, and data acquisition procedure for M-PEA-2 can be found in Strasser et al. (). PPFD and duration of actinic illumination were set to 1300 μmol photons m−2 s−1 and 3 s, respectively. In the M-PEA-2, the DF induction curves are constructed from the DF intensities at certain delay time-point in the kinetics of DF decay recorded during the dark interruptions of the actinic light, which induces OJIP (intermediate levels of the light-induced fluorescence curve) rise. In our experiments, the delay time point in the DF decay curve was set to 50 μs. It is assumed that submillisecond DF components are generated from PSII reaction centers in the state Z+P680QA− (Goltsev et al. ), where Z is a tyrosine Z residue, P680 is a primary electron donor, and QA is a primary quinone acceptor in PSII.
Fluorescence was measured by using Water-PAM fluorometer (Heiz Walz, Effeltrich, Germany). Ratio F V /F M is the maximum quantum yield of PSII photochemistry was measured in dark-adapted sample, where F V = F M -F O . Measurements in the light were performed with progressive increase of light intensity from 0 to 800 μmol photons m−2 s−1. At the end of each illumination session, F M ’ and fluorescence yield in light F(t) were measured using saturating flash (0.8 s, 3000 μmol photons m−2 s−1) (Schreiber ; Herlory et al. ). These parameters were used to calculate non-photochemical quenching of chlorophyll fluorescence NPQ = (F M - F M ’)/F M ', the quantum yield of photochemical conversion of light energy in PSII, as Y = (F M ’- F(t))/F M ', and the relative yield of noncyclic electron transport at given light intensity rETR = Y × E i × 0.5, where E i is illumination intensity (μmol photons m−2 s−1) (Lippemeier et al. ). Parameters are designated according to generally accepted nomenclature (Schreiber ).
Light absorption spectrum of samples was measured by using HITACHI-557 spectrophotometer. Fluorescence spectra, excitation spectra, and fluorescence decay times in algae were measured by using Fluorolog-3 (Horiba Jobin Yvon) spectrofluorometer with the TCSPC (time correlated single photon counting) option (O’Connor and Philips ). Fluorescence was measured at 685 nm under excitation with pulse LED at 390 nm. Fluorescence decay kinetics was approximated with the sum of two exponential curves.
All measurements were repeated no less than five times. The figures present data of three replicate experiments at least.
Results and discussion
In this study mutations were induced by UV-C radiation. UV-C mutagenesis offers many advantages such as less pollution, simple operation, and sterile cultivation conditions. After UV-C irradiation, some colonies appeared with phenotypic characteristics that differed in color and size from the control colonies. The colonies were selected based on their phenotypic appearance, transferred into liquid medium and then subcloned several times to insure that the mutation was stable and no changes in their phenotypic characteristics occurred. The major cellular targets of UV-C are different biomolecules, which directly absorb this radiation, or which are indirectly affected by various UV-C induced photochemical reactions (Charles ). In the present paper two mutant strains of P. kessleri after irradiation during 3 and 10 min respectively were selected and designated as PC Mut2 and PC Mut4. It was shown microscopically that mutant cells are smaller, than wild type cells. All strains were grown in the liquid TAP medium under phototrophic conditions. The algal cultures of the wild and mutant strains were different in color.
Figure 3 shows fast increase of algal cells of wild type (curve 1) after the lag-phase (1st day), as measured by F O . PC Mut2 (curve 2) and PC Mut4 (curve 3) mutants demonstrate a lower growth rate during investigation period. Photosynthetic activity (F V /F M ratio) of wild type cells slightly increased during growth, reaching the maximum value at the 3rd day. After prolonged culturing (6th day) photosynthetic activity, judged by F V /F M ratio, was decreased, possibly due to exhaustion of mineral nutrients in the growth medium. Mutants PC Mut2 and PC Mut4 had a lower F V /F M values.
