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Alteration in photosystem II photochemistry of thylakoids isolated from senescing leaves of wheat seedlings
197
I. Photochem. Photobiol. B: Bioi., 20 (1993) 197-202
Alteration in photosystem II photochemistry from senescing leaves of wheat seedlings P.N. Joshi”, N.K. Rarnaswamyb,
of thylakoids isolated
M.K. Ravalb, T.S. Desaic, P.M. Nairb and U.C. Biswal”p+
“School of Life Sciences, Sambalpur University lyotivihar, Sambalpur (India) bFood Technology and Enzyme Engineering Division, Bhabha Atomic Research Centre, Bombay (India) ‘Molecular Biology and Agriculture Division, Bhabha Atomic Research Centre, Bombay (India) (Received
March 31, 1993; accepted
May 12, 1993)
Abstract Wheat seedlings, grown for 7 days in the light, were allowed to senesce in the light or dark, and the change in the photosystem II (PS II) photochemistry of chloroplasts isolated from the primary leaves of these seedlings was investigated. The decrease in oxygen evolution and the fast fluorescence results indicated that the impairment of PS II in the leaves of seedlings senescing in the light was different from that in the leaves of seedlings senescing in the dark. Thermoluminescence studies showed a structural modification in the Qa protein of chloroplasts isolated from leaves senescing in the light and an alteration in the S state transition of chloroplasts isolated from leaves senescing in the dark.
Key words: Fluorescence
induction;
Photosystem
II; Senescence;
1. Introduction
During leaf senescence the structural integrity and functional activity of the photosynthetic apparatus are altered [l, 21. These alterations include a decrease in pigment and protein contents [3], disorganization of the thylakoid membrane [4], lipid peroxidation [5] and a decline in photochemical reaction [2]. The restoration of the senescence-mediated loss in Hill activity, as measured by 2,6-dichlorophenol indophenol (DCIP) photoreduction, with an exogeneous electron donor such as diphenylcarbazide (DPC) and a decline in oxygen evolution indicate the inactivation of the oxygen evolving system [6]. Although aginginduced release of manganese atoms from chloroplasts has been reported [2, 7, 81, the exact site of inactivation has not been located. Therefore, to examine the alteration in the oxygen evolving complex (OEC), we have analysed thermoluminescence (TL) bands. These bands are known to arise mostly from photosystem II (PS II) [9] via the recombination of positive charges (holes) on different S states of the OEC and negative charges on primary electron acceptors ‘Author
to whom
loll-1344/93/$6.00
correspondence
should
be addressed.
Thermoluminescence;
Oxygen
evolution
(Q,) or secondary electron acceptors (Q,) on the reducing side of PS II. In this paper, we examine the senescence-mediated modification of the OEC of chloroplasts and the alteration of the charge pairs responsible for the different TL peaks associated with PS II to obtain information on the reducing and oxidizing components of reaction centre II (RC II).
2. Materials
and methods
Wheat (Triticum aestivum L. CV. Sonalika) seedlings were grown in Petri dishes for 7 days in continuous white light (fluence rate, 12 W m-‘) obtained from a fluorescent tube lamp at 25 f 2 “C without any external nutrients as described previously [lo]. In one set of experiments the seedlings were kept under the same conditions until day 15. In a second experiment the seedlings grown for 7 days in light were transferred to the dark and allowed to senesce until day 15. Pigments were extracted from the primary leaves of wheat seedlings in ice cold 80% acetone in dim green light and the extract was used for spectrophotometric determination of chlorophyll (Chl) as described by Arnon [ll]. 0 1993 - Elsevier
Sequoia. All rights reserved
198
P.N. Joshi et al. I PS II photochemistry of thylakoids porn senescing wheat leaves
Chloroplasts were isolated from the primary leaves of wheat seedlings by different treatments according to the method described by Izawa and Good [12]. The grinding medium used contained 0.3 M NaCl, 30 mM Tricine (pH 7.8) and 1 mM ethylenediaminetetraacetic acid (EDTA). The washing medium contained 0.2 M sucrose, 20 mM NaCl and 20 mM MOPS (3-[N-morpholinolpropanesulfonic acid) (pH 7.4). The chloroplasts were finally suspended in a medium containing 300 mM sucrose, 50 mM NaCl and 50 mM Na/K phosphate buffer (pH 6.9) with a Chl concentration of 2 mg Chl ml-‘. Oxygen evolution was measured with a Clarktype oxygen electrode at 21 “C in rate-saturating red light. The basic assay buffer consisted of 30 mM Na/K phosphate (pH 7.2) with 30 mM NaCl and 200 mM sucrose. The electron acceptor was 0.4 mM K,Fe(CN),. The reaction mixture (2 ml) for assay contained 40 pg equivalent of chloroplasts. When used as an uncoupler, the gramicidin concentration was 2.5 PM. The fluorescence transient of the primary leaf was followed, as described by Kulandaivelu and Daniel [13], by exciting the leaf with a broad-band blue light at saturating intensity. Leaves were dark adapted for 20 min before fluorescence measurement. The signal was stored in a digital oscilloscope (Iwatsu SS-5802) and then transferred to a recorder. The glow curves were obtained following the method of Desai et al. [14]. Isolated chloroplasts in suspension medium containing 100 pg Chl ml-’ were frozen to liquid nitrogen temperature under illumination at a fluence rate of 1 W m-*. TL experiments were run at a heating rate of 0.22 “C s-l. 3-(3,4-D’ic hl orophenyl)-1,1-dimethyl urea (DCMU) was added at a concentration of lo-’ M. The activation energy E was calculated using an Arrhenius plot [15, 161. The mean lifetime 7r was calculated using the relation T,= eElkT/S following Tatake et al. [17], where T is the absolute temperature, S is the preexponential frequency factor and k is the Boltzmann constant. 3. Results Leaf senescence was assessed on the basis of Chl loss. Figure 1 depicts the change in Chl content per gram of fresh weight in the primary leaves of wheat seedlings senescing in the light or dark on day 15. The Chl content decreased by 42% in the light and 76% in the dark.
