plasto globules

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with petrol (4o-6o °C)/die thyl ether /a cetone (1o/2/1) ;phytol , s terols , and quinone s with CHCI~; methyl-

o Jes ters of FAs with petrol (4o-6o C)/die thyl ether(35/2o); FAs with CHCl~ /e thy laee ta t e / fo rma te (4o/6/3;cf. Kati ng et al . 197o~; and the polar l ip i d f ract ionus ing the 2-dimens ional sys tem of Allen and Good (1971) .Rou t in e ly th e l ip id s po t s w er e de tec ted by s p r ay ingwith Rhodamin- Tinop al (Popov and Stefan ov 1968),w her eas th e p la s toqu in on es w er e v i s ua l i s ed by theEmmer ie-Eng els re agent (Barr and Crane 1971) , and thes teroids and phytol by the Car t-P r ice rea gent (Neherand Wetts tein 1951) . Separat ion of caroten oids (af terhydrol s is of carotenoi d es ters) took place on cel lu-lose th in layers (Merck, impreg nated by a t r iacyl g ly-cerol phase) with methano l / ace tone /HgO (15/5/i) assolvent (Egger 1962) . Individual l ip i ds were ident i-f i ed by co - ch r omatog r aph y o f au then t i c r e f e r ence s ub -s t ances an d b y comb in a t io n o f TLC and G LC. G LC w asus ed to id en t i f y the me thy l e s t e r s o f co r r e s po nd ingFAs ( io EGSS-X, Gasch rom P, 125-15o mesh) as wellas phytol (3 Poly A io3, Gaschrom Q, Ioo/12o mesh) .

Q uan t i t a t iv e A na ly s es : Co mmon FA s w er e de te r minedaccord ing to Duncomb e (1963) , whereas the amount of~ - hyd r o x y FA s w as ca lcu la t ed f r om the i r r e l a t ive p r o -po r t io n s on gas - l iqu id ch r o matog r ams . The amoun t s o f

po la r l i p id s w er e de te r min ed v ia the i r f a t ty ac idmo ie t i e s . Th e f o l lo w in g co mpounds w er e eva lua ted quan -t i t a t ive ly acco r d ing to s t and a r d me thods : G lyce r o l(Renkonen 1962); ste rols (Clark et al. 1968); caro-

tenoids (Liaaen-Jensen and Jensen 1971) ; p las t oqui-none 45 (Lichtenthaler 1968a) . The relat i ve amountso f ~ - toco p her o l an d a lk anes w er e e s t ima ted a f t e rv i s ua l i za t ion on TLC p la t e s w i th e i the r Emmer ie - En -gel reagent (Barr and Crane 1971) or Rhodam in-Tin o-pal reagent (Kleinig and Lempe r t ]97o) . The amountof phytol was determined by GLC.

Elec t r o n M ic r os copy: S amp les o f u l t r a th in s ec t ionsaf ter GA/ OsO 4 or KMnOa f i xat ion as well as of f ro-zenetche d mater i al ( je tals and isolate d globules )f o r TEM w er e p r epa r ed a s d es c r ibed p r ev ious ly ( L ied -

vogel et al . 1976) . Electron microg raphs we re takenwith a Zeiss EM 9 at 6o kV.

Res u l t s

M os t o f th e pe ta l ch r o mo p las t s o f V~o~a t2~ooloP arefound in the basal ( inner) pa r t of epidermal cel ls .The eh r omo p las t s a r e t ig h t ly f i l l ed w i th p la s tog lo -bules (Fig. i ) . The plas t ids of mesoph yll cel ls aremuch less numerous , smaller, and contain thylakoidsas well as some plas togl obules . Oleoso mes are ex-t r emely r a r e in p e ta l ce l l s . F o l low ing f ixa t ion w i thei ther GA/OsO~ or KMnO4, the chromoplas t g lobules

4exh ib i t an e l ec t r on - t r ans lu cen t in t e r io r and a dens esurface lay er 3 to 4 nm thick, corre spond ing to a'half uni t membr ane' (Figs. 1 and 2). Ther e is noind ica t ion o f a t r i l amin a te un i t membr ane . I n f r eeze -f r ac tu r e and f r eeze - e tch p r ep a r a t ions , t he in t e r io ro f the g lobu les l ikew is e app ea r s homogeneous w her easat their surface there is apparent ly a layer of d iffe-r en t f r ac tu r ing p r ope r t i e s w h ich does no t , how ever,corre spond to a typical b io membr ane (Fig. 3) .