Parameters of OJIP kinetics of fluorescence induction curve of wild type and three mutant strains Parachlorella kessleri cells
F V /F M
Maximum quantum yield of charge separation in PS2
0.610 ± 0.01
0.607 ± 0.01
0.506 ± 0.01
Relative amplitude of the O-J phase
0.38 ± 0.1
0.47 ± 0.01
0.47 ± 0.003
Relative amplitude of the J-I phase
0.96 ± 0.02
0.96 ± 0.01
0.94 ± 0.004
Initial slope of the O-J fluorescence phase
0.60 ± 0.04
0.73 ± 0.02
0.87 ± 0.03
Area between the fluorescence kinetic curve (O–J–I–P) and the level of Fm normalized against the Fv value
40.97 ± 4.24
38.72 ± 2.32
30.06 ± 1.01
Average value of absorbed photon flows in PS2 RC (of apparent size of the active antenna in PS2)
2.57 ± 0.2
2.85 ± 0.1
3.51 ± 0.2
Capacity for pH-induced non-photochemical fluorescence quenching
0.14 ± 0.01
0.12 ± 0.01
0.10 ± 0.01
Capacity of the quinone pool for fluorescence quenching
0.24 ± 0.04
0.20 ± 0.01
0.19 ± 0.02
It is assumed that the O–J phase of fluorescence induction reflects the accumulation of both reducing and non-reducing PSII (Antal et al. ; Strasser et al. ). The hypothesis is supported by the fact that DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), which inhibits electron transport between QA and QB, leads to fast growth of fluorescence to the maximum level within 2 ms, corresponding to the appearance of the J peak in the control. Analysis demonstrated that, in cells of the strains obtained, the number of non-QB-reducing centers of PSII, which are incapable of the quinone pool reduction, was increased. This was supported by a number of other parameters (Table 1). Accordingly, values of the parameter М O , which reflects the initial slope of the induction curve, increased. Total flow of photons, absorbed by the pigments of the PSII antenna and normalized against the RC number (ABS/RC), increased in the cultures of mutant strains P. kessleri, as compared to the control values. At the same time, the relative amplitude of the J–I phase (VI) was practically unchanged.
Prompt fluorescence induction curves also demonstrated the suppression of fluorescence decay after reaching the maximum due to ΔpH dependent nonphotochemical quenching (qE = (F M – F 6s )/F V )). The value of this parameter decreased in mutant strains, which evidences a decrease in the membrane energization. The ability of the quinone pool to quench fluorescence qPQ = (F M – F I )/F V in algae of mutant strains was also reduced.
Changes of amplitude and duration of fast (A1, τ 1 ) and slow (A2, τ 2 ) components of fluorescence decay kinetics of wild type and mutant strains Parachlorella kessleri cells
1.0 ± 0.13
2.24 ± 0.03
PC Mut 2
0.97 ± 0.14
2.18 ± 0.03
PC Mut 4
1.09 ± 0.23
2.47 ± 0.04
Slower fluorescence decay and an increase in the contribution of the slow component, generally, reflect a higher reduction of the PSII QA acceptor. It is clearly seen in the presence of DCMU, an inhibitor of electron transport. The fact of slower fluorescence decay in PC Mut4 agrees well with the increase in the concentration of non-QB-reducing centers of PSII in cells of this mutant strain.
Reflectance changes at 820 nm (MR) are assigned to the redox transitions of P700, a primary electron donor in PSI, and plastocyanin, an electron donor for P700+. For the simplicity, further in the text we refer MR signal to P700. In the dark adapted state, P700 is in the neutral state and the reflectance at 820 nm is high. After the onset of illumination MR value rapidly declines, reaching a dip at about 10 ms, followed by the slow recovery to the initial level. These changes reflect the rapid oxidation of P700 by the acceptor side of PSI, and the subsequent reduction of P700+ by electrons arriving from PSII (Figure 4B) shows MR transients normalized to the initial value at 20 μs (MR/MR0) recorded simultaneously with the OJIP curves described above.
