Indian Journal of Biochemi stry & Biophysics Vol. 36, October 1999, pp. 289-295

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Thermoluminescence (TL) from photosynthetic materials

Sarah Thomas, Jyoti U Gaikwad and P B Vidyasagar*

Department of Physics , University of Pune, Pune 411 007

Received 4 December 1998; revised 2 Auglls/ /999

Introduction When a thermoluminescent material is supplied

energy by electromagnetic radiations, some of this energy is stored in the form of electrons trapped into so called "forbidden levels", "metastable states" or "traps". During the de-excitation process, this energy is released in the form of luminescence. In case of thermoluminescence thi s de-exc itation is thermally stimulated. The usefulness of the thermoluminescence (TL) technique as a sensitive monitor of defect concentrations in material s and for applications in areas like geological dating and radiation dosimetry is well known l-4• TL studies provide vital information about the mechanism of generation and modification of traps and nature of traps 5,6. TL systems used for studies on phosphors, alkali halides etc_ record high temperature TLI. Because of the simplicity in des ign, such systems are commercially availab le. However, these are not suitable for the study of biological spec imens like photosynthetic materials, proteins, enzymes and nucleic acids, where sample degradation may occur at high temperatures 7.8. Low temperature TL studies have unique advantages over high temperature studies. They form one of the sensitive techniques to study post irradiation events where the excited states are investigated by lowering the temperature to appropriate levels. Their interactions can then be explored further by allowing the temperature to ri se at controlled rates9,IO.

TL can be observed in photosynthetic bacteria, algae and various higher plant preparations, ranging from intact leaves to purified PS II RC complexes . TL curves have been investigated in the temperature region -1 96DC to 100°C and about 13 TL bands have been shown to ex ist in th is temperature region ll. The mai n peaks are denoted as A(peak f) (-30°C - -20°C) ,

*Aulhor for correspondence

D(peak II) (_IODC - ODC) , Q(peak IIJ) (OnC - 10°C), B(peak IV) (20DC - 35 DC) and C(peak V) (45"C -55DC) (ref. 12) . Under low pH conditions the B band splits into BI (20DC) and B2 (30nC) bands I'. It has been established that the charge storage states of the water oxidation complex (S states) and the primary (Qa) and secondary (Qb) quinone acceptors participate in the generation of the maj or TL bands. Among the well assigned peaks are, peak IV originating from Qh and peak II originating from Qal l - I ~.

TLset up Though TL is genera ll y recorded as a functi on of

temperature l5, in some cases TL is measured as a function of wavelength . Such studi es are useful in understanding the dependence of the TL emiss ion spectrum on temperature. However, since in thi s case experimental problems ari se due to transient nature of the signals involved, relatively few groups have tt d b · h .. 16 a empte to 0 tam suc emt SS lon spectra . In thi s

review, we shall be dealing with onl y the former TL systems.

One of the earlier systems for recording low temperature TL was developed by Tatake el of.

16 A simple liquid nitrogen gas fl ow cryos tat for variab le temperature laser luminescence studi es has been developed by Fairman el 01.

17. Compari son of laser

and conventional heating in TL studi es has been done IX . by Jones et af. . Computeri zed TL systems are

convenient in re lation to the storage and analysis of 19 data. Baba et af. have deve loped a compact cryostat

and simple photon - countin g unit for a micro computer controlled TL measuring system. Very few computerized TL systems ex ist for recording TL from photosynthetic material s. ]n a TL-cum- fluo rescence set up for photosynthetic material developed by D ?O ucruet el af.- , the phOIOl1 coulilci data are transmitted to a 2086 micro computer using a GPIB, IEEE card. An applica ti on of a 2-dimensional photon

290 INDIAN 1 BIOCHEM BIOPHYS, VOL. 36, OCTOBER 1999

counter for the determination of emission spectra of TL from photosynthetic materials was de monstrated by Sonoike21.