A
8
C
Fig. 1. The content of Chl per gram of fresh weight in primary wheat leaves: (A) 7 day old seedlings; (B) 15 day old lightsenescing seedlings; (C) 15 day old dark-senescing seedlings. Chl content of 7 day old leaf is taken as 100%. Experimental details are given in Section 2. TABLE 1. Or evolution as a measure of PS II activity in isolated wheat chloroplasts. Each value is a mean of three independent results Treatment
Or evolution with K,Fe(CN), as electron acceptor (pmol (mg Chl-‘) h-l)
7 days light 15 days light 15 days dark
420 (100) 212 (50) 156 (37)
Values in parentheses indicate percentage. Dark treatment starts from day 7 until day 15. Other experimental details are given in Section 2.
The inactivation of the OEC was monitored by measuring the oxygen evolution of chloroplasts isolated from senescing leaves (Table 1). The rate of oxygen evolution was reduced by 50% and 63% during senescence in the light and in the dark respectively, where the rate of oxygen evolution from the chloroplasts of 7 day old primary leaves was taken as 100%. The changes in the parameters associated with the fast fluorescence transient of primary wheat leaves are shown in Table 2. The F0 level remained unchanged in leaves senescing in the light, whereas it decreased by 23% in leaves senescing in the dark. The variable fluorescence (Fv = FM - F0 where Fhl is the maximum fluorescence and F,, is the constant fluorescence) and the F,/F, ratio decreased in leaves from both light- and darkincubated seedlings, the decrease being 13% and
199
P.N. Joshi et al. / PS II photochemisOy of thylakoids from senescing wheat leaves TABLE 2. Effect of senescence on different parameters associated with fast fluorescence induction in primary leaves of wheat seedlings. Seedlings grown for 7 days in the light were transferred to the dark and senescence was studied until day 15. Each value is a mean of three independent calculations Treatment
Fv’
&/FM
T,,
I
CS;0;)
2.7 2.7 2.1
6.3 5.9 4.1
3.6 3.2 2.0
0.58 0.53 0.48
15d Light
-.--
15d Dark
/ .*‘-.A
.r\ \ /a
170 300 390
il
1’
I-
!I
!/
\
\\
‘\
I,
.\
\,‘,,
i
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‘F,, mtttal fluorescence. bFM, maximum fluorescence. ‘Fv = FM-F,, variable fluorescence. F,, FM and Fv are in arbitrary units. For other details, see Section 2.
7d Light
;\L
of FM rise
(@I 7 days light 15 days light 15 days dark
---
\
‘..A
\
,./ //
\_A’
//’
TL
‘1\ ‘.
/’
\
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.i// / /
‘./’
Ii
1 ! 1 I
I
I__*! :
I
I -12
t10 Temperature
I
I
48
(“Cl
Fig. 3. Glow curves of isolated chloroplasts obtained from primary wheat leaves senescing in the light or dark in the presence of DCMU.
Temperature
1°C)
Fig. 2. Glow curves of isolated chloroplasts from 15 day old primary wheat leaves senescing in the light or dark.