In PC Mut2 mutant strain, the initial oxidation, as well as reduction of P700 was similar to those in the wild type. In PC Mut4 mutant strain, P700 oxidation was reduced, indicating an inhibition of electron transport via PSI.
Both DF and PF emission originates from radiative deactivation of the singlet excited state of antennae Chl in PSII (Goltsev et al. ). In the case of PF, excited states are created mainly through the absorption of photons by photosynthetic pigments, whereas DF emission is coupled to excited states formed as a result of the backward reactions of electron transport. In order to separate a low yield DF emission from high yield PF signal, DF is recorded in the dark after the excitation pulse offset. DF intensity is proportional to the rates of the corresponding recombination reactions which are strongly affected by the energization of a thylakoid membrane (Wraight and Crofts ). Thus, changes of DF yield during the induction period depend on the formation of electrical and proton differences across the membrane. Fast and slow phases of the DF induction kinetics recorded with M-PEA in plant leaves have recently been analyzed (Goltsev et al. ; Oukarroum et al. ). It is assumed that the fast component (I1) (~10-100 ms) reflects transient formation of the electrical gradient across the membrane, likely, due to generation of charge pairs in PSI (P700+[Fe-S]−). The presence of the second DF peak, I2, in the seconds range, is associated with the light induced formation of the proton transmembrane gradient (∆pH) that increases the constant of rate of emission transitions in the RC of PSII.
Peaks on DF curve at 20–50 ms, and 1 s were reduced in mutant strains, indicating a decrease in the formation of electrical and proton potential differences across the thylakoid membrane. However, the fast component of DF was reduced to a greater extent in PC Mut4 strain. The slow phase of the DF induction kinetics (∆pH) was reduced more significantly in PC Mut2, compared to the wild type. The last fact agrees with the reduced non-photochemical quenching qE = (F M – F 6s )/F V in PC Mut2 (Table 1).
Changes of fluorescence parameters of wild type and mutant strains of Parachlorella kessleri cells
F V /F M
0.58 ± 0.01
0.5 ± 0.01
0.45 ± 0.02
0.179 ± 0.02
0.133 ± 0.003
0.083 ± 0.004
15.5 ± 1.3
12.06 ± 0.4
8.3 ± 1.4
Thus, simultaneous registration of the kinetics of prompt, delayed fluorescence and redox state of P700 made it possible to monitor the individual reactions of accumulation of reduced carriers between the photosystems and kinetics of electrochemical proton gradient on a thylakoid membrane of mutant strains P. kessleri.
It is known that the constant fluorescence level F O highly correlates with the total content of pigments in the photosynthetic apparatus of algal cells that harvest light energy (Matorin et al.). The chlorophyll a and b content in the wild type were 53.3 ± 5.7, 30.2 ± 2.4 mg/l respectively while in the PCMut2 were 48.3 ± 2.3, 25.2 ± 2.3 mg/l respectively and in the PCMut4 were 33.3 ± 3.2, 10.2 ± 3.6 mg/l respectively. Fluorescence parameters are used long ago in hydrobiology and oceanology for an assessment of the concentration of microalgae in water and their photosynthetic activity. There is a large number various the submersible fluorometers of different firms for these purposes. Researchers have therefore been trying to find a method of determining the chlorophyll a concentration from in situ fluorescence measurements. These would cover not only fluorescence induced naturally by sunlight but also that induced by artificial light sources. Measurements of the latter are either contact measurements carried out in situ with submersible fluorometers or remote methods using lidars (Pogosyan and Matorin ).
