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Localisation of chlorophyll within the chloroplast
OROPHYLL WITHIN THE (
LAST
by S*, L. C. POST AND N. V E R T R E G T ;trecht/Delft under the Direction of A. J. ~¢. W. Milatz, Utrecht (Netherlands)
ft,
INTRODUCTION
For a detailed study of the way of functioning of the th~ photosy aratus it cessary to penetrate into the micro-localisation of the phot( pigments. resent it seems most likely that, in chloroplasts containing con g Lded in a la substance, the chlorophylls are concentrated in the 1 grana eption is t on light microscopical examination of various chloroplasl c] observed .red grana surrounded by a colourless stroma within withil chlorop ny plant rGGER4 chloro I es. According to HEITZ ~, METZNER 3, and STRUGGE~ scence is cted to the grana. However, in many cases the dimensions of the grana are belo~ oelow tight microscopic ~ical statements concerning the Ooccu ltion. This fact may account for contradictory state ccurrence of grana in chloroplasts, and, consequently, with :regard to the distribution of the photosynthetic refer the reader to the followii hotosynthetic pigments. As to this, it may suffice to re owing pertaininl FREY-WYssLING 7, GRANICKs, a n d WEI~ i n i n g r e v i e w s : W E I E R 5, RABINOWITCH6, FREY-WYSS] rEIER AND STOCKING9.
ma in various chloroplasts that th~ ['he electron microscope revealed the presence of gran The looked d homogeneous under the light microscope. In fact, these observations render¢~d grana. Even in chlor oit highly ;hly probable that all chlorophyll-bearing plastids contain co various algae, grana were we~ plast-free -free organisms, such as photosynthesizing bacteria and a f o u n d - - C A L V I N AND LYNCH 1°O, THOMASll. -sections However, WOLKEN AND PALADE 12 published electron micrographs of cross-sectiol
mstrate [ng plastids in two flagellates, which clearly demonstra through chlorophyll-containin 1re free from the "usual" grana. STEINMANN18 showed in i a regularly lamellate structure the same way that this also holds for plastids of Spirogyra and Mougeotia. micr M o r e o v e r , STEINMANN14 and FREv-WYssLING AND STEINMANN15 published a mlcrosho~ graph of a cross-section throuugh a tulip chloroplast in which lamellate grana are shown to be suspended in the stroma. ;ture ltain grana, while other ones do not. However, the structm So, some chloroplasts contain ral'lum [1-bearing ~rin~ plastids strikingly resembles that of a granm of the grana-free chlorophyll-bearir :ther itself. If one compares them e.~.g. with the bacterial and algal grana, one may ask wheth~ loting the latter as grana-free chloroplasts too, in spite of there is any objection to denotin f * This investigation has been made possible by a grant from the Netherlands Org~.nisation for Pure Research (Z.W.O.). Re]erences p. 3 o.
:ase it m a y be preferable to define 111 ~ J. %.~11 in grana; the grana m a y occur ei tually or w, one can state that, in all cases till now, ms to be restricted to lamellate st investigation to render support t¢ ption b y llae are the bearers of chlorophyll. : purpose r an adequate tool, it was necess )duce an r, which could be observed under n microaction seemed most suitable. This nsists of )roplasts in the presence of silvel e to the silver. Vlany investigations concerning this reduction have been cam ese have MOLISCIt reviewed by WEBER 1~ and METZNERls. However, the th, mechan tion was ion has not yet been fully elucidated. Originally the silver n zed to occur in living cells only. METZNERls, however, howeve demor t the rea took place in killed cells as well. The MOLISCI~react he dark; eaction also r FI of the however, increases its rate considerably. METZNER showed fiver; at tckened by th~ am determines which cell constituents are blackem reaction, ~articular-4 the c h l o r o p l a s t s - - t h e i r surface and the grana in p~ range in at p H 7-8 the precipitate is mainly found in the cytoplasm. c) 1 the chloroplasts react this author examined the wavelengtt w :y of the SCH reaction. This was done by counting the silver precipitate pl s and by tically evaluating the figures obtained. The action s;pectrum I in living ~hyll )hyll absorpt absorption spectrum. shows some features in common with the chlorophyl m. Ho~ Howthat obtained from killed cells hardly admits any conclusion. cc In order to examine whether chlorophyll occurs in isolated isol~ tecessary In lamellae it was necessa: w~ necessary to study wheth whether to use grana suspensions instead of tissues. Therefore, it was [OLISCH reaction can be established in such a suspensl the M( ent )ension. Consequently the presel study deals with: re( IE. . demonstration of a light-induced silver nitrate reduction in grana suspensior )enslons, 2. ,'. demonstration of the dependency of the MOLISCH reaction sensitization on the t] presence nce of. excited chlorophyll molecules by establishin~g an action spectrum of this t~ reaction, llorophyll. 3. localisation of the chl, MATERIAL AND METHODS
Preparation. G r a n a suspensions Lsions were p r e p a r e d from fresh leaves of Hibiscus rosa sinews szne~sis. U s u a l l y a b o u t 4 g of w a s h e d leaves yes were g r o u n d in a W a r i n g blendor in 75 ml of a pre-cooled o.2 M glucose or, m o s t l y , sucrose solution ~n in glass-distilled w a t e r for 2 minutes. T h e brei w a s filtered t h r o utgh c o t t o n wool a n d centrifuged at I2oo g for 5 minutes. The s u p e r n a t a n t w a s diluted w i t h an eqtual v o l u m e of a o. 4 M s o d i u m a c e t a t :e e buffer in o.2 M sucrose. The p H values will be mentioned togetl2 ether w i t h the e x p e r i m e n t s . This suspenmon ension will be referred to as " g r a n a s u s p e n s i o n " . L a m e l l a e suspensions w e r e p r e p a r e d b y weakly disintegrating the g r a n a suspensions w i t h tthe aid of a m a g n e t o s t r i c t i o n oscillator. Ltor. The energy delivered to the m a g n e t o s t r i c t i v e t u b e w a s abo ~out 3o0 w a t t s a t a frequency of 7ooo )o Hz. A p p r o x i m a t e l y i o % of the energy w a s effective. T h e gra rana were t r e a t e d for i m i n u t e u n d e r: nitrogen. D u r i n g operation t h e v i b r a t i o n cup w a s cooled w i t h ttap w a t e r . O u r t h a n k s are due t o D r J. A. NII~MEIJER, w h o kindly placed t h e oscillator of the L aabboorraattoo r y for Physiological Chemistry, U t r •eecht, c h t , a t o u r disposal. T h r o u g h o u t the p r e p a r a t i o nx the suspensions were k e p t a t low t e m p e r a t u r e as m u c h as possib ~ible. Occasionally s h o r t - l a s t i n g storagq;e occurred in a refrigerator. Care w a s t a k e n to keep t h e suspensic mnslons Relerences p. 30.