11% respectively in the former and 45% and 18% respectively in the latter. The glow curves of isolated chloroplasts from wheat leaves showed five well-resolved peaks, two in the absence of DCMU and three in the presence of DCMU in the temperature range of interest. These peaks appeared at around 236 K (I), 261 K (II), 283 K (III), 298 K (IV) and 321 K (V) which agree well with earlier findings [9]. These peaks are named following Tatake et al. [ 171. Peaks I and IV were obtained in the TL curve of normal chloroplasts. On poisoning the chloroplasts with DCMU, peaks I and IV disappeared and peaks II, III and V were observed. The glow curves obtained from isolated chloroplasts of primary wheat leaves of seedlings senesting in the light and dark are depicted in Fig. 2. The height of peak I decreased and peak IV was shifted by 10 “C towards a lower temperature in the light. The heights of peaks I and IV were
decreased significantly during dark-induced senescence without any alteration in peak temperature. Figure 3 shows the glow curves of DCMUpoisoned chloroplasts isolated from the primary leaves of wheat seedlings senescing in the light or dark. The intensity of the TL bands II, III and V decreased and peaks III and V were shifted marginally towards higher temperature in the sample senescing in the light. However, in the sample senescing in the dark, the intensity of peak II decreased and peak III was modified and enhanced. Table 3 shows the changes in activation energy (E) and mean lifetime at room temperature (7,) calculated from the glow peaks. 4. Discussion PS II-mediated oxygen evolution decreases during the senescence of wheat seedlings. The loss in oxygen evolving capacity is attributed to damage of the OEC, since the loss has been shown to be restored by exogenous electron donors such as DPC [2]. The damage to the OEC has been correlated with the depletion of extrinsic polypeptides associated with the complex [8] leading to RC II degradation. In order to understand the mechanism of RC II degradation, fluorescence induction was examined. The ratio F,/F,, a measure of the photochemical potential, decreases in the leaves of both light-
200
P.N. Joshi et al. / PS II photochemistry
of thylakoids from senescing wheat leaves
TABLE 3. Different energetic parameters calculated from the TL peaks of isolated chloroplasts treatment starts from 7 days. Each value is the mean of three independent glow curves
with different
treatments.
Dark
Treatment
Parameter (unit)
I (236 K)
II (261 K)
III (283 K)
IV (298 K)
V (321 K)
7 days light
E (eV) rr (s)
0.49 0.259
0.62 1.19
0.74 5.86
1.22 28.47
1.4 28.83
15 days light
E (eV) rr (s)
0.489 0.263
0.61 1.28
0.73 8.8
1.19 28.00
1.42 28.38
15 days dark
E (eV) rr (s)
0.471 0.33
0.589 1.498
0.67 9.85
1.25 27.78
-
E, activation
energy in electron
volts (ev).
r,, mean lifetime
at room temperature
and dark-senescing seedlings but in two distinctly different modes. In the former, the decrease in Fv is solely due to a decrease in FM since F. tends to remain unchanged, whereas in the latter the decrease is due to a simultaneous decrease in FM and F. (Table 2). Furthermore, the decrease in F,, the enhancement of T,, of the FM rise and the constancy of F. in the light-senescing sample suggest that the impairment is at the level of RC II, the electron transfer chain between QA and Qg or both [18] and is photoinhibitory [19]. On the other hand, the decrease in F,, Fv and FM and the enhancement of T,,z of the FM rise (Table 2) may indicate an impairment on the donor side of PS II [20]. These results suggest that RCs II are rendered inactive for the trapping of excitons with a concomitant increase in thermal deactivation in the antenna complex. The possible changes in F. and Tin of the FM rise due to variation in the antenna size have not been emphasized, since senescence-mediated loss of Chl is accompanied by a decrease in the number or active RCs II as evident from the TL studies described below. Glow curves of chloroplasts, isolated from primary leaves of senescing seedlings, were examined to locate the exact site(s) of impairment. Sane et al. [21] proposed that the origin of peak I was a recombination of charges stabilized on Z+ and Qg -. The absence of a Z,, band (equivalent to TL band I under our experimental conditions) in the glow curves of etiolated leaves and isolated subchloroplast particles [22], its presence in the glow curves of wheat leaves greened under intermittent flash illumination [23] and its removal on DCMU treatment provide enough evidence in support of this proposition. However, the origin of the peak changes is not well understood. A decrease in peak I height may indicate a decrease in the number of charge pairs or the number of active RCs II during senescence either in the light
in seconds
(s).