Microalgae, which use sun energy for synthesis of organic matter, are the main energy source in aquatic systems and serve as the food for other organisms. The linear chain of photosynthetic electron transport comprises two sequentially operating photosystems (PSII and PSI) interconnected through intermediary quinone pool of electron carriers. According to the Z-scheme of electron-transport chain (ETC.), activities of each photosystem affect the redox state of the other system. Such a relation between PSII and PSI is evident from chlorophyll fluorescence, whose level depends on the redox state of the quinone acceptor QA, as well as from the redox state of chlorophyll P700, the primary electron donor of PSI. The photoreaction of PSII reduces QA, thus raising chlorophyll fluorescence and the extent of P700 reduction, while the PSI reaction converts P700 into its oxidized form (P700+), which promotes QA oxidation and fluorescence lowering. Simultaneous chlorophyll fluorescence and 820 nm transmission measurements by using М-РЕА-2 allow evaluating the functional state of PSII and PSI. Analysis of fluorescence induction curves demonstrated inhibition of electron transport in PSII and an increase fraction of non-QB-reducing centers. This manifested itself also in an increase of the slow component in decay curve of fluorescence with half-time 2 ns in PC Mut4. In addition, analysis of induction curves of the delayed fluorescence and a decrease of NPQ revealed a decrease in the energization of photosynthetic membranes in mutant cells. Our study showed that the most sensitive parameters are induction curves of prompt and delayed fluorescence in mutant algal cells. Selected parameters are indeed viable tools for diagnostic alga state in biotechnological studies during obtaining mutant forms.
Secondary quinone acceptors of electron
J, I, P, Intermediate levels of the light-induced fluorescence curve
Delayed Chl fluorescence
Pigment of the RC of PSI
The authors acknowledge department of biotechnology, Al-Farabi Kazakh National University which granted materials used in this research and department of biophysics, Moscow State University for supporting material analysis.
- Antal TK, Matorin DN, Ilyash LV, Volgusheva AA, Osipov VA, Konyuhov IV, Krendeleva TE, Rubin AB: Probing of photosynthetic reactions in four phytoplanktonic algae with a PEA fluorometer. Photosynth Res 2009, 102: 67–76. 10.1007/s11120-009-9491-6View ArticlePubMedGoogle Scholar
- Bulychev AA, Osipov VA, Matorin DN, Vredenberg WJ: Effects of far-red light on fluorescence induction in infiltrated pea leaves under diminished ΔpH and Δφ components of the proton motive force. J Bioenerg Biomembr 2013, 45: 37–45. 10.1007/s10863-012-9476-6View ArticlePubMedGoogle Scholar
- Charles DA: Algal Chemical Ecology.ch. 13, U. Springer-Verlag Berlin Heidelberg, Karsten; 2008.Google Scholar
- Fott B, Nováková M: A monograph of the genus Chlorella . The fresh-water species. In Studies in Phycology. Edited by: Fott B. Academia, Prague; 1969:10–74.Google Scholar
- Goltsev V, Zaharieva I, Chernev P, Strasser RJ: Delayed fluorescence in photosynthesis. Photosynth Res 2009, 101: 217–232. 10.1007/s11120-009-9451-1View ArticlePubMedGoogle Scholar
- Gorman DS, Levine RP: Proc Natl Acad Sci U S A. 1965, 54: 1665–1669. 10.1073/pnas.54.6.1665View ArticlePubMedPubMed CentralGoogle Scholar
- Harris EH: The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego; 1989.Google Scholar
- Herlory O, Richard P, Blanchard GF: Methodology of light response curves: Application of chlorophyll fluorescence to microphytobenthic biofilms. Mar Biol 2007, 153: 91–101. 10.1007/s00227-007-0787-9View ArticleGoogle Scholar
- Holzinger A, Lütz C: Algae and UV irradiation: effects on ultrastructure and related metabolic functions. Micron 2006, 35: 190–207. 10.1016/j.micron.2005.10.015View ArticleGoogle Scholar