A cco r d ing to e l ec t r o n mic r o scopy, i s o la t ed ch r omo-p las t g lobu les w er e f r ee o f r ecogn i s ab le impur i t i e ssuch as cel l organel les , membr anes , or nucleo pro-teins . Presumably, o leosom es are prese nt in the f rac-t ion, a l though in very small amounts ( less than iacco r d ing to mor p homet r i c e s t ima tes o f the ~ n 8~s i tua t io n) . Con tamina t ion b y cu t i cu la r w axes cou ldbe ruled out in three diffe rent ways , e .g . rem oval ofep icu t i l a r w axes w i th ch lo r o f o r m be f o r e t i s s ue homo-genisat ion, search for w ax- s pec i f i c componen t s in

chroma togram s ( there were none detectable ) , andcompar i s on o f th e FA pa t t e r n ( w h ich d i f f e r s g r ea t ly) .

F ig . i: Chromoplas t s , pa cked with plas togl obules , inthe basal c ytopl asm of an epiderma l cel l of a

V~ o~ petal . KMnO 4. 17,ooo:i . Bar, 1 ,um. /

F ig. 2 : F r ac t ion o f i s o la t ed ch r omop la s t g lobu les .OsO4.31,ooo:i . Bar, I /um

F ig . 3 : F r eeze - f r ac tu r e appea r ance o f i s o la t ed ch r o -mopl as t g lobules . 94,ooo:I . Bar, o , I /um.

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Ta b l e I : C h e m i c a l c o m p o s i t i o n of c h r o m o p l a s t gl o b u l e s .D a t a a r e e x p r e s s e d i n p e r c e n t o f t o t a l d r y w e i g h t .

C o n s t i t u e n t s Viola Caltha a

P ro t e i n s 3 . 9 + +P o l a r l i p i d s 4 . 7 7A l k an es 3 . 3 -

Q u i n o n es i . i +S t e ro l s , e s t e r s 0 . 5 +P h y t o l , e s t e r s 2 . 5 -C a ro t e n o i d s , e s t e r s 2 3 1 4Tr i a c y l g l y c e r o l s 5 7 7 4

Sa. 96 95

as i t t e e t a l . (1980)

Ta b l e 2: P o l a r l i p i d c o m p o n e n t s o f p l a s t o g l o b u l e sa n d d i f f e r e n t m e m b r a n e f r a c t i o n s o f p l a s t i d s .

P o l a r l i p i d s I I I I I I IV V

M o n o g a l a c t o s y l d i a c y l g l y c e r o l 3 5 . 1 5 5 . 8 2 8 . 8 3 8 . 4 3 9 . 1D i g a l a c t o s y l d i a c y l g l y c e r o l 2 1 ,1 1 7 , 2 2 6 . 6 3 2 . 3 2 0 .1S u l fo l i p i d 7 , 5 3 . 9 5 , 6 9 . 8 7 . 3P h o s p h o l i p i d s 2 1 . 5 2 3 , 1 3 9 . 0 1 9 . 5 2 6 , 7F ree FA s 1 4 , 5

Sa. 1OO 1OO iOO i00 93 .1

I = C h r o m o p l a s t g l o b u l e s o f Viola tricolor

I I = C h r o m o p l a s t i n t e r n a l m e m b r a n e s o f Narcissuspseudonarcissus (Liedv ogel e t a l . 1976)

I I I : C h l o r o p l a s t e n v e l o p e m e m b r a n e s o f Narcissuspseudonarcissus (Lied vogel e t a l. 1976)

I V = C h l o r o p l a s t t h y l a k o i d m e m b r a n e s o f Narcissuspseudo~rc issus (Liedv ogel e t a l. 1976)