A TL system was fabricated earlier in our laborator/2 on the lines of the system developed by Tatake et al. 16

• The cryostat was capable of producing and maintaining low temperatures. However this system provided only a fixed heating rate of 10DC/min and the glow curves were recorded using an X/t recorder. This system was modified to obtain programmable and linear temperature-time profiles using a dedicated microprocessor-based PID temperature controller23

. The PC/AT with an ADC add-on card acquires the TL data using software developed .for the data acquisition and processing. The analysis software deve loped is described at a later part of this review.

TL measurements The dark relaxed leaf disc or a piece of filter paper

soaked with the sample suspension is fixed on the sample holder. Various types of illuminati on conditions, ranging from single fl ash illumination or continuous light illumination either at a low temperature, or at two different temperatures or during cooling, can be adopted l4. The sample is then warmed gradually at a suitable heating rate between 10 to 60DC/min and the luminescence recorded 13,24.

In contrast with one-turnover fl ashes whic h were a useful tool in the ass ignment of well defined TL bands , continuous illumination as found in natural conditions, induces complex TL signal s13. It is seen that though the overall properties of the Band Q bands remain the same from Cyanobacteria to higher plants25

, a qualitati ve survey showed that the ir apparent characteristics, namely, temperature at peak max ima T ill, intens ity, width at half he ight and peak shape ( slopes at low and high temperature edges) are dependent on a variety of factors such as species, growth conditions, pH of the med ium26

, herbicide -resistant mutations27 and for the Q band, the chemical nature of the inhibitor bound in the Qb site28

. Various stress factors also alter the charge stabilization pattern of PS II inducing the emergence of new or shifted TL bands29. These conditions call for a detailed quantitative analysis of TL bands in order to extract more information and therefore the necess ity for a method of decomposition of these complex signal s .

The parameters assoc iated with TL bands are the acti vation energy, frequency factor, entropy etc. To

determine these parameters , it is necessary to fit a h . I . h . I ,0 11 TI ' t eoretlca equatIOn to t e entIre g ow curve' " . lIS

is a difficult procedure because of the overlapping of the bands32

. A simple description of TL, arising from solid states, was developed by Randall and Wilkins .1O. Photosynthetic TL has until now been treated bas ically in the same framework, assuming that the generalized energetic schemes for a so lid state and for PS II are essentially the same24

.

Analysis of TL from photosynthetic material Arnold and Azd', were the first to apply the

Randall-Wilkins theory to explain TL g low curves. The ir calculation of the activation energy was based on the temperature of the peak maximum and peak half-heights, using an arbitrarily decided frequency fac tor. Since the frequency factor was taken from the TL of organic crys tals, the interpretat ion of the activation energies ( 0.53, 0 .60 and 0 .64 eV ) obtained by them was uncerta in . Shuvalov and Litvin '4 estimated the activation energies ( 0.35 and 0 .90 eV ) from Arrhenius plots of the rising side of the glow bands. The errors due to the low TL va lues at the ri si ng side of band ' al so makes the ac li vati on energies obtained by thi s method not dependable. Lurie and Bertsch attempted to reso lve the g low curve into three overlapping bands according to ri gorous theoretical considerations3

). They calc ulated the activation energ ies (0.48, 0 .57 and 0 .80 eV) and frequency factors from the temperatures at maximum and half maximum intens iti es of the separated bands. It could be seen that the activation energ ies based on the measurements of the initi a l ri se of the indi vidual peaks and those based on the temperature of the peak max imum and ha lf he ights were contradi ctory. Experimental errors also contri buted to the large di screpancies between the resu lts obta ined by different groups. Difficulties were also e ncoun tered in trying to obtain necessary data from overlapping peaks.'

It was later shown that the g low curve cons isted of more than three bands,5.>,7 and the re fore the

conversion of peak positi on into ac ti vat ion energy is inaccurate due to the uncertainty in the numbe r and positions of the bands. One a lso had to be care ful in accepting activation ene rgies determined by temperature jump TL measurements because of the large degree of overlapping of the bands in the g low curve38,39. The shortcomings of the methods mentioned above resulted in unre liabl e va lues fo r the

THOM AS el at.: THERMOLUMINESCENCE FROM PHOTOSYNTHETIC M ATER IALS 29 1

activation energies and several of these energies did not sati sfy the limit of the minimum activation energy, 0 .57 eV, estimated by Malkin from delayed luminescence measurements"·39.