or in the dark, and the proposition confirms the conclusions drawn on the basis of a decrease in height of peaks II, III and IV. Peak IV, consisting of two overlapping bands and resulting from SZQB- and S3QB- charge pairs [24], is the most well characterized of all the TL bands of chloroplasts. The shifting of the peak temperature towards the ascending side of the glow curve in the light-senescing sample indicates a decrease in mid-point redox potential [9, 251, suggesting a decrease in stability of SZQB- and S,QB- charge pairs which is also reflected in energetic parameters (Table 3). The modification of Qg due to the altered conformation of the D, protein of the reaction centre core complex of PS II may be the reason. Recently, Ohad et al. [26] have proposed the downshift of the B band peak temperature and the damping of its oscillation pattern as a result of the destabilization of the D1 protein conformation. The light intensity used during the growth of the seedlings may be greater than the intensity required to saturate photosynthesis during senescence, since the photosynthetic capacity decreases, as shown by the decrease in oxygen evolution (Table 1) and photochemical potential (Table 2). The system under such conditions is comparable with the photoinhibitory system of Ohad et al. [26]. Thus the structural modification of the Qg binding protein as suggested by Ohad et al. [26, 271 is induced in the light-senescing sample. Recently, Kettunen et al. [28] have also shown the modification of the D, protein prior to its degradation during photoinhibition of intact leaves, which supports our proposition. However, although there is a decrease in the peak height in the darksenescing sample reflecting a decline in the number of active charge pairs, no shift in peak temperature has been demonstrated. The drastic loss of oxygen evolution in dark-induced senescence (Table 1) may be due to a decline in the number of active
P.N. Joshi et al. 1 PS II photochemistry of thylakoids from senescing wheat leaves
reaction centres of PS II as reflected in a decrease in the peak height. These findings suggest that the damage of RC II in the light-senescing sample is linked to a light-mediated structural modification of the D1 protein as shown in the downshift of the peak IV temperature and is different from the hitherto unknown molecular mechanism of dark-induced damage of the PS II reaction centre. Peaks II and III are known to arise from S3QA[29] and SZQA- [25] charge pairs respectively. A decline in the intensities of these peaks during senescence in the light is indicative of a decrease in population of the S2 and S3 states of the OEC or QA- or both. Since the damage of QA, which is buried deep inside the D2 protein, is less probable, an alteration in the S state transition may be the major cause of decline. Our data showing a higher lifetime of the S2Qa- pair (Table 3) confirm this view. On the other hand, a decrease in peak II and an increase in peak III intensities in the dark-senescing sample reveal the critical nature of the increase in S, population suggesting a blockage of the Sz + S3 transition. Furthermore, extensive modification of peak III is suggestive of intense membrane damage and is supported by the activation energy data which decrease by 31 and 70 meV for peaks II and III respectively (Table 3). We therefore believe that the loss of oxygen evolution as demonstrated previously in different plant systems and in wheat in this study (Table 1) during leaf senescence is possibly due to an alteration in the S state transition and/or destabilization of Qg on the D1 protein of the PS II reaction centre in the thylakoid membrane.
Acknowledgment
Facilities for the measurement of fluorescence transients were made available by Professor G. Kulandaivelu, Department of Biological Sciences, Madurai Kamraj University.
References
8
9 10
11
12
13
14
15
16
17
18
19
20
21
1 H. Thomas and J.L. Stoddart, The leaf senescence,
Annu. Rev. Plant Physiol., 31 (1980) 83-111. 2 U.C. Biswal and B. Biswal, Ultrastructural modification and biochemical changes during senescence of chloroplasts, Znt. Rev. Cytol., 113 (1988) 271-321. 3 H.W. Woolhouse, Senescence in plant cells, in I. Davies and D.C. Sigee (eds.), Cell Ageing and Cell Death, Cambridge University Press, Cambridge, 1984, pp. 123-153.
22
23
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U.C. Biswal and B. Biswal, Photocontrol of leaf senescence, Photochem. Photobiol., 39 (1984) 875-879. H. Thomas, The role of polyunsaturated fatty acids in senescence, J. PZant Physiol., 123 (1986) 97-105. H.W. Woolhouse, The biochemistry and regulation of senescence in chloroplasts, Can. J. Bot., 62 (1984) 2934-2942. M.M. Margulies, Electron transport properties of chloroplasts from aged bean leaves and their relation to the manganese content of the chloroplasts, in G. Stresa, M.F. Avron and A. Melandri (eds), Proc. 2nd Int. Congr on Photosynthesis Research, N.V. Publishers, The Hague, 1971, pp. 539-545. N.K. Choudhury and H. Imaseki, Loss of photochemical functions of thylakoid membranes and PS 2 complex during senescence of barley