Volume 309, number I, 73-?? FEBS 11475 O 1992 Federation of European Biorhemi¢=} ~.~a~-= ~;.L~99~I~5.00

August 1992

Detection of the photoactive protochlorophyllide-protein complex in the light during the greening of barley Fabri¢¢ F r a n c k ' a n d K a z i m i e r z Strzalka s

•Laboratory of Photabiology, Deparnnent of Botany B23, Univer~iO,of Lilge, SarbTihuan, B.4000 I.a~ft¢. Belgium and ab~titute of Molecular Biology. Jagiellonlan Univer#ity, AI, Mtckiewit:a 3, 31-t20 Krakaw, Poland Received 8 July I992 A photoaetiv¢protoehlorophyllid=.protein¢ompleawith absorban¢¢ alld Rapt=cent maximaat 64S and 653 am was dora:ted in llr==ninilI~rley leaves without any r¢-darkeninil, T ~ variations of the amiditudeaof the ab-.orban~ and the fluor¢.~¢¢n¢¢of the photoaetiv¢protoahlorophylli~k= with g~eeningtime at two different lillht intenaitimlindicate a do= relationthip betweenthe rate of chlorophyll synthesisand the amount of the ¢oml:t~x daring the first hours, The ehlorophyllide resulting from photoreduetiondarinli greeningha~ an absorban¢©maximum at 61~ am, which

shirt towardsa shorter wav=lcnEthwithina few~conds, ladi~atinsrapid liberationof the pigmentfrom the ¢nxyme,We ¢oncladethat chlorophyll a~:umulation proceedsthroui~ continuous regenerationand phototransformation of the photoaetiv=compl¢~. Chlorophyll synth~is; Protoehlorophyllide redu=ar,e; Gr=nin$

I. I N T R O D U C T I O N

650 and 657 nm, respectively [6,7], T h e e bands disappear after a short light flash and are replaced by Chlide

In angiosperms chlorophyll (Chl) synthesis depends on light. Although much effort has been undertaken in order to elucidate the mechanism governing Chl accumulation in the light, the role o f the enzyme, protochlorophyllide (Pchlide) reductase, in this p r o ~ s s is still controversial [1-3]. This e n ~ m e is a major membrane protein o f the etioplasts o f etiolated plants where it ¢.atalyz~s the photoredaetion of Pchlide into ¢hlorophyllide (Chlide). Upon continuous illumination o f etiolatexi plants the amount and the activity of this enzyme, however, decrease to a low level within a short time (I-2 h) although the rate of Chl accumulation increases at the same time [1,2,4]. For this reason it was sometimes concluded that the function of Pchlide reductase in Chl synthesis is restricted to a short time after a sufficiently long period of darkness and that Chl synthesis in greening or green leaves might involve another, hypothetic enzymatic system [1,2].

bands. It is therefore in principal easy to detect photoa¢tire Pchlide through in viva s~ctroscopi¢ measurements.

In ctioplasts.Pchlide reductase forms a stable complex with its substrate, Pchlid¢. and the cola=tar, N A D P H [5], Upon illumination, Pehlid¢ is rapidly reduced into Chlide in the complex which then dissociates within several minutes, The Pchlide~nzym¢-NADPH complex o f etiolated leaves, or 'photoactive Pehlide', has in vivo main absorbanc¢ and fluoresc.¢n~ bands at

Corre~Fondcnc¢address: F, Franck, Laboratoryof Photobiolo~y,~:partment of Botany B22, Universityof Liege, Sart-Tilman, B-4000 l.i~s¢, Belgium.Fa~: (32)(41)562926. Abbr¢vfatlQ~: Chl, chlorophyll; Chlid¢, chlorophyllide; Pchl, protochlorophyll; Pchlid=, protoehlorophyllid~,

Pubtished by Elsevfer Science Fubl~hePs B. g

If Pghlide reduotase remains the main light-requiring enzyme involved in Chl accumulation during greening, one expects the photoactiv¢ Pchlid=.-protein complex to

be continuously regenerated and phototransformed in the light. If this is so, a small amount o f the complex should be present at any time in the ligtzt, especially when light intensity is low, In this paper we show by in viva spectroscopic measurem¢nts that this is indeed the

case. Our results also indicate that Chlide dissociation from the enzyme is much more rapid in illuminated leaves than in ¢tiolat~ ones, which can explain that rapid Chl accumulation is sustained ¢v=n at relatively low enzyme levels.

2. MATERIALS A N D METHODS Barleyt~Mlin~ (tlord~um Pulgurecv, Avilion)weregrow in darkn¢.~ for 6 days on vermiculite and tap water at 23'C. Greening was performed with intactplants undercool white fluorerccnt tubesat two different intensiti¢=of 2 and 20 W,m"= (low and medium intemitic=)

at 25*C.