Tatake et al. 40 calculated the activation energies for the glow peaks without an assumed frequency factor by using different methods. The frequency factor itself and the lifetime of the states responsible for each glow peak were calculated by using the values for the activation energies. Some of the results of the analysis by Tatake et al. are shown in Table I . It can be seen that the values of the activation energy (E) and frequency factor (s) show a large variation over the temperature range from -33°C to 47°C. The extremely large values of frequency factor and entropy of activation for peaks IV and V appeared to be impossibly large41 . Values of s larger than 1013 S-I

are outside the range of chemical reaction kinetics . 1015

S- I correspond to electronic vibrations in the valence shells of atoms and 1019 s - I to a 50,000 e V quantum. They also did not expect positive values for entropy of activation, because the activated state is presumed to be a restricted, specialized state with lower entropy than that of the reactants forming it41. Tatake et al . therefore concluded that peaks IV and V do not follow Randall-Wilkins theory .

In another study, Vass et al. 32 attempted to resolve the problem of overlapping glow peaks by using a computer assisted curve resolving technique. Using all the experimental points of the glow curve, they calculated the activation energy, frequency factor, free energy of activation and the lifetime of the states responsible for the TL, by a slightly modified version of the Randall Wilkins theory. These authors pointed out that the values of the free energies of activation were higher than 0.5geV estimated for the minimum recombination step and suggested that TL recombination involves back reactions of subsequent stabilization steps.

De Vault et al.4 1 also postulated the involvement of reversal of the charge translocation steps. They carried out a theoretical study of the recombination of charges di stributed in the e lectron transport chain in a temperature equilibrium and deri ved equations for different electron transport pathways. The simulation of a TL peak by the Randall Wilkins equation gives apparent enthalpy and entropy of acti vation, which cannot be ascribed to one particular recombination step, but depend on the whole charge stabilization system. They pointed out that the rate of

Table I-Photosynthetic glow peak parameters resu lting fro m fitting with the Randall -Wil kins theory by Tatakc el (1/. 40

Peak Tm E s t.S /ka COC) (eV ) (S- I)

- 37 0.52 2.5 x 10'1 -7.6

II -1 2 0.64 4.5 x 10 10 -4.8

III +10 0.79 2.4 x 1012 -0.9

IV + 25 1.1 0 1.0 x 1017 9.7

V +48 1.32 l .4x 1 01~ 14.6

recombination is determined not only by the rate limiting back reaction step but a lso by earlier steps which affect the concentration of charges that are ready for the rate limiting step. It was suggested that the enthalpy of the earlier steps could add to the activation energy and entropy of the earlier steps to the activation entropy of the back reaction step. Their study thus tried to justify the high values of E and s. A further study by DeVault and Govindjee24

concluded that apparent energy and entropy of activation could provide an estimate of the sum of enthalpies and of entropies of the e lectron transfer steps involved . It was also shown that in photosynthetic systems, the temperature ,T nh at peak maximum is linearly related to the tota l free energy change . In a contemporary study, Jean Ducreut et al?5 examined the practical aspects of the simulation of TL bands and the decomposition of complex TL signals . They used a computerized procedure developed to perform a graphical multicomponent simulation, refined by a numerical minimi zation procedure. They modified the Eyring equation to take into account the progressive depletion of +/- pairs.