leaves, Photosynthetica, 24 (1990) 436-445. S. Demeter and Govindjee, Thermoluminescence in Plants, Physiol. Plant., 75 (1989) 121-130. P.N. Joshi, B. Biswal and UC. B&al, Effects of UVA on aging of wheat leaves and role of phytochrome, Environ. &LX Bot., 31 (3) (1991) 267-276. D.I. Amon, Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vtdgaris, Plant Physiol., 24 (1949) l-15. S. Izawa and N.E. Good, The stoichiometric relation of phosphotylation to electron transport in isolated chloroplasts, Biochim. Biophys. Acta, I62 (1968) 380-391. G. Kulandaivelu and H. Daniel, DCMU induced increase in chlorophyll-a fluorescence intensity - an index of oxygen evolution in leaves, chloroplasts and algae, Physiol. Plant., 48 (1980) 385-388. T.S. Desai, P.V. Sane and V.G. Tatake, Thermoluminescence studies on spinach leaves and Euglena, Photochem. Photobiol., 21 (1971) 345-350. S. Lurie and W. Bertsch, Thermoluminescence studies on photosynthetic energy conversion. I. Evidence for three types of energy storage by photoreaction II of higher plants, Biochim. Biophys. Acta, 357 (1974) 42M28. S. Lurie and W. Bertsch, Thermoluminescence studies on photosynthetic energy conversion. II. Activation energies for three energy storage states associated with photoreaction II of higher plants, Biochim. Biophys. Acta, 357 (1974) 429-438. V.G. Tatake, T.S. Desai, Govindjee and P.V. Sane, Energy storage states of photosynthetic membrane: activation energies and lifetimes of electrons in trap states by TL methods, Photochem. Photobiol., 33 (1981) 243-250. R.E. Cleland, Molecular events of photoinhibitory inactivation in the reaction centre II, Aust. J. Plant Physiol., 15 (1988) 135-150. I. Setlik, S.I. Allakhverdiev, L. Nebdal, E. Setlikova and V.V. Klimov, Three types of photosystem II photoinactivation, PhotoJynth. Rex, 23 (1990) 39-48. U. Schreiber and C. Neubaver, The polyphasic rise of chlorophyll fluorescence upon onset of strong continuous illumination. II. Partial control by photosystem II donor side and possible ways of interpretation, Z. Natuq%orsch., Teil C, 42 (1987) 1255-1264. P.V. Sane, T.S. Desai, S.S. Rane and V.G. Tatake, Characterisation of glow peaks of chloroplasts membrane: part I, Indian J. Exp. Biol, 21 (1983) 396-400. T. Ichikawa, Y. Inoue and K. Shibata, Characteristics of thermoluminescence bands of intact leaves and isolated chloroplasts in relation to the water splitting activity in photosynthesis, Biochim. Biophys. Acta, 408 (1975) 228-239. Y. Inoue, T. Oku, S. Furata and K. Shibata, Multiple-flash development of thermoluminescence bands in dark grown spruce leaves, B&him. Biophys. Acta, 440 (1976) 772-776.
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P.N. Joshi et al. / PS II photochemistry of thylakoids from senescing wheat leaves
24 Y. Inoue and K. Shibata, Oscillation of thermoluminescence at medium low temperature, FEBS Lett., 85 (1978) 193-207. 25 P.V. Sane and A.W. Rutherford, Thermoluminescence from photosynthetic membrane, in Govindjee, J. Amesz and D.C. Fork (eds.), Light Emission by Plants and Bacteria, Academic Press, Orlando, 1986, pp. 329-360. 26 N. Ohad, D. Amir-Shapira, H. Koike, Y. Inoue, I. Ohad and J. Hirschberg, Amino acid substitution in the D, protein of PS II affected Qa stabilisation and accelerated turn over of D,, 2. Naturforsch., Teil C, 45 (1990) 402-408.
27 I. Ohad, D.J. Kyle and J. Hirschberg, Light dependent degradation of Qa protein in isolated pea thylakoids, EMBO J., 4 (1985) 1655-1659. 28 R. Kettunen, E. Tyystijarvi and E.M. Aro, D, protein degradation during photoinhibition of intact leaves, FEBS Lett., 290 (1991) 153-156. 29 H. Koike, Y. Siderer, T. Ono and Y. Inoue, Assignment of thermoluminescence A band to SJQA charge recombination: sequentiai stabilization of S3 and QA by a two-step illumination at different temperatures, B&him. Biophys. Acta, 850 (1986) 80-89.
I. Photochem. Photobiol. B: Bioi., 20 (1993) 197-202
Alteration in photosystem II photochemistry from senescing leaves of wheat seedlings P.N. Joshi”, N.K. Rarnaswamyb,
of thylakoids isolated
M.K. Ravalb, T.S. Desaic, P.M. Nairb and U.C. Biswal”p+
“School of Life Sciences, Sambalpur University lyotivihar, Sambalpur (India) bFood Technology and Enzyme Engineering Division, Bhabha Atomic Research Centre, Bombay (India) ‘Molecular Biology and Agriculture Division, Bhabha Atomic Research Centre, Bombay (India) (Received
March 31, 1993; accepted
May 12, 1993)
Abstract Wheat seedlings, grown for 7 days in the light, were allowed to senesce in the light or dark, and the change in the photosystem II (PS II) photochemistry of chloroplasts isolated from the primary leaves of these seedlings was investigated. The decrease in oxygen evolution and the fast fluorescence results indicated that the impairment of PS II in the leaves of seedlings senescing in the light was different from that in the leaves of seedlings senescing in the dark. Thermoluminescence studies showed a structural modification in the Qa protein of chloroplasts isolated from leaves senescing in the light and an alteration in the S state transition of chloroplasts isolated from leaves senescing in the dark.
