(Picea abies in Aurea

S T U D I A F O R E S T A L I A S U E C I C A

The Homozygous and Heterozygous Effects of an Aurea Mutation on

Plastid Development in Spruce (Picea abies (L.) Karst.)

De homozygota och heterozygota effekterna p8 plastidutvecklingen av en a u r e a-mutation

hos gran ( P i c e a a b i e s (L.) Karst.)

S K O G S H ~ G S K O L A N

ROYAL COLLEGE OF FORESTRY

STOCKHOLM

31' vcceived for publication October 6tl1, 1967 E S S E L T E A 3 . S T H L M 6 7

' i 1 % 2 9

Introduction

Semi-dominant azrrea mutations were described for t h e first time by B irrn ( 1 9 0 7 ) i n .lizfirrhinlrm i n a j u s a n d Pelargonizlm sonalc. .I zrrea forms have later been found i n s e ~ e r a l other plant species. They are characlerised by a yellow-green phenotype a n d after self-fertilisation gi\e a n F, segregation of 1 green ( - \ ~ i l d type) : 2 yellov-green ( a z r r e a ) : 1 lethal yellow or white ( x a n t h a or alhirza).

Jrrrea plants belong to t h a t group of mutants-chloroplast or chloro- phyll mutant\-mhich have a disturbed chloroplast development. By using plants ~ , h i c h a r e heterozygous and homozggous for a n aurea factor, it i \ possible to study t h e effect on plastid morphogenesis of a semi-dominant gene i n Gngle a n d double doses. T h e investigation t o be presented here ( c f . W ~ L L E S , 1966 a ) was made i n a n arzrea type of spruce, t h e genetics of which has been described by LASGNER ( 1 9 5 3 ) . The heterozygous seedlings are yellow-green n h e n they emerge, but can t u r n green with time, i.e. they are of a virescens ( c f . G V ~ T A ~ S S O Y , 1 9 4 0 ) phenotype. T h e new shoots on aurca trees are initially yellow nit11 a jireeni\h tint, and similarly delayed i n becoilling green. Seedlings h o r ~ ~ o z y g o u s for t h e azrrea gene are of a yellon ( x a n t h a ) phenotype.

vo\ 6 T ~ 1 1 S T E I N (1958 ) o b s e r ~ e d t h a t plastids of heterozygous azrrca seedlings in spruce deleloped small grana, consisting of a fern layers of double disc\, and from this fact he a r r i ~ e d at the important con- clusion t h a t pl~otosgnthesis seems possible even by suc11 a simple c h l o r o p l a ~ t structure.

Material and Methods

Offspring from a single open-pollinated ouren tree (Th-2) v e r e used.

Amon,: 162 seedlings iiere found 81 (50 per cent normal green ill- d i ~ i d u a l s , '74 (46 per cent) lleterozygotes ( a u r e a ) a n d 7 ( 4 per cent) yellon homozjgotes.

A preliminary study TI-as rnade of seedlings grown i n sand i n t h e laboratory or in t h e greenhouse. Other plants which were grown in

sand and given nutrient solution were liept i n t h e pliytotron of this college. The seedlings \\ere g i ~ e i i 16 hours of light per day (intensity of i l l ~ ~ n i i i i a t i o n : 40,000 L u x ) or liept in total darkness. T h e temperature n a s 20- C during 16 hours and 15C C during eight hours.

F r o m azuea a n d wild t j p e individuals, samples v e r e taken of t h e rotjledons and first needle? of 10-37 days old seedlings and of needles of 10-18 months old planls. F r o m homozygous zantha plants, samples mere taken of t h e coty1edo:ls of 10-21 days old seedlings. The collected material was ( 1 ) fixed i n 6.3 per cent glutaraldehyde i n 0.1 JI phos- pliate buffer (pH = 7.0) for i i l e hours, left i n buffer o \ e r night a n d postfixed for tmo hours i n buffered 2 per cent OsO, ( i n some cases 0.2 hI sugar n a s added to t h e buffer) or ( 2 ) fixed in t h e glutaraldehyde solution for 24 hours, \\aslied in buffer a n d postfixed for four hours in 3 per cent I W n O , i n mater or ( 3 ) fixed directly i n 5 pcr cent KhlnO, for two hours. I11 all cases the fixation and n a s h i n g v a s performed at 4" C. T h e dark-gromn seedlings mere fixed i n a dim green safe light.

?'lie objects were dehydrated in a n acetone series followed by propylene oxide and embedded in Epon 812 (Lr r l , 1661). T h e osmium-fixed material was stained after sectioning on t h e grids mith 4 per cent uranyl acetate at 40" C a n d then with lead citrale ( R E E A O L U S , 1963).

The sections \\ere cut on a n ultramicrotome constructed at this labora- tory or on a LIiB I'ltrotome 111 a n d collecled on f o r m ~ a r - c o a t e d grids.