It was noted that none of the above mentioned models took into consideration the probability of retrapping which may occur during the de-excitati on phase. It is well known that for retrapping to occur, the existence of metastable states , triplet states or defects is essential. In the case of PS II, the phenomenon of delayed luminescence indicates the existence of metastable states. A lso, the presence of triplet states in PS II has been reported by Yass et aL.42. The traps in such systems have a maxwelli an distribution, and for every de-excitati on process there is a f(nite retrapping probability. The decay of the reported TL peaks observed fo r photosy nthetic materials also provide e~ idence fo r the ex istence of retrapping. Taking all these points into consideration, we43 applied the general order kineti cs model proposed by Chen44 and Mazumdar et {/L. 45 fo r fitt ing

292 INDIAN J BIOCHEM BIOPHYS, VOL. 36, OCTOBER 1999

the TL peaks recorded from photosynthetic samples. The activation energy was calcu lated by the peak shape method as suggested by Mazumdar et al.45

,

while the frequency factor was calcu lated from the cond ition of maximum intensity. Since Vass et al.46

reported va lues of frequency factor so. where So = (kn/h) exp (~ S/kn) [where kn is Boltzmann constant; h is Planck's constant ; S is entropy] these values are presented in column 8 of Table 2 . It was suggested by DeVault41 that these So values must be muliplied by T ill to correspond to the s va lues that we report. The values of So calcu lated according to DeVault et ai. (so=s/TI11) are presented in the last column of Table 2. It can be seen that these two columns match closely with each other. The entropy of activation was evaluated as per the absolute reac tion rate theor/ I

.

Use of this model resulted in acceptable values of the peak parameters such as activation energy, frequency factor, entropy and free energy of activation (Table 2) . Thus apparently the non­consideration of the retrapping of e lectrons is the reason for the abnormally large activation energy and frequency factor values observed by earlier workers . Recently, Mika et al. 47 applied the general order kinetics model for the thermoluminescence study of three D I protein mutants of Synechocysti s 6803. It was also reported by them that the general order model was more su itable than the first order model.

Applications of TL The thermoluminescence technique has found wide

spread applications in biological research . TL is useful in studying the reducing and oxidising s ide of PS II alongwith studying environmental effects on the photosystems.

The reducing side of PSII has been studied in terms of the energetic stabi lity of the reduced quinones

which is affected by varIOUS fac to rs that can be monitored by TL. A 50-70 mV redox potenti a l difference between the Q" I Q,,- and Qh / Qh- coup les resu lts in about 25-30°C difference in the peak temperatures of the corresponding Q and B bands4x

.

Removal of Qa and Qb from the ir binding sites and their reconstitution with various qu inones and subsequent TL measurements is a useful meth od to study the mechani sm of quinone binding in psrr4<1 . The protolytic events at Q" and Qb be ing coupl ed with the functioning of the two e lectron gates affec ts the stability of the quinone acceptors50 and thus can also be monitored using TL. T he upshi ft of the B2 band at low pH has been ass igned to the protonati on induced stabilization of Qb-' T he peak temperature of the B I band is not increased at low pH, but rema ins a lmost constant and can be ex pl a ined by compensating e ffects due to the proton at ion induced stabili zati on of

Q 51 b .

One of the major application areas where the TL technique has proved to be useful is in understanding the effect of various chemica l modifying agents and inhibitors which may either block the photosyntheti c e lectron transport fl ow at different s ites or may in hibit photosynthetic phosphorylation . Herbi cides, are widely used in modern agricu ltura l production. TL has been used extensive ly to study the ir mode o f action and also for herbicide d isplacement studi es to investigate the proximity of binding s ites of different herbic!des52

. Many :::ommerc ia l herbic ides inhibit electron flow between Q" and Qh. These herbic ides have their binding niche on the D I protei n and act by displacing Qb from its binding site5

.1. In the absence of functional Qb, elec trons accumulate on Q" which leads to conversion of B band into the Q band. The characteri stics of the Q band depend on the chemical nature of the herbicides. The changes in the peak

Table 2-Thermoluminescence peak parameters, for the control samples, resulting from the cu rve titling proceuure based on the general order kinetics theory.