Key words: Fluorescence
induction;
Photosystem
II; Senescence;
1. Introduction
During leaf senescence the structural integrity and functional activity of the photosynthetic apparatus are altered [l, 21. These alterations include a decrease in pigment and protein contents [3], disorganization of the thylakoid membrane [4], lipid peroxidation [5] and a decline in photochemical reaction [2]. The restoration of the senescence-mediated loss in Hill activity, as measured by 2,6-dichlorophenol indophenol (DCIP) photoreduction, with an exogeneous electron donor such as diphenylcarbazide (DPC) and a decline in oxygen evolution indicate the inactivation of the oxygen evolving system [6]. Although aginginduced release of manganese atoms from chloroplasts has been reported [2, 7, 81, the exact site of inactivation has not been located. Therefore, to examine the alteration in the oxygen evolving complex (OEC), we have analysed thermoluminescence (TL) bands. These bands are known to arise mostly from photosystem II (PS II) [9] via the recombination of positive charges (holes) on different S states of the OEC and negative charges on primary electron acceptors ‘Author
to whom
loll-1344/93/$6.00
correspondence
should
be addressed.
Thermoluminescence;
Oxygen
evolution
(Q,) or secondary electron acceptors (Q,) on the reducing side of PS II. In this paper, we examine the senescence-mediated modification of the OEC of chloroplasts and the alteration of the charge pairs responsible for the different TL peaks associated with PS II to obtain information on the reducing and oxidizing components of reaction centre II (RC II).
2. Materials
and methods
Wheat (Triticum aestivum L. CV. Sonalika) seedlings were grown in Petri dishes for 7 days in continuous white light (fluence rate, 12 W m-‘) obtained from a fluorescent tube lamp at 25 f 2 “C without any external nutrients as described previously [lo]. In one set of experiments the seedlings were kept under the same conditions until day 15. In a second experiment the seedlings grown for 7 days in light were transferred to the dark and allowed to senesce until day 15. Pigments were extracted from the primary leaves of wheat seedlings in ice cold 80% acetone in dim green light and the extract was used for spectrophotometric determination of chlorophyll (Chl) as described by Arnon [ll]. 0 1993 - Elsevier
Sequoia. All rights reserved
198
P.N. Joshi et al. I PS II photochemistry of thylakoids porn senescing wheat leaves
Chloroplasts were isolated from the primary leaves of wheat seedlings by different treatments according to the method described by Izawa and Good [12]. The grinding medium used contained 0.3 M NaCl, 30 mM Tricine (pH 7.8) and 1 mM ethylenediaminetetraacetic acid (EDTA). The washing medium contained 0.2 M sucrose, 20 mM NaCl and 20 mM MOPS (3-[N-morpholinolpropanesulfonic acid) (pH 7.4). The chloroplasts were finally suspended in a medium containing 300 mM sucrose, 50 mM NaCl and 50 mM Na/K phosphate buffer (pH 6.9) with a Chl concentration of 2 mg Chl ml-‘. Oxygen evolution was measured with a Clarktype oxygen electrode at 21 “C in rate-saturating red light. The basic assay buffer consisted of 30 mM Na/K phosphate (pH 7.2) with 30 mM NaCl and 200 mM sucrose. The electron acceptor was 0.4 mM K,Fe(CN),. The reaction mixture (2 ml) for assay contained 40 pg equivalent of chloroplasts. When used as an uncoupler, the gramicidin concentration was 2.5 PM. The fluorescence transient of the primary leaf was followed, as described by Kulandaivelu and Daniel [13], by exciting the leaf with a broad-band blue light at saturating intensity. Leaves were dark adapted for 20 min before fluorescence measurement. The signal was stored in a digital oscilloscope (Iwatsu SS-5802) and then transferred to a recorder. The glow curves were obtained following the method of Desai et al. [14]. Isolated chloroplasts in suspension medium containing 100 pg Chl ml-’ were frozen to liquid nitrogen temperature under illumination at a fluence rate of 1 W m-*. TL experiments were run at a heating rate of 0.22 “C s-l. 3-(3,4-D’ic hl orophenyl)-1,1-dimethyl urea (DCMU) was added at a concentration of lo-’ M. The activation energy E was calculated using an Arrhenius plot [15, 161. The mean lifetime 7r was calculated using the relation T,= eElkT/S following Tatake et al. [17], where T is the absolute temperature, S is the preexponential frequency factor and k is the Boltzmann constant. 3. Results Leaf senescence was assessed on the basis of Chl loss. Figure 1 depicts the change in Chl content per gram of fresh weight in the primary leaves of wheat seedlings senescing in the light or dark on day 15. The Chl content decreased by 42% in the light and 76% in the dark.
A
8
C
Fig. 1. The content of Chl per gram of fresh weight in primary wheat leaves: (A) 7 day old seedlings; (B) 15 day old lightsenescing seedlings; (C) 15 day old dark-senescing seedlings. Chl content of 7 day old leaf is taken as 100%. Experimental details are given in Section 2. TABLE 1. Or evolution as a measure of PS II activity in isolated wheat chloroplasts. Each value is a mean of three independent results Treatment
Or evolution with K,Fe(CN), as electron acceptor (pmol (mg Chl-‘) h-l)
7 days light 15 days light 15 days dark
420 (100) 212 (50) 156 (37)
Values in parentheses indicate percentage. Dark treatment starts from day 7 until day 15. Other experimental details are given in Section 2.