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formed in weak green light. Before st~ for 5 m i n u t e s at least. The experime
ormed at
:ISCH reaction. Samples for illuminatioi )1 aliquots were k e p t in the d a r k unde
from the qual con-
n o u n t of metallic silver w i t h t h e aid c stop endAYE1~, were unsuccessful, t h e y m a y be )ned here )LlSCH reaction. After addition of silv utions of the g r a n a suspension w a s illuminated. atrifuged. as well as of the sediment, w a s h e d w vater and nined b y titration. I n b o t h illuminated ;pensions, a m o u n t s of silver were found to occur Lent. This , seems to indicate t h a t p a r t of the si] strongly n o t noticeably r e m o v e d b y t w o w a s ver, since ration in q u e s t i o n is r a t h e r sensitive this p h e n o m e n o n mayy suggest t llic silver • is produced b y reduction of p r i m a r i l y adsorbed silver nitrate. nitral reacti 'or t h e q u a n t i t a t i v e d e t e r m i n a t i o n of t h e effect of the MOLISCH Morn ~sed light ~tallic silver, fission of t h e suspension, owing to blackening b y metallic silve w a s mea Letrically. of the g r a n a suspension were p i p e t t e d into a plane-parallel 5.5 5 m m glm, e cuvette aunted in the p h o t o m e t e r and a v i b r a t i n g stirrer was a d j u s t e d in order to aentation in gl suspended particles. T h e n I ml of a lO% silver n i t r a t e solution soh ?ater w a s green carefully. Initially the light transmission, determined in weak w~ ed owing tions w i t h sot f o r m a t i o n of nearly white insoluble silver salts b y reaction hering to ,~anic m a t t e r or diffusing from it. After a few m i n u t e s t h e ligh t transml a steady illuminated and This value w a s called lOO%. N e x t t h e c u v e t t e was illumir nsmission as determined. E x c e p t for the illumination period, a sample, serving , s treated )roved to incr s a m e way. W i t h time the light a b s o r p t i o n of the controls pro" 11a m o u n t Lhe highest value o b t a i n e d w a s 13 % for 4 hours. green : he p h o t o m e t r i c e q u i p m e n t allowed t h e p r o j e c t i o n of a parallel para i the susn c u v e t t e on to a selenium b a r r i e r layer cell connected to a Zernike ]2 mometer. f ioo w incandescent l a m p w a s used as a light source. A g lass filter of S c h o t t x e c u t e d as q u i c k l y as possible. Check Chec and VG 9 w a s placed in front of it. The readings were eexecu ments p o i n t e d o u t t h a t , in this way, no reaction due to t h i s procedure resulted. ~ium i l l u m i n a t e d either b y sodim llumination equipment. The c o n t i n u o u s l y stirred suspensions were v Illumination )oo w Philips E 4 ° p r o j e c t i o n lainP .r b y light of various w a v e l e n g t h s obtained from a 3 ° V 9oc light or i d of c o m b i n a t i o n s of a Christiansen-Weichert dispersion m o n o c h r o m a t o r and S c h 0 t t gla~ lass w i t h t hhee aaid filters. M e a s u r e m e n t s of incident light intensity were carried o u t w i t h a s t a n d a r d i z e d thermopile. o t h grana and lamellae were made mad 7.lectron microscopy. Electron microscopic p r e p a r a t i o n s of bbet Elect~ dmitted Aously 2° described. M o u n t i n g a n d w a s h i n g occurred in weak glreen light. F u l l light w a s aadmitte as previously re eexamine, x a m i n e d in a P h i l i p s electron microscop [ter t h o r o u g h l y drying of the p r e p a r a t i o n s . T h e y were o n l y after )e,
RESULTS
Demonstration o/light-inducedi silver nitrate reduction m grana suspensions mple out of three experiments Fig. I shows an evident evider Time [actor. As an exam miincrease of absorption, owing t ° the formation of metallic silver, with time at an illum time. Lsion. Apparently the rate of increase declines with tim nated Hibiscus grana suspension. silver This phenomenon m a y be duee to three causes. First, the amount of black metallic silv( both ithin the suspension an increasing part of the light is bet increases with time. Thus, within ased. nsequently, the intensity of the effective light is decrease( screened of and absorbed. Cons( Fitration ntity of silver nitrate m a y become exhausted. Titratio Secondly, the available quantity dq experiments showed that thee amount of dissolved silver nitrate is not noticeably de:ion ted above, if merely adsorbed salt is reduced, exhaustie creased. However, as suggested ay .icture. Thirdly, the activity of the grana suspension m ma phenomena m a y enter the picture. arious decrease. This was checked bby drawing samples from the same suspension at vvarlet ncrease intervals. These samples were'e kept at room temperature and their absorption Iincrea,' nly On] per unit of t i m e - - m o s t l y 3 o m i n u t e s - - w a s measured at equal light intensity. O Re]erences p. 3o.
tction was observed. As a mean ot per hour. The question remains, light. However, because of the c
.~o
I
~,o
activity hange of
C .o
I
30
I
I
I
I
60
90
t20
150
• Time course of silver n i t r a t e reduction, m e a s u r e d as a b s o r t ~tion incre suspension in s o d i u m light. I n c i d e n t intensity: 4.4" lO4 ee~r ~ s c m - 2 s{
~cUS g r a n a
condition within the suspension, owing to the formation foi ver, this em can hardly be solved. ally, the )roceeds al )ne m a y ask whether the reaction, once started, pr ~ry silver precipitates functioning as condensation nuclei. i Tt I ms quesuon m a y be t ~red by interrupting the exposure to light of a reac ting suspension. Such an ee:x eriments periment rant is shown in Fig. 2. Evidently no after-effect occurs• occu: Moreover two experimen sc were carried out, in which the absorption increase of a suspension, pre-iUuminated for f~
co
g
1o
20
35
,b
~o
65
75
minutes
Fig. 2. Effect of darkening, indicated ieated b y the arrows, on the silver nitrate reduction, measured a s a b s o r p t i o n increase, of a Hibiscus us grana suspension. Incident sodium light intensity: 4.4" IO4 ergs c m -2 sec -x. p H : 6.6.
5 minutes, was compared with th that of a control sample. The experiments lasted 75 and ar 23o minutes, respectively. The le corresponding absorption increase values of the controls contro were 2 and 13%; those of both ~th pre-illuminated suspensions amounted to 9 %. Thus, in i these prolonged experiments no after-effect of illumination was observed. p H sensitivity o/the MOLl: .ISCH reaction. According to METZNER TM, chloroplasts withi within References p. 30.
m a l l y a t p H 3 - 4 of t h e s u r r o u n d i is are m a r k e d l y different, and, wi emonstrates that the pH depend k a l i n e p H r a n g e s p o n t a n e o u s blacl
~Lx ~ o 4 o
le o p t i m a l n g e d cont to occur,
?.
TABLE i F pkI
ON
THE
MOLISCH
REACTION
RAl
m light intensity 4.4" 1°4 ergs cm a sec )uration of lure*nation
A bsorption increase in per cen of control at p H
in minutes
75 23o
I 2
4.6
5.6
6.~
3 23
II 25
xc 44
; intensity a n d in L i g h t /actor. T h e r e l a t i o n b e t w e e n MOLISCH r e a cction tic ties a b o u t , r e s e n t e d in Fig. 3. E v i d e n t l y n o l i g h t s a t u r a t i o n is i reache( it o c c u r s 4 t i m e s h i g h e r ttlh a n t h o s aspensions in p h o t o s y n,nthesis t h e s i , of gree] d doubtful e n s i t y . So of c o m p a r a b l e dde1 ~V[oLISCH mee's are i n v w h e t h e r e n z y 'm s reaction r e a c t i o n . METZNER METZN~ Is s t a t r ent p r o c e e d s in cells t h a t w e r e k i l l e d b y p r e t r e aattrm w i t h s i l v e r n i t r a it e . H o w e v e r , t h o u g h it ssee e m s s o m e e n z y m e s m i g h t be l e f t h i g h l y i m p )robable, robabl h i s r e a g e n t . F o r t h i s r e a s o n ss(o m e u n a f f e c t e d b y tthi: I i T i ,,nsions w e r e p e r f o r m e d w i t h suspens! 6 e ~ , , e x p e r i m e n t s we~ 2 4 d In e P g s c r d ~ .10 " k i l l e d " b y h e a lt i n g t h e m in a b o i l i n g w a t e r Fig.. 3. Relation between light intensity b a t h . A f t e r ccoolJ )erature o o l i n g d o w n to r o o m tempera1 and rate of silver nitrate reduction, n i t r a t e w a s a d d e d . T h e r e s u l t s are ~sured as absorption increase per 25 the silver nitrat( measured rotes of a Hibiscus grana suspension in g i v e n in T a b l e I I . minute sodiumlight, pH : 6.6.
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TABLE II MOLISCH
REACTION
OF GRANA
pH:[ : Graua suspension
6. 5.
SUSPENSIONS ILLUMINATION
PRE-HBATED WITH
IN
SODIUM
A BOILING
WATER
A bsorption
A bsorption
Incident light increase of illumi- increase of dark Heatingperiod Illumination in-1ergs nnted suspensions control in % in minv2es period in minutes intensity cm-2 sec in %
a a
IO
b
15
b
20
b*
60
25
8.12" lO 4
34
25 3° 3° 3°
8.12" lO4 9.35" lO3 9.35" IOn 9.35" lO3
36 19 20 II
3 8 8
• At the end of the heatingg period thiss suspensionshoweda-brownish-green-colourReferences 19. 30.
BATH.