Peak Tm b E s tS / kB FE Stl So= srrm

(0C) (eV) (S- I) (eV) (S-I) (S- I)

I (A) -30 1.1 0.742 1.13 x 1014 3.1 I 0.67 4.6 x lO" 7. 1 X lO "

II -10 1.1 0.756 1.29 x 1013 0.85 0.73 4.9 x 10 111 7.4 x 1010

III (Q) 10 1.1 0.858 7.76 x 1013 2.57 0.79 2.7 x lO " 4. 1 X lO"

IV (B) 32 1.01 0.915 4.8 1 x lO" 2.02 0.86 1.6 x lO" 2.3 x lO"

V(C) 55 1.01 1.110 4.59 x 1015 6.50 0.92 1.3 x 1 () 1.1 2 .1 X 10 1.1

(Note: The standard deviat ion of the data are 1.5 for Tm. 0.03 for b, 0.01 for E, O.S for ~S/kB and 0.0003 for the FE. Since the magnitude of s is large, the standard devi ati on of the values of log s was calculated and found to be 0.2 . These values are valid fo r al l the other tables based on the general order kinetics theory.)

THOMAS e/ at.: THERMOLUMINESCENCE FROM PHOTOSYNTHETIC MATERIALS 293

posItIon of the B band in susceptible and resistant plant plant biotypes enable one to apply TL for quick determination of herbicide resistance in plants II.

Analysis of TL glow curves to study the effect of chemical modifying agents on the thermodynamical parameters related to TL glow curves was carried out using the general order kinetics (GOK) theory43. The fitting of the TL spectra from the chloroplast treated with the commercial herbicide DCMU (Diuron) , using GOK showed that the value of order of kinetics(b) for peak II, Q and the reduced B band was much lower than the respective values of b for the control sample indicating reduction in the probability of retrapping54. On the other hand, DEPC (diethylpyrocarbonate) , a histidine modifying agent was shown to affect both the acceptor and donor side of PSI! and fitting showed increase in the value of 'b' for Q, Band C indicating increase in retrapping probabili ty55 (Table 3). The high values of 'b' also contribute to increase in values of the frequency factor and entropy. 8- HQ (hydroxyquinoline), a well known non specific metal chelator, was found to have its inhibition site on the donor side of PSII using TL. Instability of the photosynthetic apparatus could also be characterized using the thermodynamical parameters namely entropy and free energy, obtained by analyzing the TL glow curves56

.

TL analysis also gives information about the oxidising side of PSII, such as the characteristics of S

states and the role of various inorganic and protein cofactors involved in photosynthetic oxygen evolutions7 . Temperature dependence of S state transitions58

, effect of chemical inhibitors like ADRY agents (acceleration of deactivation reactions of the water splitting enzyme Y) to study the turnover and stability of the higher S states59

, NH3 and other water analogues inhibiting oxygen evoluti on60 and several amines affecting S states turnovers can be studied using TL61. TL has been used to explore the role of the extrinsic proteins I ike EP 16, EPn and EP33 subunits in PSI! (ref. 62) and also the role played by M Il hl'd 61 b' b 64 d I ' 6S d n , c on e ' , Icar onate an ca clum ' an copper66.

Development of TL bands during greening of plants under different illumination conditions such as multiple flashes of 2 J1sec at uniform interval s or intermittent illumination of I ms duration at long intervals (5 min) can be studied by monitoring TL patterns 14,67.

Among the applications of TL in studying the environmental effects on PS II, those re lated to photoinhibition have been we ll documented

6g.6

<1.

Application of TL to study effect of heavy metal ions h C 2+ C 2+ N· 2+ d Z ?+ 'd d suc as u, 0, I an n- proVI e a new

approach to localize the target for this inhibition in PS II (ref. 65) . We70 have demonstrated the effect of a toxic heavy metal Pb2

+, during the early developmental stages of rice seedlings. TL from the

Table 3- Thermoluminescence peak parameters, for the control and DEPC-treated samples, resulting from the cu rve filling procedure based on the general order kinetics theory.