The inactivation of the OEC was monitored by measuring the oxygen evolution of chloroplasts isolated from senescing leaves (Table 1). The rate of oxygen evolution was reduced by 50% and 63% during senescence in the light and in the dark respectively, where the rate of oxygen evolution from the chloroplasts of 7 day old primary leaves was taken as 100%. The changes in the parameters associated with the fast fluorescence transient of primary wheat leaves are shown in Table 2. The F0 level remained unchanged in leaves senescing in the light, whereas it decreased by 23% in leaves senescing in the dark. The variable fluorescence (Fv = FM - F0 where Fhl is the maximum fluorescence and F,, is the constant fluorescence) and the F,/F, ratio decreased in leaves from both light- and darkincubated seedlings, the decrease being 13% and
199
P.N. Joshi et al. / PS II photochemisOy of thylakoids from senescing wheat leaves TABLE 2. Effect of senescence on different parameters associated with fast fluorescence induction in primary leaves of wheat seedlings. Seedlings grown for 7 days in the light were transferred to the dark and senescence was studied until day 15. Each value is a mean of three independent calculations Treatment
Fv’
&/FM
T,,
I
CS;0;)
2.7 2.7 2.1
6.3 5.9 4.1
3.6 3.2 2.0
0.58 0.53 0.48
15d Light
-.--
15d Dark
/ .*‘-.A
.r\ \ /a
170 300 390
il
1’
I-
!I
!/
\
\\
‘\
I,
.\
\,‘,,
i
/ /-
1: \\?\
. ..
‘F,, mtttal fluorescence. bFM, maximum fluorescence. ‘Fv = FM-F,, variable fluorescence. F,, FM and Fv are in arbitrary units. For other details, see Section 2.
7d Light
;\L
of FM rise
(@I 7 days light 15 days light 15 days dark
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\
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TL
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I
48
(“Cl
Fig. 3. Glow curves of isolated chloroplasts obtained from primary wheat leaves senescing in the light or dark in the presence of DCMU.
Temperature
1°C)
Fig. 2. Glow curves of isolated chloroplasts from 15 day old primary wheat leaves senescing in the light or dark.
11% respectively in the former and 45% and 18% respectively in the latter. The glow curves of isolated chloroplasts from wheat leaves showed five well-resolved peaks, two in the absence of DCMU and three in the presence of DCMU in the temperature range of interest. These peaks appeared at around 236 K (I), 261 K (II), 283 K (III), 298 K (IV) and 321 K (V) which agree well with earlier findings [9]. These peaks are named following Tatake et al. [ 171. Peaks I and IV were obtained in the TL curve of normal chloroplasts. On poisoning the chloroplasts with DCMU, peaks I and IV disappeared and peaks II, III and V were observed. The glow curves obtained from isolated chloroplasts of primary wheat leaves of seedlings senesting in the light and dark are depicted in Fig. 2. The height of peak I decreased and peak IV was shifted by 10 “C towards a lower temperature in the light. The heights of peaks I and IV were
decreased significantly during dark-induced senescence without any alteration in peak temperature. Figure 3 shows the glow curves of DCMUpoisoned chloroplasts isolated from the primary leaves of wheat seedlings senescing in the light or dark. The intensity of the TL bands II, III and V decreased and peaks III and V were shifted marginally towards higher temperature in the sample senescing in the light. However, in the sample senescing in the dark, the intensity of peak II decreased and peak III was modified and enhanced. Table 3 shows the changes in activation energy (E) and mean lifetime at room temperature (7,) calculated from the glow peaks. 4. Discussion PS II-mediated oxygen evolution decreases during the senescence of wheat seedlings. The loss in oxygen evolving capacity is attributed to damage of the OEC, since the loss has been shown to be restored by exogenous electron donors such as DPC [2]. The damage to the OEC has been correlated with the depletion of extrinsic polypeptides associated with the complex [8] leading to RC II degradation. In order to understand the mechanism of RC II degradation, fluorescence induction was examined. The ratio F,/F,, a measure of the photochemical potential, decreases in the leaves of both light-
200
P.N. Joshi et al. / PS II photochemistry
of thylakoids from senescing wheat leaves
TABLE 3. Different energetic parameters calculated from the TL peaks of isolated chloroplasts treatment starts from 7 days. Each value is the mean of three independent glow curves
with different
treatments.