LIGHT
e " a " series show that the MoL lg period. In the " b " series, at low samples heated for 15 and 20 mir ence of any enzymic character of 3ur the rate of this reaction is dec resh green to brownish-green. Th the chlorophyll results in a reduc that excited chlorophyll itself is t
Q11
L~
IIUL
intensity, vn. These reaction. colour of non m a y MOLISCH
agent in
:hlorophyll anism of the MOLISCH reaction. The idea De responslDle ior rne r e a u c u o n of oI the rne silver Sliver n i t r a t e w a s already air( RET~1. This teste¢ )r p r e p a r e d active alcoholic chlorophyll e x t r a c t s from spina( ~r w a s h i n g )inach leaves. he chlorophyll w i t h petrol ether the alcoholic p h a s e prov s activity. )roved to hay 'HERET concluded t h a t the reducing p o w e r responsible for the MOLLS instead of due to the chlorophyll, depends on the presence of a substance s u b s t a n soluble t insoluble trol ether. I n t h i s connection a few additional e x p e r i m e n t s will be bri However, ,~h t h e y m a y yield some suggestive information, t h e y are nnot Lt study. o t essential A colloidal solution of c h r o m a t o g r a p h i c a l l y p u r e chloroph] toz, Basel, phyll, o b t a i n )repared b y dissolving I m g of this p i g m e n t in 2 ml acetone. U n d e r ra l ds solution tdded to 20 ml of a o.I M sucrose containing o.i M buffer solution o this w a y a h e r u n s t a b l e - - h y d r o p h o b i c sol w a s obtained. T h e n I ml of a lO% sil lution was t. T h e light a b s o r p t i o n of t h e m i x t u r e was determined. N Ne~ Lt h e dark, ext a sampl sec - i - for t an aliquot w a s e x p o s e d to s o d i u m l i g h t - - i n c i d e n t iintensi n t e n s i t y : 3.26- I inutes. After this period t h e light a b s o r p t i o n was determin ed a g a i n . ' m increase n t r o l a n d illuminated suspension a m o u n t e d to i and 13% respectiv ,,xperiment ires t h a t t h e MOLISCH reaction directly depends on the reducing redu hlorophyll. powe: t t t e m p t s t o reproduce this result, however, were unsuccessful ; the colloi, II solutions ce showed a flocculation a t the addition of the silver Initrate. nitr~ These ll,taLC. ilig~C l i U S t , tllcttCta ~ uuspensions ~gllbl~ ot s h o w a n y light reaction. This difference in b e h a v i o u r is ni o t clear since p r e p a r a t i o n as well p e r i m e n t a l conditions did n o t noticeably deviate from those thos in the first experiment. I t m a y be a d v a n t a g e o u s to stabilise the chlorophyll sol w i t h the aid of a 1c)rotective colloid. Since, as alrea already r e m a rrked, k e d this s t u d y does n o t aim at an investigation of the m mee c h a n i s m of the MOLISCH reactfl reaction, t h e s e e x p e r i m e n t s were n o t extended a n y further. Fin Finally, a single e x p e r i m e n t w i t h a chlorophyll-free alga, Prototheca P: menZopfii, m a y also be m, tioned. ~. After addition of silver n i t r a t e up to a final concentration ~tion of 1% an algal suspension of :2 g fresh w e i g h t in IOO ml acetate buffer of p H 6. 5 w a s illuminated w i t h s o d i u m light of 9.35" IO3 eergs c m - 2 sec -1 for 30 m i n u t e s . A f t e r this period the a b s o r p t i o n w a s increased i: corre= b y 6 %, whilst the cot s p o n d iing n g value at a d a r k control sample a m o u n t e d t o 4 %- So these t figures m a y suggest t h a t lthe MOLISCH ;cH reaction does n o t proceed in t h e absence of chlorophyll. TFhe h e results resul m e n t i o n e d are c o n t r a r y to those of GAUTHERET.. This T] divergency can be explained b y assumm dng t h a t GAUTHERET m a y h a v e obtained " i n a c t i v e " chlo) chlorophyll suspensions, j u s t like 1the "inactive" chlorophyllpreparations ions described above. The a c t i v i t y of GAUTHERET'S chlorophyll-f~ rll-free alcoholic leaf extract may be due ~e to some reducing s u b s t a n c e soluble in ethanol. I n this respect Dect it may be mentioned that, in literature rature, ascorbic acid is often considered to be responsible /or the reducing power needed in the MOLl IOLISCH r e a c t i o n . METZNER ~2 r e m a r k s t h a t , a t the present, it can~ cannot yet be excluded that the MOLISCH SCH reaction in illuminated chloroplasts m a y proceed w i t h o u t t h e intermediary of ascorbic acid. This ais s t a t e m e n t seems to be confirmed b y the above results. H o w e v e r , it may be remarked explicitly that m t for f u r t h e r confirmation additional e x p e r i m e n t s are needed.
Summarising the main results results it can be stated that grana suspensions are able to show the MOLISCH reaction. Light is an essential factor. The reaction is pH-sensitiv ;ensitive. I t is non-enzymic.
Action spectrum o/ the MOLISC} SCH reaction. Direct proof of the dependency of tt HOLISCH this reaction
in grana
determining wavelengths
s u s p e n s i o nns s
on the presence of chlorophyll m a y be obtained tb y was done b y measuring the reaction rate at vario~ various it in per cents of this rate at sodium light. The resultii ling Fig. 4. Since the selectivity of the Christiansen-Weiche Weichert
a n a c t i o n s p e c t r uam. m. T hi This and computing
.action spectrum
References p. 3o.
is s h o w n i n
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region of the spectrum is relati ~tion with Schott glass filters as in he legends. ,trum is drawn in three parts. Es t ;5° m/~ the he relative ~xpected to occur since, when de RG filters, efficiency wit some highly ~/x light is of the very / ~ also transmitt avelengths low light int~ lts in this below 49 ° m spectral regioi inaccurate. epresented Nevertheless "ly demontoo, the dive1 g. strating their strat eniences it Despite t e MOLISCH may be evid( m closely reaction acti L spectrum rAJ ~mbles the reset For the of a grana r spectrum ~,stablishing c ' ~ esta~ Miss L. J. o our thanks a es that the BARTELS. The BAR: ds on the M O L I S C H rea( MOLl It may be )resence of el rrema emarKea i n a c K t K l o n t n aat ~ the ~ n e acremarked in pres~ curacy , cura~ of the determinations does tsions not allow to draw any conclus foJ concerning a possible carotenoid activity in this reaction. ~ a l o
~
'
~
'
' '
~t'm~ ~-a~.. 1~.__
a ~ l u i , v
o
faction MOLISCH rea~ ° -: --+~2 Since it is shown that, under unde~ the • -- ÷~5 ....... ~e chosen chos, experimental conditions, silreduction is coupled'with ver nitrate i ~o ~o ~o ~o ~o the presence of excited chlorop])hyll, lsed as MOLISCH as well the MOLISCHreaction may be use I S C H rreaction eac Fig. 4. A c t i o n s p e c t r u m of t h e MOLISCt as a b s o r p t i o n s p e c t r u m established , l i s h e d a t Hibiscus grana a toolin localising these pigment:moll suspensions. nlcroscope. ecules under the electron microsc The precipitated metallic silver causes a strong electron scattering; in this way it is s! easily recognised. Since it is necessary to remove buffer salts as well as excessive silver nitrate from preparations of illuminated suspensions, washing is required. Though this not be ed water was carried out very cautiously, it could no washing with glass-distilled prevented that part of the,~silver deposits were ruptured. These broken fragments,were ~m the eighbourhood of silver covered chloroplasts. Apart from always observed in the nei [ectron scattering layers were absent in non-illuminated SUSfact that these strongly elec it they consisted of silver by establishing electron diffrac:tion pensions it was proved thai :kened" diagrams. Fig. 5 shows thazt the pattern of silver occurs in the diagrams of "blackel grana.
/
Re/erences p. 30.
•o c n . .~ ., ~. -.m . ~+ l e,m, 6 % C u $ 0 4 •
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.
+~2* ~20
,alisation o / t h e Loca
b
C
i
I
• 5. E l e c t r o n d i f f r a c t i o n d i a g r a m s o f at : s i l v e r - c o a t e d g r a n u m , b : c o n t r o l g r a n u m , c : b l a n k c o l l o d i o n film, d : s i l v e r :ipitate. F i g . 6. Hibiscusgranum. F i g . 7. S i l v e r d e p o s i t s o n Hibiscus c h l o r o p l a s t . A d v a n c e d 1V[oLISCH 1r e a c t i o n . • 8. I n i t i a l s t a g e o f MOLISCH r e a c t i o n a t a Hibiscus g r a n u m . F i g . 9. A d v a n c e d MOLISCH r e a c t i o n a t a Hibiscus num. F i g . i o . S i l v e r p r o t r u s i o n s f!rtoom m h e a v i l y c o a t e d Hibiscus g r a n a . P a r t l y s h a d o w e d . F i g . I I. S i l v e r p r o t r u s si oi onns s fro~ f r o m Hibiscus g r a n a a n d l a m e l l a e .
