Preillumination effects on chloroplast structure and photochemical activity

Plant Science Letters, 4 (1975) 115--123 115 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

PREILLUMINATION EFFECTS ON CHLOROPLAST STRUCTURE AND PHOTOCHEMICAL ACTIVITY

C. VERNOTTE, J.-M. BRIANTAIS, P. ARMOND* and C.J. ARNTZEN** Laboratoire de Photosynth~se du C.N.R.S., 91190 Gif-sur- Yvette (France) and *Department of Botany, 289 Morrill Hall, University of lllinois, Urbana, IlL 61801 (U.S.A.) (Received October 21st, 1974)

SUMMARY

The effects of in vivo preillumination with short wavelength red light (647 nm) and long wavelength red light (710 nm) on fluorescence and photochemical characteristics of algal and higher plant chloroplasts were analyzed and data are correlated to previous studies of divalent ion effects on isolated chloroplasts. The 647 nm light increased the sensitization of photosystem I as determined from fluorescence emission at low temperature, decreased room temperature variable yield fluorescence, decreased the sigmoidal aspect of fluorescence rise, and increased the rate of photosystem I reactions measured in low intensity 647 nm light. DCMU blocked 647 preillumination effects. These data are consistent with a previous suggestion that preillumination effects on chloroplast fluorescence are related to light, induced ionic redistributions in the organelle. Structural organization of pea chloroplasts preilluminated with either 647 or 710 nm light were not significantly different with respect to the extent of grana stacking observed. The differences between membrane structural reorganization observed after preillumination and those observed with chloroplasts in the presence or absence of salts is discussed.

INTRODUCTION

It is now generally agreed that there exists in chloroplast membranes a mechanism which regulates excitation energy distribution between the two photosystems [1]. One major line of evidence for this concept developed from studies by Murata [2] on Porphyridium and by Bonaventura and Myers [3] on Chlorella. During sample illumination with light I (near 710 nm), slow changes resulted in an increased sensitization of photosystem II whereas Abbreviations: Chl, chlorophyll; DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethylurea; DPIP, 2,6-dicblorophenolindophenol; MV, methyl viologen; Tricine, N-Tris(hydroxymethyl)methylglycine.

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light II (at 650 nm) gave a time-dependent redistribution of excitation to p h o t o s y s t e m I (known as the State I--State II p h e n o m e n o n [1] ). More recently Murata and others [4--9] have also demonstrated that changing divalent cation concentrations in suspensions of isolated chloroplasts markedly alters the distribution of absorbed quanta between the t w o photosystems. Gross and Hess [10] have shown that the presence of monovalent salts is essential for the divalent cation effect. It is known that illumination of chloroplasts results in ion movements across the thylakoid membranes [ 1 1 - - 1 3 ] . Murata [4] has therefore hypothesized that saltAnduced changes in transfer of excitation energy in vitro are phenomenologically related to the lightinduced (State I--State II) changes observed in vivo. Recent data b y Krause [14] and Barber et al. [15] add credulence to this idea since t h e y have shown that time-dependent variations of p h o t o s y s t e m II fluorescence (an indirect measure of exciton~ arrival at p h o t o s y s t e m II) correlate closely with ion transport phenomena. To date, light-induced state changes in chloroplasts have only been shown for algal cells whereas most salt-induced state change studies were with isolated higher plant chloroplasts. In this study we will demonstrate that State I--State II changes can be demonstrated in vivo for chloroplasts of higher plants. These findings will be related to the state changes occurring in vivo in ChloreUa. MATERIALS AND METHODS