V : C h l o r o p l a s t t h y l a k o i d m e m b r a n e s o f Spin~ciaoleracea (H as h i m o t o an d M u ra k am i 1 9 7 5)

T h e r e s u l t s o f c h r o m o p l a s t g l o b u l e a n a l y s i s a r e s h o w ni n Tab l e 1 . A m o n g s t t h e p ro t e i n s , t w o s p ec i e s (3 0 O O ~6 8 0 0 0 ) p r e v a i l a c c o r d i n g t o S D S - PA G E . T h e p a t t e r n o fp o l a r l i p i d s s t r i k i n g l y r e s e m b l e s t h a t of d i f f e r e n tp l a s t i d m e m b r a n e s ( cf . Ta b l e 2 ). P h o s p h a t i d y l i n o s i t o lh a s b e e n f o u n d i n t r a c e s o n ly, p h o s p h a t i d y l e t h a n o l -a m i n e i s v i r t u a l l y a b s e n t . - A c c o r d i n g t o L i c h t e n -t h a l e r ( 1 9 6 8a ) , 5 q u i n o n e s p ec i e s can b e fo u n d i np e t a l s o f Viola tricolor n a m e l y p l a s t o q u i n o n e 4 5 ,p l a s t o h y d r o q u i n o n e 4 5 , ~ - t o c o p h e r o l , ~ - t o c o q u i n o n e ,a n d v i t a m i n K I . I n i s o l a t e d c h r o m o p l a s t g l o b u l e s , w ec o u l d d e t e c t t w o o f t h e m i n a p p r e c i a b l e a m o u n t s ,( e - t o c o p h e ro l a n d p l a s t o q u i n o n e 4 5, m a s s r a t i o 2 . 2

t o 1 ) . - T h e FA p a t t e rn i s co m p l ex (Tab l e 3 ) . I n ad d i -t i o n t o c o m m o n l y o c c u r i n g FA s , 8 - h y d r o x y s p e c i e s h a v eb e e n f o u n d i n a p p r e c i a b l e a m o u n t s ( m as s p r o p o r t i o n2 : 1 , H an s m an n an d K l e i n i g 1 9 8 2 ) .

Tab l e 3 : F a t t y ac i d (FA ) co m p o s i t i o n ( ) o f t o t a lp l a s t o g l o b u l e l i p i d s f r o m Viola tricolor.

FA s R e g u l a r FA s 8 - h y d r o x y FA s

3

12:0 3 .1 1109:1 1,9 -:2 9.9 -

1 4 : O 7 °0 1 9 . 8

:1 4,4 -:2 25 .2 -

16:O 7 ,9 1 .3:I + -: 2 1 . 2 -

18:O -:i

:2 1 . 8 -:3 4.6 -

Sa. 1OO

T h e m e a n FA m o l . w e i g h t i s 2 3 5 . FAs c o n s t i t u t e 6 3 o ft h e c h r o m o p l a s t g l o b u l e d r y w e i g h t ; L e s s t h a n 1 o ft h e FA s h av e b ee n fo u n d i n f r ee fo rm . - 9 4 o f t h ec a r o t e n o i d s a r e x a n t h o p h y l l e s t e r s . A f t e r s a p o n i f i c a -t i o n, 4 p r i n c i p a l c a r o t e n o i d s ( N e o x a n t h i n 4 , Vi o l a -x a n t h i n 7 2 , L u t e i n e p o x i d e 1 3 , L u t e i n 7 ) a n d s o m em i n o r c o m p o n e n t s c a n b e d e t e c t e d . T h e p a t t e r n o f f a t t ya c i d e s t e r s o f t h e x a n t h o p h y l l s i s r e p o r t e d e l s e w h e r e(H an s m an an d K l e i n i g 19 8 2 ) .