() shows an u n t r e a t e d g r a n u m . ~ted with silver as a result of th~ p a r t l y indicates contours of grar qnired to i n t e r r u p t the silver nitJ i l l u m i n a t i n g the suspensions at with a g r o u n d glass plate adjust(
reaction. to draw an at all f z 5 cm ~f it, for
of the reaction at a g r a n u m . As is deposited in a homogeneous 1~
rated by :anum is
m
Fig. 12. Supersonically disintegrated Hibiscus m. Heavily silver-coated lamellae, arranged in granum. a fan-like way. 3. initial stage of the MOLISCH reaction at a Fig. '3. single Hibiscus lamella. ~action at a single Fig. 14. Advanced MOLlSCH reaction Hibiscus lamella. increa d a m a g e d ; lipids are p r o t r u d iing n g in m y e l i n figures. Since the l a t t e r do not show increase ~ems probable t h a t silver deposits are restricted to the tl of electron scattering, it seems proteinaceous p a r t s of the g r a n u m . ~OUS ion is represented in Fig. 9. A p a r t from the homogeneo~ A more a d v a n c e d reaction lsions are shown. I n Fig. IO such-like structures are more mo coating, typical silver protrusions ,oundary pronounced. I t m a y be n o t e d that, in this figure, the g r a n a are s i t u a t e d at a bounda~ b e t w e e n a shadowed a n d a n o n - s h a d o w e d region. I n this way it is possible to get a n on scattering power of the silver coating of a single g r a n u m impression of the high electron as well as of its t h r e e - d i m e n ssional i o n a l shape. )osited cription m a y suggest that, at first, the silver is deposit( Possibly the above descri initial he protrusions arise. This, however, is not true. At initi homogeneously and, next, the Re/erences p. 30.
ther homogeneous blackening or 1 influencing the formation of on M~-TZNERlS--noticed the irregular ced reactions protrusions were alv reatment of grana, lamellae suspei reagent. Fig. I I represents a pre from the shadow length, and apar shown. Apparently the latter are silver nitrate reduction it is often r, identification remains possible ~k supersonic treatment often cau rate into a fan-like cluster of lamellae. Such a cluster, cluste heavil~ Fi~ Iwn here. The contours of the lamellae are slightly visible. v: nstrate the occurrence of silver deposits on single lamellae. lal :n conclusion it m a y be stated that lamellae are able abIc to bring ion.
ox~11o
¢ x x K;
ypes de: precipi~d.
prepared such an e thickly red with :ecognise E'his m a y .n to disth silver, 4 clearly
MOLISCH
DISCUSSION
and size in a preceding paper 23 a correlation between activityr in the H lr. These m a fragments, prepared by supersonic disintegratiol 'ration, was sh :ed from iments suggested t h a t the photochemically active active fragmel in these lae. Consequently, it seemed most likely t h a t chloro]phyll is c tures. Fhe present investigation confirms this deduction in a imore direct way. The MOLISC )LISCH reaction ction ion is shown to depend on the presence of excited chlorophyll. c~ Since this reactk is demon, nonstrated to occur in lamellae, it seems justified to t conclude that lamellae are a the bearers of chlorophyll. A few remarks m a y be made concerning the MOLISCI-I MoLISq reaction as it is obserw observed in the above experiments. M E T Z N E R ls stated that silver nitrate nit~ flasts reduction by chloroplas i n "iE~tact" cells occurred within 35 seconds in monochr~ ~chromatic light, the intensity of whichI amounted to, probably, about IOO times that used in the present investigatio ~tlon. The consider~ onsiderable divergence in both exposure time and ligh ht intensity m a y be understoc~od m l r n i n g fthat, h ~ t nowing w i n ~ fto n the ~'h~ n r e c : P n e e of a{ reducing r ~ r t l w q n ¢ s¢nh¢¢ b y assuming .cid Lhe presence u b s t a n c e s - - s u c h as ascorbic ac - - w i t h i n the cell, this re~ctioi :ion with chloroplasts in situ proceeds more rapidly than th~ t h a t with isolated grana. In fact !act, a few additional experiments showed that the MOLIS( ~LISCH reaction in grana suspensionss could be considerably increased by adding ascorbic aci cid. This seems to indicate that excited c chh xcitedd chlorophyll m a y act either as a catalyst of a reaction reactic between silver nitrate and a reducing agent, or as the reducing substance itself. Since, Sinc however, the present investiigation does not aim at an elucidation of the mechanis '~chanism of the MOLISCH reaction, the ,, validity of this suggestion was examined no further. SUMMARY Silver n i t r a t e r e d u c t i o n w a s3 s h o w n to o c c u r in i l l u m i n a t e d s u s p e n s i o n s of Hibiscus g r a n a . T h e a c t i o n s p e c t r u m of t h i s r e d u c t i o n , t h e MOLISCH reaction, p r o v e d to coincide satisfactori torily w i t h t h e c h l o r o p h y l l a b s o r p t i o n s rp e c t r u m . E l e c t r o n m i c r o g r a p h s r e v e a l[ t h a t t h i s r e a c t i o n occurs in single lameUae. F r o m t h i s it is conclud concluded t h a t lamellae are t h e b e a r e r s of c~hlorophylL hloro
Re/erences p. 30.
R]~SUM~; n s i o n s de g r a n a illumin~es de Hibiscus
trgent eat
tion, la r~action de MOLISCH, r e s s e m b l e
bsorption
~ntre, q u e des lamelles isol~es s o n t ca t e q u e les lamelles c o n t i e n n e n t la chlorc
m e r cette
ZUSAMMENFASSUNG te S u s p e n s i o n e n y o n Hibiscus-granen d u k t i o n ,• die MOLISCH R e a k t i o n , ist den . . . . o -r dorophylls iihnlieh. ~ e o b a e h t u n g e n m i t t e l s des E l e k t r o n e n m i k r o s k o p s zeigten, d a s s diese a s s die L a m len v e r a n l a s s t w e r d e n k a n n . E s wird h i e r a u s geschlossen, dda: sind.
ad Silberspektrum isolierten es Chloro-
REFERENCES {EITZ, Planta, 18 (i932) 616• tEITZ, Planta, 26 (1936) 134. {ETZNER, Ber. deutsch, bot. Ges., 55 (1937) 16. TRUGGER, Ber. deutsch, bot. Ges., 64 (1951) 69. ATEIER, Bot. Rev., 4 (1938) 497i. RABINOWlTCH,
Photosynthesis and related Processes. I. I n t e r s c i e n c e
{ew Y o r k
5).
vier Publ. its Der :REY-WYSSLING, Submicroscopic Morphology o[ Protoplasm and a A m s t e r d a m , N e w Y o r k (1948). A m e s (1949) 113. ~RANICK, Photosynthesis in Plants, I o w a S t a t e Coll. Press, An ". WEIER AND C. R. STOCKING, Bot. Rev., 18 (1952) 14. l ° M . C~ALVIN AND V. LYNCH, Nature, 169 (1952) 455. n J . B .k THOMAS, Proc. Kon. Ned. Ahad. Wet. Amsterdam, Ser. C, 555 (1952 ) 207. l~j. j.• WOLKEN AND G. E . PALADE, Nature, 17o (1952) 114. 13 E . STEINMANN ;TEINMANN, Exp. Cell Research, 3 (1952) 367 . 14 E . STEINMANN ;TEINMANN, Experientia, 8 (1952) 3oo• ~REY-WYSSLING AND E. STEINMANN, Viertel]ahrsschr. Natur[. Ges. Ziirich, 98 (1953) 2o. is A . FREY-WYss ¢IOLISCH, Sitzungsber. A kad. Wiss. Wien, math.-naturwiss. Kl. K l Abt. I, 127 (1918) 449. 18 H . MOLISCB 17 F. WEBER, Protoplasma, 29 (1938) 427 • 18 H . METZNER FIETZNER, Protoplasma, 41 (1952) 129. 19 p . DELAHAYE )ELAHAYE, Anal. Chim. Acta, 4 ( I 9 5 O) 635. }hys. Acta, 8 (1952) 9o. 20j. B.~. THOMAS, M. BUSTRAAN AND C. H. PARIS, Biochim. Biophy. 21 t . J . GAUTHERET, Rev. gan. Bot., at., 47 (I935) 484 • iss. Gdttingen math.-phys. K1. I I b (1952) i. =2 H. METZNER, Nachr. Akad. Wiss. 23 j . B. THOMAS, O. H . BLAAUW AND L. N. M. DUYSENS, Biochim. Biophys. Acta, Io (1953) 23 o.