Chlorella pyrenoidosa cells were grown according to the procedures of Delrieu and De Kouchkovsky [16]. The concentration of Chl in the final suspension used was 50 ~g/ml. Chloroplasts were isolated as previously described [17] from pea, lettuce or spinach leaves with the exception that the grinding medium contained: Sorbitol, 0.4 M; Na-Tricine, 0.1 M; and glutaraldehyde at 0.5% final concentration. Chloroplasts were washed once in NaC1, 0.01 M [17]. Preilluminations of algae or leaves were performed using a 647 n m + 6.2 nm interference filter or a set of 2 filters (RG 8 cut off plus K 7 interference filter) to give 710 n m + 17 nm illumination. The intensities used were respectively 2500 and 55 000 erg. crn- 2. s- 1. Leaf samples were floated on a water bath during illumination. Algal suspensions were preilluminated directly in the fluorimeter vessel. The thickness of the suspension in the vessel was 1 mm. For these latter experiments, the excitation m o n o c h r o m a t o r had a half-bandwidth of 20 nm. The intensities received at 480, 650 and 710 nm were, respectively, 50 000, 32 000, 25 000 erg. cm- 2. s- 1. Emission spectra at liquid nitrogen temperature were determined using a Dewar flask on the b o t t o m of which a 1 ml volume of the suspension was absorbed on 2 layers of cheese-cloth as described b y Cho and Govindjee [ 1 8 ] . The half-bandwidth of the analytic m o n o c h r o m a t o r was 1.6 nm. Fluorescence inductions were recorded with an oscilloscope. P h o t o s y s t e m

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I activity was determined by measuring, in limiting light, the rate of the oxygen uptake of chloroplast suspensions containing 25 pg of Chl/ml, 10- s M D C M U , 10- 4 M azide, 10- 3 M sodium ascorbate, 10- 4 M DPIP, 2.5.10- 4 M M V , 10- a M sorbitol and 0.05 M Tricine (pH 7.8). Oxygen exchanges were detected using a Clark electrode. RESULTS

As has previously been demonstrated [2,3,19], prefll~mination conditions markedly influence the Chl fluorescence emission spectra of algal cells (Fig. 1A). The ChloreUa sample used in these experiments was preilluminated for 10 rain with either 710 or 647 nm light before freezing at 77°K. The spectrum of dark-pretreated cells was intermediate between the two curves shown in Fig. 1A. A decrease of the 685 and 695 nm emission bands and an increase of the 720 nm peak as compared to the control (dark) spectra were induced b y 647 nm preillumination. Opposite effects were observed after 710 nm preirradiation. We have attempted to detect similar changes in the chloroplasts of higher plants following preillumination of whole leaves. This was accomplished by quickly grinding the illuminated leaves in the presence of glutaraldehyde, a chemical fixative which maintains the structural organization of the membranes. As is shown in Fig. 1B, these fixed membranes isolated from preilluminated leaves show changes in the fluorescence emission spectra that are qualitatively very similar to those seen in preilluminated whole algae: short wavelength fluorescence (at 685 nm) increased but long wavelength fluoresi

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Fig.1. Liquid nitrogen temperature fluorescence emission spectra o f Chlorella (A) a n d isolated pea chloroplasts (B). Samples were preilluminated for 15 min with either 710 n m or 647 nm light (dotted and solid lines, respectively), prior to freezing Of Chlorella celia or prior to chloroplast isolation from pea leaves.

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cence (at 730 nm) decreased following 710 nm preillumination whereas 647 nm illumination elicited opposite effects. The spectrum of dark-pretreated samples was intermediate b e t w e e n the curves shown in Fig. lB. It should be noted that glutaraldehyde fixation reduces the 730 nm emission band as compared to unfixed controls. Isolated, fixed membranes retain the same fluorescence characteristics over long periods (hours) of storage at 4 ° . It has previously been reported that divalent salts primarily affect the variable yield fluorescence of isolated chloroplasts [8]. We have analyzed the light-induced fluorescence increase of Chlorella following either 647 or 710 nm preillumination and compared this to the variable fluorescence of preilluminated pea chloroplasts. In preilluminated algae, the Fo (immediate fluorescence) level was changed b y less than 4% from that of the dark-adapted cells. In samples of isolated chloroplasts adjusted to equal Chl concentrations, there also seemed to be no change in Fo in preilluminated as compared to dark-adapted preparations. As is shown in Table I, however, the preillumination treatment markedly affected the variable yield (AF) fluorescence. The pattern of change of fluorescence characteristics was qualitatively identical for b o t h the algal and higher plant material. It was of interest to determine the effect of DCMU on State I--State II adaption in Chlorella. It is shown in Table II that if DCMU was present during the pretreatment b o t h 647 and 710 nm preillumination cause an increase in variable fluorescence and a decrease of the 7 2 0 / 6 8 5 nm ratio of the liquid nitrogen temperature emission spectra. Thus DCMU blocks State II adaption whereas State I is reached. This is consistent with previous studies of TABLE I COMPARISON OF F L U O R E S C E N C E INDUCTION CHARACTERISTICS OF C H L O R E L L A CELLS (EITHER DARK-ADAPTED OR PREILLUMINATED F O R 10 MIN) TO CHARACTERISTICS OF G L U T A R A L D E H Y D E - F I X E D PEA CHLOROPLASTS (ISOLATED FROM EITHER DARK-ADAPTED OR PREILLUMINATED LEAVES) The algae were resuspended in 30 rnM phosphate buffer (pH 6.5) containing 100 mM KC1. DCMU was added just before fluorescence measurements to give a final concentration of 10-s M. Chloroplasts were suspended in 0.2 M sorbitol containing 0.01 M Na-Tricine (pH 7.8), 0.01 M NaC1 and 0.01 M NH, C1. The excitation beam was 480 nm + 10 nm. Analysis of fluorescence was through a Corning 2-64 filter plus a Wratten 70 filter. A fast trace storage oscilloscope was used to record the fluorescence rise curves. Sample