Flaor__~__n¢~and fluoresccnt¢excitation spc=tm w=r¢ rccord=dat 77 K usinil a Perkin.Elmer LSSOfluorimeter. Fair r=cordin~ of at> =orbanc¢spc=trawere ~rformed at room temperaturewith an optical

multichanmlanalyz=r(OMA II, Prino=tonInstruments).In this car,¢ Srgcnin$was carriedout in the experimentalr~t.upwiththe measuring light (Sylvania 21 V projector lamp. type DKM) at the same time actinic and analytic,at an intensitywhichcould be fixedby tim.gin8 the voltage applied to the lamp, The flash used to r,aturat= Pchlidc photorcdumtionwas providedby a Strobe Multiblitzflash lamp(duration of flash, 2 ms), Flash.inducedabsorbanc¢ variations at fised



Volume 309. number I whh the. ~rrm {1~, _=


0 20'~m '1 A,









Greening time

4 (hour)









664 I





FiB. 4, (A) Time-cou~ of th¢ increa~ of the in vivo Chl absorbancc (measured around 678 nm) at room temperature during ~'¢cninB. (B) Room t¢mpcrature a b s o r b a n ~ dilTcrcn¢~ spectra (unflash~-fla,=hed) obtain¢~ with =tiolated l=av~ (I) or with the same leave5 under greening light of 2 W,m "~ after 3 h (2) or after a farther 5 rain dark p=riod (3), Spc¢trum I has l~¢n normalized ~vith spectrum 3, (C) Time-couri¢ of the increase oi" ,dA(,'48 with l~rc=nin8 at 2 W,m':,



Volume 309,number 1

and in the same leaves after 5 h of greening under low light intensity (2) or after a further 5 rain re-darkening (3). They all show a positive part, around 648 nm corresponding to photoactive Pchlide, and a negative part, around (548 nm, corresponding to its photoreduction product, Chlide, The data show that the actually existing photoactiv¢ complex after 5 h ofsreening under low light intensity represents 6% of its amount in etiolated leave. A 5 rain re.darkening of the leaves raised this level to 15%. Under high light intensity the photoactive complex could not be detected using this technique due to background noise. Changes of AA648 as a function of greening time under low light intensity are represented in Fig. 4C. The absorbance of photoactivc Pchlide was clearly detected only after 2 h, and its amplitude then increased gradually in the course of greening. In order to explain the rapid Chl accumulation at relatively low levels of enzyme [1-4], we hypothesized that the turn-over rate of the photoactive complex is much faster in greening plants than in ¢tiolated ones. To verify this we followed the kinetics of the transtbrmation of Chlide 684 to Chlide 672 (Shibata shift, [10]). which reflects the liberation of Chlide from the enzyme and its¢sterifieationto Chl a [I 1,12].Original traces of flash-inducedabsorbance variationsat 665 and 695 n m during this shiR are reproduced in Fig, 5 for leaves illuminated for 5 h at medium intensity. The measured half-time of the process (8 s) proved to be several orders of magnitude shorter than the corresponding half-time observed in 6-day.old etiolated barley leaves after a short flash(6 rain,[12],and data not shown). 4. D I S C U S S i O N U p to now the photoactivc Pchlid= complex has b ~ n detected in green or greening plants only after transfer-


I O'~ll


- . . . . . . . . . . . . . . . . .


30 Tim0

after flaSl~

60 (=)

Fi/l.S. Kineticsof theflash-induce..d absorbanc~variationsat66fland 695 nm after3 h of gr~ningat 20 W.m "=followedby a 1 raindark period, 76