Received July 2nd, 19:)53
LAST
by S*, L. C. POST AND N. V E R T R E G T ;trecht/Delft under the Direction of A. J. ~¢. W. Milatz, Utrecht (Netherlands)
ft,
INTRODUCTION
For a detailed study of the way of functioning of the th~ photosy aratus it cessary to penetrate into the micro-localisation of the phot( pigments. resent it seems most likely that, in chloroplasts containing con g Lded in a la substance, the chlorophylls are concentrated in the 1 grana eption is t on light microscopical examination of various chloroplasl c] observed .red grana surrounded by a colourless stroma within withil chlorop ny plant rGGER4 chloro I es. According to HEITZ ~, METZNER 3, and STRUGGE~ scence is cted to the grana. However, in many cases the dimensions of the grana are belo~ oelow tight microscopic ~ical statements concerning the Ooccu ltion. This fact may account for contradictory state ccurrence of grana in chloroplasts, and, consequently, with :regard to the distribution of the photosynthetic refer the reader to the followii hotosynthetic pigments. As to this, it may suffice to re owing pertaininl FREY-WYssLING 7, GRANICKs, a n d WEI~ i n i n g r e v i e w s : W E I E R 5, RABINOWITCH6, FREY-WYSS] rEIER AND STOCKING9.
ma in various chloroplasts that th~ ['he electron microscope revealed the presence of gran The looked d homogeneous under the light microscope. In fact, these observations render¢~d grana. Even in chlor oit highly ;hly probable that all chlorophyll-bearing plastids contain co various algae, grana were we~ plast-free -free organisms, such as photosynthesizing bacteria and a f o u n d - - C A L V I N AND LYNCH 1°O, THOMASll. -sections However, WOLKEN AND PALADE 12 published electron micrographs of cross-sectiol
mstrate [ng plastids in two flagellates, which clearly demonstra through chlorophyll-containin 1re free from the "usual" grana. STEINMANN18 showed in i a regularly lamellate structure the same way that this also holds for plastids of Spirogyra and Mougeotia. micr M o r e o v e r , STEINMANN14 and FREv-WYssLING AND STEINMANN15 published a mlcrosho~ graph of a cross-section throuugh a tulip chloroplast in which lamellate grana are shown to be suspended in the stroma. ;ture ltain grana, while other ones do not. However, the structm So, some chloroplasts contain ral'lum [1-bearing ~rin~ plastids strikingly resembles that of a granm of the grana-free chlorophyll-bearir :ther itself. If one compares them e.~.g. with the bacterial and algal grana, one may ask wheth~ loting the latter as grana-free chloroplasts too, in spite of there is any objection to denotin f * This investigation has been made possible by a grant from the Netherlands Org~.nisation for Pure Research (Z.W.O.). Re]erences p. 3 o.
:ase it m a y be preferable to define 111 ~ J. %.~11 in grana; the grana m a y occur ei tually or w, one can state that, in all cases till now, ms to be restricted to lamellate st investigation to render support t¢ ption b y llae are the bearers of chlorophyll. : purpose r an adequate tool, it was necess )duce an r, which could be observed under n microaction seemed most suitable. This nsists of )roplasts in the presence of silvel e to the silver. Vlany investigations concerning this reduction have been cam ese have MOLISCIt reviewed by WEBER 1~ and METZNERls. However, the th, mechan tion was ion has not yet been fully elucidated. Originally the silver n zed to occur in living cells only. METZNERls, however, howeve demor t the rea took place in killed cells as well. The MOLISCI~react he dark; eaction also r FI of the however, increases its rate considerably. METZNER showed fiver; at tckened by th~ am determines which cell constituents are blackem reaction, ~articular-4 the c h l o r o p l a s t s - - t h e i r surface and the grana in p~ range in at p H 7-8 the precipitate is mainly found in the cytoplasm. c) 1 the chloroplasts react this author examined the wavelengtt w :y of the SCH reaction. This was done by counting the silver precipitate pl s and by tically evaluating the figures obtained. The action s;pectrum I in living ~hyll )hyll absorpt absorption spectrum. shows some features in common with the chlorophyl m. Ho~ Howthat obtained from killed cells hardly admits any conclusion. cc In order to examine whether chlorophyll occurs in isolated isol~ tecessary In lamellae it was necessa: w~ necessary to study wheth whether to use grana suspensions instead of tissues. Therefore, it was [OLISCH reaction can be established in such a suspensl the M( ent )ension. Consequently the presel study deals with: re( IE. . demonstration of a light-induced silver nitrate reduction in grana suspensior )enslons, 2. ,'. demonstration of the dependency of the MOLISCH reaction sensitization on the t] presence nce of. excited chlorophyll molecules by establishin~g an action spectrum of this t~ reaction, llorophyll. 3. localisation of the chl, MATERIAL AND METHODS
Preparation. G r a n a suspensions Lsions were p r e p a r e d from fresh leaves of Hibiscus rosa sinews szne~sis. U s u a l l y a b o u t 4 g of w a s h e d leaves yes were g r o u n d in a W a r i n g blendor in 75 ml of a pre-cooled o.2 M glucose or, m o s t l y , sucrose solution ~n in glass-distilled w a t e r for 2 minutes. T h e brei w a s filtered t h r o utgh c o t t o n wool a n d centrifuged at I2oo g for 5 minutes. The s u p e r n a t a n t w a s diluted w i t h an eqtual v o l u m e of a o. 4 M s o d i u m a c e t a t :e e buffer in o.2 M sucrose. The p H values will be mentioned togetl2 ether w i t h the e x p e r i m e n t s . This suspenmon ension will be referred to as " g r a n a s u s p e n s i o n " . L a m e l l a e suspensions w e r e p r e p a r e d b y weakly disintegrating the g r a n a suspensions w i t h tthe aid of a m a g n e t o s t r i c t i o n oscillator. Ltor. The energy delivered to the m a g n e t o s t r i c t i v e t u b e w a s abo ~out 3o0 w a t t s a t a frequency of 7ooo )o Hz. A p p r o x i m a t e l y i o % of the energy w a s effective. T h e gra rana were t r e a t e d for i m i n u t e u n d e r: nitrogen. D u r i n g operation t h e v i b r a t i o n cup w a s cooled w i t h ttap w a t e r . O u r t h a n k s are due t o D r J. A. NII~MEIJER, w h o kindly placed t h e oscillator of the L aabboorraattoo r y for Physiological Chemistry, U t r •eecht, c h t , a t o u r disposal. T h r o u g h o u t the p r e p a r a t i o nx the suspensions were k e p t a t low t e m p e r a t u r e as m u c h as possib ~ible. Occasionally s h o r t - l a s t i n g storagq;e occurred in a refrigerator. Care w a s t a k e n to keep t h e suspensic mnslons Relerences p. 30.
.......
,
.
.
.
.
.
.
.
.
.
j
. . . . . . . . . . . . . . .