Dark
Treatment
Parameter (unit)
I (236 K)
II (261 K)
III (283 K)
IV (298 K)
V (321 K)
7 days light
E (eV) rr (s)
0.49 0.259
0.62 1.19
0.74 5.86
1.22 28.47
1.4 28.83
15 days light
E (eV) rr (s)
0.489 0.263
0.61 1.28
0.73 8.8
1.19 28.00
1.42 28.38
15 days dark
E (eV) rr (s)
0.471 0.33
0.589 1.498
0.67 9.85
1.25 27.78
-
E, activation
energy in electron
volts (ev).
r,, mean lifetime
at room temperature
and dark-senescing seedlings but in two distinctly different modes. In the former, the decrease in Fv is solely due to a decrease in FM since F. tends to remain unchanged, whereas in the latter the decrease is due to a simultaneous decrease in FM and F. (Table 2). Furthermore, the decrease in F,, the enhancement of T,, of the FM rise and the constancy of F. in the light-senescing sample suggest that the impairment is at the level of RC II, the electron transfer chain between QA and Qg or both [18] and is photoinhibitory [19]. On the other hand, the decrease in F,, Fv and FM and the enhancement of T,,z of the FM rise (Table 2) may indicate an impairment on the donor side of PS II [20]. These results suggest that RCs II are rendered inactive for the trapping of excitons with a concomitant increase in thermal deactivation in the antenna complex. The possible changes in F. and Tin of the FM rise due to variation in the antenna size have not been emphasized, since senescence-mediated loss of Chl is accompanied by a decrease in the number or active RCs II as evident from the TL studies described below. Glow curves of chloroplasts, isolated from primary leaves of senescing seedlings, were examined to locate the exact site(s) of impairment. Sane et al. [21] proposed that the origin of peak I was a recombination of charges stabilized on Z+ and Qg -. The absence of a Z,, band (equivalent to TL band I under our experimental conditions) in the glow curves of etiolated leaves and isolated subchloroplast particles [22], its presence in the glow curves of wheat leaves greened under intermittent flash illumination [23] and its removal on DCMU treatment provide enough evidence in support of this proposition. However, the origin of the peak changes is not well understood. A decrease in peak I height may indicate a decrease in the number of charge pairs or the number of active RCs II during senescence either in the light
in seconds
(s).
or in the dark, and the proposition confirms the conclusions drawn on the basis of a decrease in height of peaks II, III and IV. Peak IV, consisting of two overlapping bands and resulting from SZQB- and S3QB- charge pairs [24], is the most well characterized of all the TL bands of chloroplasts. The shifting of the peak temperature towards the ascending side of the glow curve in the light-senescing sample indicates a decrease in mid-point redox potential [9, 251, suggesting a decrease in stability of SZQB- and S,QB- charge pairs which is also reflected in energetic parameters (Table 3). The modification of Qg due to the altered conformation of the D, protein of the reaction centre core complex of PS II may be the reason. Recently, Ohad et al. [26] have proposed the downshift of the B band peak temperature and the damping of its oscillation pattern as a result of the destabilization of the D1 protein conformation. The light intensity used during the growth of the seedlings may be greater than the intensity required to saturate photosynthesis during senescence, since the photosynthetic capacity decreases, as shown by the decrease in oxygen evolution (Table 1) and photochemical potential (Table 2). The system under such conditions is comparable with the photoinhibitory system of Ohad et al. [26]. Thus the structural modification of the Qg binding protein as suggested by Ohad et al. [26, 271 is induced in the light-senescing sample. Recently, Kettunen et al. [28] have also shown the modification of the D, protein prior to its degradation during photoinhibition of intact leaves, which supports our proposition. However, although there is a decrease in the peak height in the darksenescing sample reflecting a decline in the number of active charge pairs, no shift in peak temperature has been demonstrated. The drastic loss of oxygen evolution in dark-induced senescence (Table 1) may be due to a decline in the number of active
P.N. Joshi et al. 1 PS II photochemistry of thylakoids from senescing wheat leaves
reaction centres of PS II as reflected in a decrease in the peak height. These findings suggest that the damage of RC II in the light-senescing sample is linked to a light-mediated structural modification of the D1 protein as shown in the downshift of the peak IV temperature and is different from the hitherto unknown molecular mechanism of dark-induced damage of the PS II reaction centre. Peaks II and III are known to arise from S3QA[29] and SZQA- [25] charge pairs respectively. A decline in the intensities of these peaks during senescence in the light is indicative of a decrease in population of the S2 and S3 states of the OEC or QA- or both. Since the damage of QA, which is buried deep inside the D2 protein, is less probable, an alteration in the S state transition may be the major cause of decline. Our data showing a higher lifetime of the S2Qa- pair (Table 3) confirm this view. On the other hand, a decrease in peak II and an increase in peak III intensities in the dark-senescing sample reveal the critical nature of the increase in S, population suggesting a blockage of the Sz + S3 transition. Furthermore, extensive modification of peak III is suggestive of intense membrane damage and is supported by the activation energy data which decrease by 31 and 70 meV for peaks II and III respectively (Table 3). We therefore believe that the loss of oxygen evolution as demonstrated previously in different plant systems and in wheat in this study (Table 1) during leaf senescence is possibly due to an alteration in the S state transition and/or destabilization of Qg on the D1 protein of the PS II reaction centre in the thylakoid membrane.
Acknowledgment
Facilities for the measurement of fluorescence transients were made available by Professor G. Kulandaivelu, Department of Biological Sciences, Madurai Kamraj University.
References
8
9 10
11
12
13
14
15
16
17
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21
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