August 1992

ring tl;em to darkness [9013-16].The resultspresented in thispaper demonstrate that thiscomplex existsin the light.This finding strongly su~,~.Se.sts that Chl accumulation proceeds through continuous regeneration and phototransformation of a Pchlidc--cnzyme-NADPH complex with a red absorbanc¢ band at 648 nm, and is in agreement with a previous study showing that the action spectrum of Chl accumulation during greening has a red maximum around 650 n m it./],According to our measurements, the complex has slightlydifferent emission and excitationmaxima in stoning leaves than in edolamd ones (653 and 452 n m insteadof 65"]and 45"/ nm). Such a shiR was also observed in re-darkened green leaves [9],and its nature is not well undt:rstood. "Pne Chlide which rc,sulmfrom Pchlid¢ photoreduction during greening has a red absorbancc m a x i m u m at 684 nm, wl~ich is found also upon Pchlid¢ photoreduction in etiolatedleaves [6,8,10]. The amount of the complex depends on stoning time and on lightintensity.Shortly (= l5 rain)aRer the onset of illuminationthe complex could not be detected, but aRtr l h it, fluorcsc.cnc¢at 653 n m could be easily measured. The intensityof this fluorescence increased then up to about 3 h and then sharply declined.O n the other hand detection of the complex by measuring its absorbance was possible only after 2 h. This apparent discrepency can ~ explained by differentsensitivitiesof both methods. Unlike im fluorcw,cnc¢ intensity,the absorbance of the compl~ exhibitsa continuous increase approaching a plateau around S h of illumination.The different bchaviour of fluorescence intensity and absorbanc~ during the period 3-5 h after the onset of illumination is most probably related to an increase of energy transfer efficiency from Pchlide to Chl, as it exists in re-darkened sr~n leaves [14].A n increase of the amount of photoactiv¢ complex during the first hours of greening is consistent with the accelerationof Chl synthesis and with the increase of the amount of Pchlide observed in this period [18]. The actual amount of the photoactive complex depends on light intensity. This is clearly evidenced by our fluorcsctnc~ data (Fig. 3B) which show an inverse proportionality with lightintensityin an intensityrange at which Chl accumulation stilloccurs at the same rate (Fig. 4A). This observation can be explained by the linear dependence of the rate constant of Pchlide photoreduction on light intensity[19], ifone assumes that Pchlide and NADPH availability is not limited, In other words, the turn-over of the complex increases with light intensity,which ensures efficientsynthesisof Chl at low steady-state levels of the photoacfiv= complex, Such rapid turn-over would not be possibleifthe releasetime of Chlide was long, as observed for the classicalShibata shiftin e'.iolatedi~aves[I0,12].A shortening of thisshift would be a necessary condition for the observed rate of Chl synthesis. Our kinetics measurements indeed revealed a considerable -ccclerationof the process.

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In conclusion, the presented results clearly indicate that Pchlide photoreduction in 8reening leaves is operated by the enzyme Pchlide reductase as it is in etiolated leave, but that the turn-over of the photoactive compl~ is Srcatly accelerated, Ackno.'led&ements: The authors thank the Ministry of National IEducation of Bclllium for financial support. Thb work was partly fi. nanccd by a iirant from the Belllian National Fund of Scientific P,esearch to F,F,. who is a _rc~__.rchassociate at this institution, The authors thank Dr. ikla Beddi (University of Budapest) for helpful disc,uions.

REFERENCES ill Santcl, H.3. and Ar,cl, K. (1981) Ear. J. Biochem. 120, %-!03. [2] Forfeiter, C., van Cleve, B., Schmidt, A. and Apcl, K. (1990) Pinata 183. 126--132. [3] Griffiths, W.T., Kay, S.A. and Oliver, R.P. (1985) Plant Mol. Biol. 4, 13-22, [4] Kay. S.A, and Grifliths. W,T, (1983) Plant Physiol, 72, 229-236, [$] Oriffiths, W.T. (1978) Biochem. J. 174, 6t11-~92.

Au~st 1992

[6] Sironvul, C. (Igfti) in: Photo~nthcsls. V. Chloroplast D~vclopmcnt (Akoyunollou, G, ed,) pp, 3-14, Balaban Inccmutior~l Scien~ Service. Philadelphia. [7] i~]tddi, B.. Ryberl. M, and Sundo.vi,~t,C, (if,-~i) ,L Pllotochgm. Photobiol. D: Biol. 12, 389..403. [8] Michel, J,M, and Sironval. C, (1977) Plant Cell Physiol. 18,12231234. [91 I.,cbedev,N.N,.Siffel, P. and Krasnovskii, A,A. (1985) Photownthetlca 19, 183-187. [10] $hibata, K. (1957) 3, Biochcm. 44, 143-173, [1 !] Oliver, R,P. and Grtmths, W.T. (1982) Plant PhysioL 70, 1019-1025. [12] Hennilptcn, K.W. and Thorns, S.W. (1974) Phyrdol. Plant. 30, 82-89. [13] Cohen, C.E. and Rebciz, C.A. (1981) Plant Ph~iol, 67, 98-103. [14] Frndkin. L.I., Domznskuyu, i.N. and ~hlyk, A.A. Ci g82) Doklad. Biophys. 261,189-192. [IS] Virtin, H,[, (1984) Physiol. Plant, 61,303-307. [16] Minkov, [.N,, Rybcril, M, and Sundqvist, C. (lg~) Physlol. Plant, 72, 725-732. [17j Virgin. H.[. (1986) Physiol. Plant. 66, 277-282. [18] Shlyk, A.A., Chalks, M.T., Fradkin, L.[., R.udoi, A.B., Avcdna, N.G. and 5nvchenko, G.E. 0986) Photobiochcm. Photobiophys. 12, $7-96, [19] Simn~--,I. C., Broueru, M., Mich~l, J.-M. and Kuypcr, Y. (Igf;8) Photosynthctica 2, 268-21t7.


Detection of the photoactive protochlorophyllide-protein complex in the light during the greening of barley.

A photoactive protochlorophyllide-protein complex with absorbance and fluorescence maxima at 648 and 653 nm was detected in greening barley leaves wit...
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