formed in weak green light. Before st~ for 5 m i n u t e s at least. The experime
ormed at
:ISCH reaction. Samples for illuminatioi )1 aliquots were k e p t in the d a r k unde
from the qual con-
n o u n t of metallic silver w i t h t h e aid c stop endAYE1~, were unsuccessful, t h e y m a y be )ned here )LlSCH reaction. After addition of silv utions of the g r a n a suspension w a s illuminated. atrifuged. as well as of the sediment, w a s h e d w vater and nined b y titration. I n b o t h illuminated ;pensions, a m o u n t s of silver were found to occur Lent. This , seems to indicate t h a t p a r t of the si] strongly n o t noticeably r e m o v e d b y t w o w a s ver, since ration in q u e s t i o n is r a t h e r sensitive this p h e n o m e n o n mayy suggest t llic silver • is produced b y reduction of p r i m a r i l y adsorbed silver nitrate. nitral reacti 'or t h e q u a n t i t a t i v e d e t e r m i n a t i o n of t h e effect of the MOLISCH Morn ~sed light ~tallic silver, fission of t h e suspension, owing to blackening b y metallic silve w a s mea Letrically. of the g r a n a suspension were p i p e t t e d into a plane-parallel 5.5 5 m m glm, e cuvette aunted in the p h o t o m e t e r and a v i b r a t i n g stirrer was a d j u s t e d in order to aentation in gl suspended particles. T h e n I ml of a lO% silver n i t r a t e solution soh ?ater w a s green carefully. Initially the light transmission, determined in weak w~ ed owing tions w i t h sot f o r m a t i o n of nearly white insoluble silver salts b y reaction hering to ,~anic m a t t e r or diffusing from it. After a few m i n u t e s t h e ligh t transml a steady illuminated and This value w a s called lOO%. N e x t t h e c u v e t t e was illumir nsmission as determined. E x c e p t for the illumination period, a sample, serving , s treated )roved to incr s a m e way. W i t h time the light a b s o r p t i o n of the controls pro" 11a m o u n t Lhe highest value o b t a i n e d w a s 13 % for 4 hours. green : he p h o t o m e t r i c e q u i p m e n t allowed t h e p r o j e c t i o n of a parallel para i the susn c u v e t t e on to a selenium b a r r i e r layer cell connected to a Zernike ]2 mometer. f ioo w incandescent l a m p w a s used as a light source. A g lass filter of S c h o t t x e c u t e d as q u i c k l y as possible. Check Chec and VG 9 w a s placed in front of it. The readings were eexecu ments p o i n t e d o u t t h a t , in this way, no reaction due to t h i s procedure resulted. ~ium i l l u m i n a t e d either b y sodim llumination equipment. The c o n t i n u o u s l y stirred suspensions were v Illumination )oo w Philips E 4 ° p r o j e c t i o n lainP .r b y light of various w a v e l e n g t h s obtained from a 3 ° V 9oc light or i d of c o m b i n a t i o n s of a Christiansen-Weichert dispersion m o n o c h r o m a t o r and S c h 0 t t gla~ lass w i t h t hhee aaid filters. M e a s u r e m e n t s of incident light intensity were carried o u t w i t h a s t a n d a r d i z e d thermopile. o t h grana and lamellae were made mad 7.lectron microscopy. Electron microscopic p r e p a r a t i o n s of bbet Elect~ dmitted Aously 2° described. M o u n t i n g a n d w a s h i n g occurred in weak glreen light. F u l l light w a s aadmitte as previously re eexamine, x a m i n e d in a P h i l i p s electron microscop [ter t h o r o u g h l y drying of the p r e p a r a t i o n s . T h e y were o n l y after )e,
RESULTS
Demonstration o/light-inducedi silver nitrate reduction m grana suspensions mple out of three experiments Fig. I shows an evident evider Time [actor. As an exam miincrease of absorption, owing t ° the formation of metallic silver, with time at an illum time. Lsion. Apparently the rate of increase declines with tim nated Hibiscus grana suspension. silver This phenomenon m a y be duee to three causes. First, the amount of black metallic silv( both ithin the suspension an increasing part of the light is bet increases with time. Thus, within ased. nsequently, the intensity of the effective light is decrease( screened of and absorbed. Cons( Fitration ntity of silver nitrate m a y become exhausted. Titratio Secondly, the available quantity dq experiments showed that thee amount of dissolved silver nitrate is not noticeably de:ion ted above, if merely adsorbed salt is reduced, exhaustie creased. However, as suggested ay .icture. Thirdly, the activity of the grana suspension m ma phenomena m a y enter the picture. arious decrease. This was checked bby drawing samples from the same suspension at vvarlet ncrease intervals. These samples were'e kept at room temperature and their absorption Iincrea,' nly On] per unit of t i m e - - m o s t l y 3 o m i n u t e s - - w a s measured at equal light intensity. O Re]erences p. 3o.
tction was observed. As a mean ot per hour. The question remains, light. However, because of the c
.~o
I
~,o
activity hange of
C .o
I
30
I
I
I
I
60
90
t20
150
• Time course of silver n i t r a t e reduction, m e a s u r e d as a b s o r t ~tion incre suspension in s o d i u m light. I n c i d e n t intensity: 4.4" lO4 ee~r ~ s c m - 2 s{
~cUS g r a n a
condition within the suspension, owing to the formation foi ver, this em can hardly be solved. ally, the )roceeds al )ne m a y ask whether the reaction, once started, pr ~ry silver precipitates functioning as condensation nuclei. i Tt I ms quesuon m a y be t ~red by interrupting the exposure to light of a reac ting suspension. Such an ee:x eriments periment rant is shown in Fig. 2. Evidently no after-effect occurs• occu: Moreover two experimen sc were carried out, in which the absorption increase of a suspension, pre-iUuminated for f~
co
g
1o
20
35
,b
~o
65
75
minutes
Fig. 2. Effect of darkening, indicated ieated b y the arrows, on the silver nitrate reduction, measured a s a b s o r p t i o n increase, of a Hibiscus us grana suspension. Incident sodium light intensity: 4.4" IO4 ergs c m -2 sec -x. p H : 6.6.
5 minutes, was compared with th that of a control sample. The experiments lasted 75 and ar 23o minutes, respectively. The le corresponding absorption increase values of the controls contro were 2 and 13%; those of both ~th pre-illuminated suspensions amounted to 9 %. Thus, in i these prolonged experiments no after-effect of illumination was observed. p H sensitivity o/the MOLl: .ISCH reaction. According to METZNER TM, chloroplasts withi within References p. 30.
m a l l y a t p H 3 - 4 of t h e s u r r o u n d i is are m a r k e d l y different, and, wi emonstrates that the pH depend k a l i n e p H r a n g e s p o n t a n e o u s blacl
~Lx ~ o 4 o
le o p t i m a l n g e d cont to occur,
?.
TABLE i F pkI
ON
THE
MOLISCH
REACTION
RAl
m light intensity 4.4" 1°4 ergs cm a sec )uration of lure*nation
A bsorption increase in per cen of control at p H
in minutes
75 23o
I 2
4.6
5.6
6.~
3 23
II 25
xc 44
; intensity a n d in L i g h t /actor. T h e r e l a t i o n b e t w e e n MOLISCH r e a cction tic ties a b o u t , r e s e n t e d in Fig. 3. E v i d e n t l y n o l i g h t s a t u r a t i o n is i reache( it o c c u r s 4 t i m e s h i g h e r ttlh a n t h o s aspensions in p h o t o s y n,nthesis t h e s i , of gree] d doubtful e n s i t y . So of c o m p a r a b l e dde1 ~V[oLISCH mee's are i n v w h e t h e r e n z y 'm s reaction r e a c t i o n . METZNER METZN~ Is s t a t r ent p r o c e e d s in cells t h a t w e r e k i l l e d b y p r e t r e aattrm w i t h s i l v e r n i t r a it e . H o w e v e r , t h o u g h it ssee e m s s o m e e n z y m e s m i g h t be l e f t h i g h l y i m p )robable, robabl h i s r e a g e n t . F o r t h i s r e a s o n ss(o m e u n a f f e c t e d b y tthi: I i T i ,,nsions w e r e p e r f o r m e d w i t h suspens! 6 e ~ , , e x p e r i m e n t s we~ 2 4 d In e P g s c r d ~ .10 " k i l l e d " b y h e a lt i n g t h e m in a b o i l i n g w a t e r Fig.. 3. Relation between light intensity b a t h . A f t e r ccoolJ )erature o o l i n g d o w n to r o o m tempera1 and rate of silver nitrate reduction, n i t r a t e w a s a d d e d . T h e r e s u l t s are ~sured as absorption increase per 25 the silver nitrat( measured rotes of a Hibiscus grana suspension in g i v e n in T a b l e I I . minute sodiumlight, pH : 6.6.
./
TABLE II MOLISCH
REACTION
OF GRANA
pH:[ : Graua suspension
6. 5.
SUSPENSIONS ILLUMINATION
PRE-HBATED WITH
IN
SODIUM
A BOILING
WATER
A bsorption
A bsorption
Incident light increase of illumi- increase of dark Heatingperiod Illumination in-1ergs nnted suspensions control in % in minv2es period in minutes intensity cm-2 sec in %
a a
IO
b
15
b
20
b*
60
25
8.12" lO 4
34
25 3° 3° 3°
8.12" lO4 9.35" lO3 9.35" IOn 9.35" lO3
36 19 20 II
3 8 8
• At the end of the heatingg period thiss suspensionshoweda-brownish-green-colourReferences 19. 30.
BATH.