Peak Tn! b E s t.S/ku FE (0C) (eV) (S-I) (eV)

(a) Control

I (A) -30 1.1 0,742 1.13 x 10 14 3.11 0.67

II -10 1.1 0.756 1.29 x 10" 0.S5 0.73

IJJ(Q) 10 1.1 0.858 7.76 x 10 " 2.57 0.79

IV (B) 32 1.01 0.9 15 4.81 x 10" 2.02 0.)-:6

V (C) 55 1.01 1.11 4.59 x 10" 6.5 0.92

(b) DEPC treated chloroplasts (5 min incubation)

-15 1.I 0.951 2.IS x 1017 10.61 0.72

IJJ(Q) 12 1.1 0.879 1.45x10 14 3.IS 0.)-:0

IV (B) 32 1.1 0.792 3.93 x 10" -2.n 0.)-:6

V (C) 55 1.I 1.088 1.97 x lOl.l 5.66 0.93

(c) DEPC treated chloroplas ts ( 15 min incubation)

-15 1.1 0.808 2.77 x 1014 3.97 0.72

III(Q) 10 1.4 1.055 2.97 x 10 17 10.S2 0.79

IV (B) 32 1.06 1.059 1.34 x 1016 7.65 0.g5

V (e) 54 1.I 1.147 1.86x 1016 7.91 o.n

294 INDIAN J BIOCHEM BIOPHYS, VOL. 36, OCTOBER 1999

control seedlings showed the existance of three phases during the developmental process, while a lag was observed in the emergence of the TL peaks of the treated samples, Evidence for Pb2

+ effect on the donor side of PS IT was also obtained70

. Acclimatization to high temperatures by plants and cyanobacteria has b d· d . TL I I 7 1 een stu Ie uSll1g "

In a recent work, Gaikwad et al. 72 have used TL to compare different varieties from Vitis (grape) species . It was observed that the vinifera varieties showed an entirely different TL pattern consisting of only one prominent and highly narrow p,eak at around -5°C as compared to the other two varieties of V.labrusca and V.champini. The TL peaks were also analysed using the general order kinetics theory . The highly narrow peak in the case of V. vinifera varieties was found to show extremely hi gh values of thermodynamical parameters viz. activation energy, entropy and free energy72 The observations also formed the basis fo r stud ies related to possible modifications in the photosynthetic electron transport properties induced by indiscriminate application of pesticides which are being used to control various diseasesD

. Moreover, the information obtained on the electron transport of the species examined would be of help in further definitive work on the breeeding and genetic analysis of the species examined. Infact, many workers in recent years have used TL advantageously to study PS II functioning in mutants. In addition to characterization of various mutant strains74

.76

, TL has been useful in investigations related to determining the amino acid binding environment of anions77

, study f I h b· 'd' 7879 I' f o nove er ICI es res Istant mutants . , ana YSIS 0

d· . PS II . 80·8? genes enco II1g varIOUS protell1s -, understanding the role played by specific regions of the D I protein sequenceS3 and also the role of disulfide linkage and intermolecular binding residues in the stability of PS II proteins84

.

Concluding remarks Fitting of complex thermoluminescence glow

curves which was started with the application of the well known Randall Wilkins theory developed in 1945 mainly for inorganic crystals and solid state materials was the most widely used method for fitting photosynthetic TL glow curves also. We achieved further improvement to the analysis of the TL glow peaks by the application of the general order kinetics theory. The application of this model resulted in peak parameter values within tolerable limits in addition to

giving a good fit to the experimental datl points. It also brought out the importance of the probabi I ity of the retrapping of e lectrons during the deexc itati on phase. TL indeed holds out great promises in so lving some of the intricate mysteries of pho1:osynt hesis and improying our understanding of som of nature's puzzling problems .

Acknowledgement The authors are gratefu l to the earlier members of

the research group, V David, Dr M Banelj ee and Mr S Nikum for their contributions. S Thomas and J Gaikwad acknowledge CSIR for the fin ancial support. We are also thankful to Prof P Mohanty and Prof Govindjee for their timely inputs and useful discussions.

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