- Krienitz L, Hegewald EH, Hepperle D, Huss VAR, Rohr T, Wolf M: Phylogenetic relationship of Chlorella and Parachlorella gen. nov. (Chlorophyta, Trebouxiophyceae). Phycologia 2004, 43: 529–42. 10.2216/i0031-8884-43-5-529.1View ArticleGoogle Scholar
- Lazár D: Modelling of light-induced chlorophyll a fluorescence rise (O-J-I-P transient) and changes in 820 nm-transmittance signal of photosynthesis. Photosynthetica 2009, 47: 483–498. 10.1007/s11099-009-0074-8View ArticleGoogle Scholar
- Lippemeier S, Harting P, Colijn F: Direct impact of silicate on the photosynthetic performance of the diatom Thalassiosira weissflogii assessed by on and off-line PAM fluorescence measurements. J Plankton Res 1999, 21: 269–283. 10.1093/plankt/21.2.269View ArticleGoogle Scholar
- Matorin DN, Rubin AB: Chlorophyllа fluorescence in higher plants and algae Moscow. IKI-RKhD, Izhevsk; 2012.Google Scholar
- Matorin DN, Antal TK, Ostrowska M, Rubin AB, Ficek D: Chlorophyll fluorometry as a method for studying light absorption by photosynthetic pigments in marine algae. Oceanologia 2004, 46: 519–531.Google Scholar
- Matorin DN, Todorenko DA, Seifullina NK, Zayadan BK, Rubin AB: Effect of silver nanoparticles on the parameters of chlorophyll fluorescence and P700 reaction in the green alga Chlamydomonas reinhardtii . Microbiology 2013, 82: 862–867. 10.1134/S002626171401010XView ArticleGoogle Scholar
- Merchant SS, Simon EP, Olivier V: The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions. Sci 2007, 318: 245–251. 10.1126/science.1143609View ArticleGoogle Scholar
- O’Connor DV, Phillips D: Time correlated single photon counting. Academic Press, London; 1984.Google Scholar
- Oukarroum A, Goltsev V, Strasser RJ: Temperature effects on pea plants probed by simultaneous measurements of the kinetics of prompt fluorescence, delayed fluorescence and modulated 820 nm reflection. PLoS One 2013, 8: 1–10. 10.1371/journal.pone.0059433View ArticleGoogle Scholar
- Pogosyan SI, Matorin DN: Variability in the state of the photosynthetic system of the Black Sea phytoplankton. Oceanology 2005, 45(1):139–148.Google Scholar
- Schansker G, To’th SZ, Holzwarth AR, Garab G: Chlorophyll a fluorescence: beyong the limits of the Qa model. Photosynth Res 2014, 120: 43–58. 10.1007/s11120-013-9806-5View ArticlePubMedGoogle Scholar
- Schreiber U: Pulse-Amplitude (PAM) fluorometry and saturation pulse method: an overview. In Chlorophyll fluorescence: A signature of Photosynthesis and Respiration. Edited by: Papageorgiou G. Springer, Dordrecht; 2004:79–319.Google Scholar
- Strasser RJ, Tsimilli-Michael M, Srivastava A: Analysis of chlorophyll a fluorescence transient. In Papageorgiou GC and Govindjee (eds) Chlorophyll fluorescence: a signature of photosynthesis. Kluwer Academic Publishers, Dordrecht/Boston/London; 2005:321–362.Google Scholar
- Strasser RJ, Tsimilli-Michael M, Sheng Q, Goltsev V: Simultaneous in vivo recording of prompt and delayed fluorescence and 820-nm reflection changes during drying and after rehydration of the resurrection plant Haberlea rhodopensis . Biochim Biophys Acta 2010, 1797: 1313–1326. 10.1016/j.bbabio.2010.03.008View ArticlePubMedGoogle Scholar
- Volgusheva AA, Zagidullin VE, Antal TK, Korvatovsky BN, Krendeleva TE, Paschenko VZ, Rubin AB: Examination of chlorophyll fluorescence decay kinetics in sulfur deprived algae Chlamydomonas reinhardtii . Biochim Biophys Acta 2007, 1767: 559–564. 10.1016/j.bbabio.2007.04.006View ArticlePubMedGoogle Scholar
- Wraight CA, Crofts AT: Delayed fluorescence and the high-energy state of chloroplasts. Eur J Biochem 1971, 19: 386–397. 10.1111/j.1432-1033.1971.tb01328.xView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.