LIGHT
e " a " series show that the MoL lg period. In the " b " series, at low samples heated for 15 and 20 mir ence of any enzymic character of 3ur the rate of this reaction is dec resh green to brownish-green. Th the chlorophyll results in a reduc that excited chlorophyll itself is t
Q11
L~
IIUL
intensity, vn. These reaction. colour of non m a y MOLISCH
agent in
:hlorophyll anism of the MOLISCH reaction. The idea De responslDle ior rne r e a u c u o n of oI the rne silver Sliver n i t r a t e w a s already air( RET~1. This teste¢ )r p r e p a r e d active alcoholic chlorophyll e x t r a c t s from spina( ~r w a s h i n g )inach leaves. he chlorophyll w i t h petrol ether the alcoholic p h a s e prov s activity. )roved to hay 'HERET concluded t h a t the reducing p o w e r responsible for the MOLLS instead of due to the chlorophyll, depends on the presence of a substance s u b s t a n soluble t insoluble trol ether. I n t h i s connection a few additional e x p e r i m e n t s will be bri However, ,~h t h e y m a y yield some suggestive information, t h e y are nnot Lt study. o t essential A colloidal solution of c h r o m a t o g r a p h i c a l l y p u r e chloroph] toz, Basel, phyll, o b t a i n )repared b y dissolving I m g of this p i g m e n t in 2 ml acetone. U n d e r ra l ds solution tdded to 20 ml of a o.I M sucrose containing o.i M buffer solution o this w a y a h e r u n s t a b l e - - h y d r o p h o b i c sol w a s obtained. T h e n I ml of a lO% sil lution was t. T h e light a b s o r p t i o n of t h e m i x t u r e was determined. N Ne~ Lt h e dark, ext a sampl sec - i - for t an aliquot w a s e x p o s e d to s o d i u m l i g h t - - i n c i d e n t iintensi n t e n s i t y : 3.26- I inutes. After this period t h e light a b s o r p t i o n was determin ed a g a i n . ' m increase n t r o l a n d illuminated suspension a m o u n t e d to i and 13% respectiv ,,xperiment ires t h a t t h e MOLISCH reaction directly depends on the reducing redu hlorophyll. powe: t t t e m p t s t o reproduce this result, however, were unsuccessful ; the colloi, II solutions ce showed a flocculation a t the addition of the silver Initrate. nitr~ These ll,taLC. ilig~C l i U S t , tllcttCta ~ uuspensions ~gllbl~ ot s h o w a n y light reaction. This difference in b e h a v i o u r is ni o t clear since p r e p a r a t i o n as well p e r i m e n t a l conditions did n o t noticeably deviate from those thos in the first experiment. I t m a y be a d v a n t a g e o u s to stabilise the chlorophyll sol w i t h the aid of a 1c)rotective colloid. Since, as alrea already r e m a rrked, k e d this s t u d y does n o t aim at an investigation of the m mee c h a n i s m of the MOLISCH reactfl reaction, t h e s e e x p e r i m e n t s were n o t extended a n y further. Fin Finally, a single e x p e r i m e n t w i t h a chlorophyll-free alga, Prototheca P: menZopfii, m a y also be m, tioned. ~. After addition of silver n i t r a t e up to a final concentration ~tion of 1% an algal suspension of :2 g fresh w e i g h t in IOO ml acetate buffer of p H 6. 5 w a s illuminated w i t h s o d i u m light of 9.35" IO3 eergs c m - 2 sec -1 for 30 m i n u t e s . A f t e r this period the a b s o r p t i o n w a s increased i: corre= b y 6 %, whilst the cot s p o n d iing n g value at a d a r k control sample a m o u n t e d t o 4 %- So these t figures m a y suggest t h a t lthe MOLISCH ;cH reaction does n o t proceed in t h e absence of chlorophyll. TFhe h e results resul m e n t i o n e d are c o n t r a r y to those of GAUTHERET.. This T] divergency can be explained b y assumm dng t h a t GAUTHERET m a y h a v e obtained " i n a c t i v e " chlo) chlorophyll suspensions, j u s t like 1the "inactive" chlorophyllpreparations ions described above. The a c t i v i t y of GAUTHERET'S chlorophyll-f~ rll-free alcoholic leaf extract may be due ~e to some reducing s u b s t a n c e soluble in ethanol. I n this respect Dect it may be mentioned that, in literature rature, ascorbic acid is often considered to be responsible /or the reducing power needed in the MOLl IOLISCH r e a c t i o n . METZNER ~2 r e m a r k s t h a t , a t the present, it can~ cannot yet be excluded that the MOLISCH SCH reaction in illuminated chloroplasts m a y proceed w i t h o u t t h e intermediary of ascorbic acid. This ais s t a t e m e n t seems to be confirmed b y the above results. H o w e v e r , it may be remarked explicitly that m t for f u r t h e r confirmation additional e x p e r i m e n t s are needed.
Summarising the main results results it can be stated that grana suspensions are able to show the MOLISCH reaction. Light is an essential factor. The reaction is pH-sensitiv ;ensitive. I t is non-enzymic.
Action spectrum o/ the MOLISC} SCH reaction. Direct proof of the dependency of tt HOLISCH this reaction
in grana
determining wavelengths
s u s p e n s i o nns s
on the presence of chlorophyll m a y be obtained tb y was done b y measuring the reaction rate at vario~ various it in per cents of this rate at sodium light. The resultii ling Fig. 4. Since the selectivity of the Christiansen-Weiche Weichert
a n a c t i o n s p e c t r uam. m. T hi This and computing
.action spectrum
References p. 3o.
is s h o w n i n
. . . . . . . . .
j
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.
.
.
.
.
.
,
.
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.
region of the spectrum is relati ~tion with Schott glass filters as in he legends. ,trum is drawn in three parts. Es t ;5° m/~ the he relative ~xpected to occur since, when de RG filters, efficiency wit some highly ~/x light is of the very / ~ also transmitt avelengths low light int~ lts in this below 49 ° m spectral regioi inaccurate. epresented Nevertheless "ly demontoo, the dive1 g. strating their strat eniences it Despite t e MOLISCH may be evid( m closely reaction acti L spectrum rAJ ~mbles the reset For the of a grana r spectrum ~,stablishing c ' ~ esta~ Miss L. J. o our thanks a es that the BARTELS. The BAR: ds on the M O L I S C H rea( MOLl It may be )resence of el rrema emarKea i n a c K t K l o n t n aat ~ the ~ n e acremarked in pres~ curacy , cura~ of the determinations does tsions not allow to draw any conclus foJ concerning a possible carotenoid activity in this reaction. ~ a l o
~
'
~
'
' '
~t'm~ ~-a~.. 1~.__
a ~ l u i , v
o
faction MOLISCH rea~ ° -: --+~2 Since it is shown that, under unde~ the • -- ÷~5 ....... ~e chosen chos, experimental conditions, silreduction is coupled'with ver nitrate i ~o ~o ~o ~o ~o the presence of excited chlorop])hyll, lsed as MOLISCH as well the MOLISCHreaction may be use I S C H rreaction eac Fig. 4. A c t i o n s p e c t r u m of t h e MOLISCt as a b s o r p t i o n s p e c t r u m established , l i s h e d a t Hibiscus grana a toolin localising these pigment:moll suspensions. nlcroscope. ecules under the electron microsc The precipitated metallic silver causes a strong electron scattering; in this way it is s! easily recognised. Since it is necessary to remove buffer salts as well as excessive silver nitrate from preparations of illuminated suspensions, washing is required. Though this not be ed water was carried out very cautiously, it could no washing with glass-distilled prevented that part of the,~silver deposits were ruptured. These broken fragments,were ~m the eighbourhood of silver covered chloroplasts. Apart from always observed in the nei [ectron scattering layers were absent in non-illuminated SUSfact that these strongly elec it they consisted of silver by establishing electron diffrac:tion pensions it was proved thai :kened" diagrams. Fig. 5 shows thazt the pattern of silver occurs in the diagrams of "blackel grana.
/
Re/erences p. 30.
•o c n . .~ ., ~. -.m . ~+ l e,m, 6 % C u $ 0 4 •
.
.
.
.
+~2* ~20
,alisation o / t h e Loca
b
C
i
I
• 5. E l e c t r o n d i f f r a c t i o n d i a g r a m s o f at : s i l v e r - c o a t e d g r a n u m , b : c o n t r o l g r a n u m , c : b l a n k c o l l o d i o n film, d : s i l v e r :ipitate. F i g . 6. Hibiscusgranum. F i g . 7. S i l v e r d e p o s i t s o n Hibiscus c h l o r o p l a s t . A d v a n c e d 1V[oLISCH 1r e a c t i o n . • 8. I n i t i a l s t a g e o f MOLISCH r e a c t i o n a t a Hibiscus g r a n u m . F i g . 9. A d v a n c e d MOLISCH r e a c t i o n a t a Hibiscus num. F i g . i o . S i l v e r p r o t r u s i o n s f!rtoom m h e a v i l y c o a t e d Hibiscus g r a n a . P a r t l y s h a d o w e d . F i g . I I. S i l v e r p r o t r u s si oi onns s fro~ f r o m Hibiscus g r a n a a n d l a m e l l a e .