z~F/Fo Preillumination conditions

Chlorella Pea chloroplasts

Dark

647 nm light

710 nm light

1.62 2.32

1.53 2.06

1.90 2.58

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TABLE II E F F E C T O F D C M U (I0-s M) P R E S E N T D U R I N G P R E I L L U M I N A T I O N O N C H A N G E S O F T H E 77 ° K F L U O R E S C E N C E EMISSION S P E C T R A A N D V A R I A B L E F L U O R E S C E N C E A M P L I T U D E (AT R O O M T E M P E R A T U R E ) O F C H L O R E L L A C E L L S Algae were treated as in Table I. DCMU was present in all samples during fluorescence measurement. Preillumination conditions

Ratio o f fluorescence intensity of 720 and 685 nm emission peaks at 77 ° K

(F,~0)/(F,s, )

Dark 6~7 n m light;I0 min 710 n m light;10 min

Maximal fluorescence intensity at 685 nm (room temperature; expressed as % of dark adapted control value)

DCMU concentration during preillumination

DCMU concentration during preillumination

0

10 -5 M

0

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1.04 1.22 0.99

1.19 1.02 1.04

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100 110 110

Mohanty and Govindjee [20]. It should be noted that D C M U alone had an effect on the emission spectra of dark-treated cells. It was noticed that the kinetics of fluorescence rise in both algal cellsand pea chloroplasts which had been preirradiated at 710 n m showed a marked sigmoidal pattern of fluorescence increase. This was less evident in 647 n m pretreated samples. To emphasize this point, the amplitude of the variable yield of fluorescence versus oxidized Q [21] are plotted in Fig. 2. (The amount of Q was estimated by the complementary area according to Malkin [22].) The figure~ points out the decreased sigmoidal kinetics in State II as dompared to State I-adapted algae. Glutaraldehyde-fixed chloroplasts from 647 n m preilluminated leaves also show less sigmoidal kinetics than those from dark-adapted or 710 preilluminated samples. It has previously been shown that the presence of M g ~+ affects the relative sensitization of photosystem I. In Table III,the rate of photosystem I activity (DPIPH2 to M V electron transport) of glutaraldehydefixed plastids from preiUuminated pea leaves is characterized. The rate of electron flow was measured in either 647 or 710 n m light,both at limiting intensities,and the ratio of these rates is presented. The 647/710 reaction rate ratio was always higher in samples preilluminated at 647 n m as c o m p ~ e d to those samples preilluminated at 710 nm, thus indicating an increased distribution of excitons to photosystem I in the short wavelength (State If) treated tissues.It should be noted that plastidsfrom dark-adapted tissueswere sometimes similar to State I and sometimes similar to State If-adapted chloroplasts. Previous structural studies of chloroplasts isolated in divalent-cation free