D i s c u s s i o n

I n c h l o r o p l a s t g l o b u l e s p r o t e i n s , s q u a l e n e , s t e r ol sa n d t h e i r e s t e r s , p l a s t i d q u i n o n e s , g l y c o l i p i d s , a n dt r a c e s o f 8 - c a r o t e n e h a v e b e e n f o u nd , b u t n o c h l o r o -p h y l l s n o r x a n t h o p h y l l s o r t h e i r e s t e r s ( G r e e n wo o d e ta l . 1 9 6 3; B a i l e y a n d W h y b o r n 1 9 63 ; L i c h t e n t h a l e r a n dS p r e y 1 9 6 6 ) . I n c h r o m o p l a s t g l o b u l e s , s o m e a d d i t i o n a lc l a s s e s o f c o m p o u n d s h a v e b e e n d e t e c t e d ( L i c h t e n t h a l e r1 9 7 0 ; Wu t t k e 1 9 7 7 ; s ee a l s o S i t t e e t a l . 1 9 8 0 ) , e . g .c a r o t e n o i d s , p h o s p h o l i p i d s , s u l f o l i p i d s , f r e e FA s a n da c y l a t e d g l y c e r o l s . I n t h e p r e s e n t s t u dy, t h e s e r e -s u l t s h a v e b e e n l a rg e l y c o n f i rm e d . Tr i a c a l g l y c e r o l s h a v eb e e n e s t a b l i s h e d a s t h e m a i n c o n s t i t u e n t o f c h r o m o p l a s tg l o b u l e s o f t h e p a n s y, f o l l o w e d b y c a r o t e n o i ds . A d d i -t i o n a l t o t h e c o m p o u n d c l a s s e s g e n e r a l l y f o u n d i np l a s t o g l e b u l e s , a l k a n e s a n d p h y t y l e s t e r s h av e b e e nd i s c o v e r e d i n t h e c h r o m o p l a s t g l o b u l e s o f Vibla. A I -k a n e s h a v e b e e n d e t e c t e d i n c h l o r o p l a s t s b y G 0 1 z ( 1 96 8 ).S t e ff e n s e t a l. ( 1 97 6 ) h av e s h o w n t h a t p h y t o l i s n o tn e c e s s a r i l y e s t e r i f i e d w i t h c h l o r o p h y l l i d e s i n s e e d s .C s u p o r ( 19 70 ) d e m o n s t r a t e d t h e p r e s e n c e o f p h y t y le s t e r s o f l i n o l e n i c a c i d i n g e r o n t o p l a s t s o f A c e rp l a t a n o i d e s .

T h e a p o l a r c o m p o u n d s o f t h e p l a s t o g l o b u l e s r e p r e s e n t av e r y h e t e r o g e n e o u s m i x t u r e. A s t h e a c y l r e s i d u e s b e -

l o n g l a rg e l y t o s h o r t - c h a i n e d a n d u n s a t u r a t e d FA s , i tap p ea r s l i k e l y t h a t t h i s m i x t u re i s i n a f l u i d s t a t e .T h i s a s s u m p t i o n i s b o r n e o u t b y t h e a m o r p h o u s a p p e a r a n -ce o n t h e i n t e r i o r o f t h e g l o b u l e a s s een i n t h e E M ,a s w e l l . a s b y t h e s p h e r i c a l s h a p e o f p l a s t o g l o b u l e sa n d t h e i r a b i l i t y t o b e c o m e d e f o r m e d u n d e r e x o g e n o u sp res s u re ( c f. , e . g . , S i t t e 1 9 6 3 ; T h o m s o n an d P l a t t1 9 7 3) . O n t h e b a s i s o f p h y s i c o - c h e m i c a l c o n s i d e r a t i o n s ,S i t t e ( 19 7 7) an d Wu t t k e (19 7 7) h av e p ro p o s ed t h a t t h ea p o l a r c o m p o n e n t s a r e b u r i e d i n t h e i n t e r i o r o f t h ep l a s t o g l o b u l e s , a n d a r e c o v e r e d b y p o l a r l i p i d s a n dp r o t e i n s a t t h e s u r f a c e o f t h e g l ob u l e . S u c h a n a r r a n g e -m e n t c o u l d a c c o u n t f o r a m a x i m u m o f e n t r o p i c i n t e r -ac t i o n s (Tan fo rd 1 9 78 ) a s w e l l a s fo r t h e l i m i t ed m i s -c i b i l i t y o f a p o l a r a n d a m p h i p o l a r l i p i d s ( S ma l l 1 9 70 ) .

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