() shows an u n t r e a t e d g r a n u m . ~ted with silver as a result of th~ p a r t l y indicates contours of grar qnired to i n t e r r u p t the silver nitJ i l l u m i n a t i n g the suspensions at with a g r o u n d glass plate adjust(
reaction. to draw an at all f z 5 cm ~f it, for
of the reaction at a g r a n u m . As is deposited in a homogeneous 1~
rated by :anum is
m
Fig. 12. Supersonically disintegrated Hibiscus m. Heavily silver-coated lamellae, arranged in granum. a fan-like way. 3. initial stage of the MOLISCH reaction at a Fig. '3. single Hibiscus lamella. ~action at a single Fig. 14. Advanced MOLlSCH reaction Hibiscus lamella. increa d a m a g e d ; lipids are p r o t r u d iing n g in m y e l i n figures. Since the l a t t e r do not show increase ~ems probable t h a t silver deposits are restricted to the tl of electron scattering, it seems proteinaceous p a r t s of the g r a n u m . ~OUS ion is represented in Fig. 9. A p a r t from the homogeneo~ A more a d v a n c e d reaction lsions are shown. I n Fig. IO such-like structures are more mo coating, typical silver protrusions ,oundary pronounced. I t m a y be n o t e d that, in this figure, the g r a n a are s i t u a t e d at a bounda~ b e t w e e n a shadowed a n d a n o n - s h a d o w e d region. I n this way it is possible to get a n on scattering power of the silver coating of a single g r a n u m impression of the high electron as well as of its t h r e e - d i m e n ssional i o n a l shape. )osited cription m a y suggest that, at first, the silver is deposit( Possibly the above descri initial he protrusions arise. This, however, is not true. At initi homogeneously and, next, the Re/erences p. 30.
ther homogeneous blackening or 1 influencing the formation of on M~-TZNERlS--noticed the irregular ced reactions protrusions were alv reatment of grana, lamellae suspei reagent. Fig. I I represents a pre from the shadow length, and apar shown. Apparently the latter are silver nitrate reduction it is often r, identification remains possible ~k supersonic treatment often cau rate into a fan-like cluster of lamellae. Such a cluster, cluste heavil~ Fi~ Iwn here. The contours of the lamellae are slightly visible. v: nstrate the occurrence of silver deposits on single lamellae. lal :n conclusion it m a y be stated that lamellae are able abIc to bring ion.
ox~11o
¢ x x K;
ypes de: precipi~d.
prepared such an e thickly red with :ecognise E'his m a y .n to disth silver, 4 clearly
MOLISCH
DISCUSSION
and size in a preceding paper 23 a correlation between activityr in the H lr. These m a fragments, prepared by supersonic disintegratiol 'ration, was sh :ed from iments suggested t h a t the photochemically active active fragmel in these lae. Consequently, it seemed most likely t h a t chloro]phyll is c tures. Fhe present investigation confirms this deduction in a imore direct way. The MOLISC )LISCH reaction ction ion is shown to depend on the presence of excited chlorophyll. c~ Since this reactk is demon, nonstrated to occur in lamellae, it seems justified to t conclude that lamellae are a the bearers of chlorophyll. A few remarks m a y be made concerning the MOLISCI-I MoLISq reaction as it is obserw observed in the above experiments. M E T Z N E R ls stated that silver nitrate nit~ flasts reduction by chloroplas i n "iE~tact" cells occurred within 35 seconds in monochr~ ~chromatic light, the intensity of whichI amounted to, probably, about IOO times that used in the present investigatio ~tlon. The consider~ onsiderable divergence in both exposure time and ligh ht intensity m a y be understoc~od m l r n i n g fthat, h ~ t nowing w i n ~ fto n the ~'h~ n r e c : P n e e of a{ reducing r ~ r t l w q n ¢ s¢nh¢¢ b y assuming .cid Lhe presence u b s t a n c e s - - s u c h as ascorbic ac - - w i t h i n the cell, this re~ctioi :ion with chloroplasts in situ proceeds more rapidly than th~ t h a t with isolated grana. In fact !act, a few additional experiments showed that the MOLIS( ~LISCH reaction in grana suspensionss could be considerably increased by adding ascorbic aci cid. This seems to indicate that excited c chh xcitedd chlorophyll m a y act either as a catalyst of a reaction reactic between silver nitrate and a reducing agent, or as the reducing substance itself. Since, Sinc however, the present investiigation does not aim at an elucidation of the mechanis '~chanism of the MOLISCH reaction, the ,, validity of this suggestion was examined no further. SUMMARY Silver n i t r a t e r e d u c t i o n w a s3 s h o w n to o c c u r in i l l u m i n a t e d s u s p e n s i o n s of Hibiscus g r a n a . T h e a c t i o n s p e c t r u m of t h i s r e d u c t i o n , t h e MOLISCH reaction, p r o v e d to coincide satisfactori torily w i t h t h e c h l o r o p h y l l a b s o r p t i o n s rp e c t r u m . E l e c t r o n m i c r o g r a p h s r e v e a l[ t h a t t h i s r e a c t i o n occurs in single lameUae. F r o m t h i s it is conclud concluded t h a t lamellae are t h e b e a r e r s of c~hlorophylL hloro
Re/erences p. 30.
R]~SUM~; n s i o n s de g r a n a illumin~es de Hibiscus
trgent eat
tion, la r~action de MOLISCH, r e s s e m b l e
bsorption
~ntre, q u e des lamelles isol~es s o n t ca t e q u e les lamelles c o n t i e n n e n t la chlorc
m e r cette
ZUSAMMENFASSUNG te S u s p e n s i o n e n y o n Hibiscus-granen d u k t i o n ,• die MOLISCH R e a k t i o n , ist den . . . . o -r dorophylls iihnlieh. ~ e o b a e h t u n g e n m i t t e l s des E l e k t r o n e n m i k r o s k o p s zeigten, d a s s diese a s s die L a m len v e r a n l a s s t w e r d e n k a n n . E s wird h i e r a u s geschlossen, dda: sind.
ad Silberspektrum isolierten es Chloro-
REFERENCES {EITZ, Planta, 18 (i932) 616• tEITZ, Planta, 26 (1936) 134. {ETZNER, Ber. deutsch, bot. Ges., 55 (1937) 16. TRUGGER, Ber. deutsch, bot. Ges., 64 (1951) 69. ATEIER, Bot. Rev., 4 (1938) 497i. RABINOWlTCH,
Photosynthesis and related Processes. I. I n t e r s c i e n c e
{ew Y o r k
5).
vier Publ. its Der :REY-WYSSLING, Submicroscopic Morphology o[ Protoplasm and a A m s t e r d a m , N e w Y o r k (1948). A m e s (1949) 113. ~RANICK, Photosynthesis in Plants, I o w a S t a t e Coll. Press, An ". WEIER AND C. R. STOCKING, Bot. Rev., 18 (1952) 14. l ° M . C~ALVIN AND V. LYNCH, Nature, 169 (1952) 455. n J . B .k THOMAS, Proc. Kon. Ned. Ahad. Wet. Amsterdam, Ser. C, 555 (1952 ) 207. l~j. j.• WOLKEN AND G. E . PALADE, Nature, 17o (1952) 114. 13 E . STEINMANN ;TEINMANN, Exp. Cell Research, 3 (1952) 367 . 14 E . STEINMANN ;TEINMANN, Experientia, 8 (1952) 3oo• ~REY-WYSSLING AND E. STEINMANN, Viertel]ahrsschr. Natur[. Ges. Ziirich, 98 (1953) 2o. is A . FREY-WYss ¢IOLISCH, Sitzungsber. A kad. Wiss. Wien, math.-naturwiss. Kl. K l Abt. I, 127 (1918) 449. 18 H . MOLISCB 17 F. WEBER, Protoplasma, 29 (1938) 427 • 18 H . METZNER FIETZNER, Protoplasma, 41 (1952) 129. 19 p . DELAHAYE )ELAHAYE, Anal. Chim. Acta, 4 ( I 9 5 O) 635. }hys. Acta, 8 (1952) 9o. 20j. B.~. THOMAS, M. BUSTRAAN AND C. H. PARIS, Biochim. Biophy. 21 t . J . GAUTHERET, Rev. gan. Bot., at., 47 (I935) 484 • iss. Gdttingen math.-phys. K1. I I b (1952) i. =2 H. METZNER, Nachr. Akad. Wiss. 23 j . B. THOMAS, O. H . BLAAUW AND L. N. M. DUYSENS, Biochim. Biophys. Acta, Io (1953) 23 o.
Received July 2nd, 19:)53