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Fig.2. Variable yield of fluorescence as a function of oxidized Q. Values of AF were measured from fast trace oscilloscope recordings of room temperature fluorescence induction of Chlorella cells which were pretreated as described in Table I. Oxidized Q w a s m e a s u r e d by the complementary area of the induction according to procedures of Malkin [22 ]. Fluorescence measurement conditions were as in Table I. TABLE

III

THE E F F E C T OF LEAF PREILLUMINATION ON THE SENSITIZATION OF PHOTOSYSTEM I BY 647 AND 710 n m LIGHT The ratio of rates of MV reduction in limiting light is presented for glutaraldehyde-fixed p e a chloroplasts isolated from leaves with different p r e t r e a t m e n t s . Rate of MV reduction in 647 n m light Rate of MV reduction in 710 n m light

Conditions for leaf preillumination prior to chloroplast isolation Dark

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medium have demonstrated that these plastids lose all grana stacking [ 2 3 ] . Addition of divalent salts to the chloroplast suspension results in reestablishment of regions o f membrane fusion [24]. Since some aspects of the preillumination-inducect state changes are similar to the salt-mediated state changes, we have examined the chloroplast structure of preilluminated pea leaves. Chloroplast membranes isolated from 647 nm and 710 nm preilluminated leaves are shown in Fig. 3 A and B. Grana stacks and connecting stroma lameUae of these washed preparations are still evident. There was no significant difference in the number of thylakoids per grana stack in the t w o samples.

121

Fig.3. Thin sections of isolated chloroplast preparations which were preilluminated prior to, isolation. (A) 710 nm pretreatment; (B) 647 nm pretreatment. Glutaraldehyde was included in the isolation media as described in METHODS. Following the initial centrifugation at 1000 g t o obtain a pellet, membranes were washed four times with 0.05 M phosphate buffer pH 7.2, and then post-fixed for 12 h in 1% OsO 4 in 0.05 M phosphate buffer, pH 7.2. Samples were embedded in Epon and sectioned with a diamond knife. (× 35 000).

122

In general, the overall structural organization of both membrane preparations was nearly identical. DISCUSSION We have isolated chloroplasts from leaf tissue using a grinding solution containing glutaraldehyde. This has allowed the preparation of plastid samples which are "fixed" in the structural state induced by preillumination conditions immediately prior to tissue disruption. Characterization of the fluorescence emission of these plastids revealed the same type of in vivo State I--State II changes that occur in Chlorella (Fig. 1). Furthermore, we have demonstrated that the decrease in room temperature fluorescence in State II-adapted chloroplasts is due to a decrease in AF whereas Fo is not significantly changed. These data indicate that the preillumination-induced, in vivo plastid state changes are of identical nature in both algae and higher plants, at least with respect to the fluorescence characteristics of the chloi oplasts. It was suggested [4] that plastid State I--State II changes are phenomenologically similar to the effects of divalent cations on photoreactions of isolated chloroplasts, with State II being comparable to chloroplasts isolated in the absence of divalent salts. The results of the present study, when compared to a previous analysis of divalent salt effects, are consistent with thi~ idea. We have previously demonstrated that addition of divalent cations to "low-salt" chloroplasts changes AF but not Fo [8]. As discussed above, this is also true for the preillumination treatments. Fig. 2 showed that State IIadapted plastids show a less sigmoidal rise in AF than State I-adapted. It was also demonstrated that "low-salt" chloroplasts have the same relationship to samples incubated with divalent cations [8]. In experiments with isolated plastids, Mg2÷ increased the relative sensitization of photosystem I when activity was measured in 647 nm light [8]. The same pattern of excitation distribution occurs in State II-adapted chloroplasts (Table III). It was shown in Table II that the presence of DCMU during preillumination blocks State II adaption but not State I adaption in Chlorella. In other experiments (not reported) we have confirmed the time-dependent decline in fluorescence yield in isolated chloroplasts which Krause [14] and Barber et al. [15] have related to ion movements. This fluorescence decrease is also blocked by DCMU. It remains to be determined why photosystem II electron transport, but not photosystem I electron flow, is apparently effective in causing the release of divalent ions from some "effector site". The only significant aspect of our studies which showed a marked difference between isolated "low-salt" chloroplasts and State II-adapted chloroplasts was th.e electron microscopic examination of preilluminated membranes. It is now well accepted that chloroplasts isolated in the absence of cations have largely unstacked lamellae [23--25]. Although the pea chloroplasts from 647 nm preilluminated leaves had fluorescence characteristics similar to preparations free of divalent cations, the light.treated membranes showed

123 normal a m o u n t s o f m e m b r a n e stacking. It seems likely t h a t if divalent cation fluxes are involved in t he State I-State II p h en o mena, as outlined above, t hen t he illumination must result in changes in ion binding sites which alter internal protein-pigment associations b u t do n o t significantly m o d i f y t he m e m b r a n e surface factors which regulate grana stacking. I t would t h e r e f o r e appear t hat f u r t h e r study must be directed toward light, induced transitory substructural m e m b r a n e reorganizations t h a t regulate excitation distribution. It is necessary to recognize t h a t study of salt-free chloroplasts m a y n o t be a means of looking at exactly t he same changes as those which occur in vivo during illumination, s i n c e t h e salt removal produces a broader range of modifications, including unstacking of lamellae which is apparently n o t directly involved in regulating excitation distribution. ACKNOWLEDGEMENTS Research was supported in part by CNRS and D G R S T grant No. 7 2 7 0 1 7 4 to J.-M.B. and funds f r o m t he Agricultural E x p e r i m e n t Station, University o f Illinois to C.J.A. REFERENCES 1 J. Myers, Ann. Rev. Plant Physiol., 22 (1971) 289. 2 N. Murata, Biochim. Biophys. Acta, 172 (1969) 242. 3 C.J. Bonaventura and J. Myers, Biochim. Biophys. Acta, 189 (1969) 366. 4 N. Murata, Biochim. Biophys. Acta, 189 (1969) 171. 5 N. Murata, H. Tashiro and H. Takamiya, Biochim. Biophys. Acta, 197 (1970) 250. 6 N. Murata, Biochim. Biophys. Acta, 245 (1971) 365. 7 P. Homann, Plant Physiol., 44 (1971) 932. 8 J.-M. Briantais, C. Vernotte and I. Moya, Biochim. Biophys. Acta, 325 (1973) 530. 9 E.L. Gross and S.C. Hess, Arch. Biochem. Biophys., 159 (1973) 832. 10 P. Mohanty, B. Braun and Govindjee, Biochim. Biophys. Acta, 292 (1973) 459. 11 R.A. Dilley and L.P. Vernon, Arch. Biochem. Biophys., 1J1 (1965) 365. 12 P.S. Nobel, Biochim. Biophys. Acta, 172 (1969) 134. 13 G. Hind, H.Y. Nakatani and S. Izawa, Proc. Natl. Acad. Sci. (U.S.), 71 (1974) 1484. 14 G.H. Krause, Biochim. Biophys. Acta, 333 (1974) 301. 15 J. Barber, A. Teller and J. Nicolson, Biochim. Biophys.Acta, 357 (1974) 161. 16 M.J. Delrieu and Y. de Kouchk.ovsky, Biochim. Biophys. Acta, 226 (1971) 409. 17 C.J. Arntzen, C. Vernotte, J.-M. Brantais and P. Armond, Biochim. Biophys. Acta, 368 (1974) 39. 18 F. Cho and Govindjee, Biochim. Biophys. Acta, 205 (1970) 371. 19 G. Papageorgio and Govindjee, Biophys. J., 8 (1968) 1316. 20 P. Mohanty and Govindjee: Plant Cell Physiol., 14 (1973)611. 21 L.N.M. Duysens and H.E. Sweers in Microalgae and Photosynthetic Bacteria, Univ. Tokyo Press, Tokyo, 1963, p.353. 22 S. Malkin, Biophys. J., 7 (1967) 629. 23 S. Izawa and N.E. Good, Plant Physiol., 41 (1966) 544. 24 S. Murakami and L. Packer, Arch. Biochem. Biophys., 146 (1971) 337. 25 R. Ohki, R. KUnieda and A. Takamiya, Biochim. Biophys. Acta, 226 (1971) 144.