They were i n ~ e s t i g a t e d i n a Siemens Elmishop I operated at 80 k\' (Figs. 1-2, 5- 11, 13-14) or in a Hitachi HS-7S electron microscope.

Chlorophy11 delerminatioris \\ere made according to the rnetliod de- scribed preT iously (WALLES, 1963 1 .

Observations

1. Wild type spruce

Chloroplasts of \\ild type spruce are s h o w ~ i i n Figs. 1 a n d 4. They are lens-shaped bodies enclosed by a double envelope. T h e chloroplasts contain a lamellar system embedded i n a matrix, t h e stroma, ~vliich after fixation appears granular aiid of moderate electron density. Fixa- tion nit11 glutaraldehyde, i'ollo\~-ed by OsO,, reveals seleral different inclnsioiis i n Llie stroilla (Fig. 1 ) . l'arious arnounts of so-called plasto- globuli (LICHTESTIHALCIZ aiid SPRCT, 1966) occur regularly. Plaslo- glohuli are usually ~ i s i h l e also after IihInO, fixation ( c f . Fig. 2 ) al-

though they are not me11 preserxed. Sucleoplasm-like regions are seen as electr 111-transparent zones containir~g D S d fibrils ( c f . GVVNIXG, 1963; K I S L I V , S ' C V I ~ I a n d B o ~ o n i u , 1965; R I ~ , 1962; J Y A L L E ~ , 1966 b ) . Numeroas eieclron-dense granules, representing ribosomes, occur all oxer the stroina x h e r e this is free from olher inclusions. Starch graills are often seen both after glutaraldehyde- OsQ, a n d ICUnO, fixation (Fig. 4 ,I.

Spruce cotyledon7 can synthesize chlorophyll and dexelop chloro- plasts, not only i n the light, but also i n t h e d a r k ( v o ~ J Y E ~ r s r r r h , 1958;

LALDI, 1964). T h e chloroplasts i n cotjledons of dark-grown seedlings contain sexeral prolamellar bodies i n addition to a lamellar \ystem with grana. Illuminalion of darb-gronn seedlings canres a progressive disappearance of' prolamellar bodies a n d a stimulation of lamellar

grov t h a n d d ~ l f erentiation.

Figs. 1, 3 a n d 4 s h o n h o v t h e lainellar systenl of chloroplasts from dark-gromn or light-gronn indix iduals consists of layers of thylalioids

( d ~ s c s ) n h i c h i n places are aggregated into grana. Prolamellar bodies (Fig. 2 ) are o h s e n ed in t h e chloroplasts of dark-gron 11 seedlings.

2. The xantha plants

The lethal zantha seedlings h a l e a life span of about four weeks.

They possess pure yellow cotyledons both i n the d a r k a n d i n t h e light.

I n acetone extracts of m u t a n t seedlings no chlorophyll pealis could be o h s e r ~ ed in the spectropliotomeler.

The plastids of xanilla seedlings are of irregular shape a n d their mternal o r g a n i ~ a t i o n reinains at a n early proplastid stage (Figs. 5-7).

The structural material consists of vesicles and a f e v non-aggregating ihylakoids. There is no formation of prolamellar bodies a n d there seem to be no structural differences between the plastids of dark-grovn (Fig. 7) a n d of light-gronn (Figs. 5-6) seedlings. There is a notice- able accumulation of plastoglobuli.

3. The aurea (virescens) plants

The anlounl of chlorophyll i n dark-gronn arzrea seedlings is about -50 per cent of tllal i n corresponding ~ v i l d type seedlings (Table 1 ) . I n both genotypes illumination promotes chloropl~yll synthesis. Light- grown 3-5 weelis old aurea seedlings contain 40 per cent of t h e amount of chlorophyll found i n corresponding n i l d type seedlings. T h e figures given i n Table 1 \\-ere obtained from plant material cultivated i n t h e phytotron.

EG61 1np

Chlorophyll content

1,

1

The figure for dark-grown aurea seedlings is based on 12 samples; in the other cases five samples were analjsed.

Dark Light

Wild type . . . 4a.7 & 0.37 9.1 -1 0.84 Atcrea . . . 2.4 & 0.17 3.6

,

The cl~loroplasts in cotyledons of dark-grov-n arzrca seedlings contain s e ~ e r a l prclamellar bodies ( F i g . 8 ) . T h e lamellar system of these chloroplasts is reduced i n comparison wilh corresponding organelles of the wild type. T h e grana c:)ntain a smaller number of thylaltoids t h a n i n the wild type, often a s few as twu or three. T h e thylalioids may, how- m e r , be aggregated along a considerable part of their length a n d may thus obscure the differentiation between grana a n d intergrana regions i n t h e lamellar system. The chloroplasts of young arzrea s e e d l i q s growing in light ha^ e their lamellar system organised i n the same way as h a l e seedlings grown in the dark, but lack prolanlellar bodies. They usually contain large starch grains. I11 their subsequent ontogeny the chloroplasts of the m u t a n t d e ~ i a t e from Lhe norinal process of plaslid inorphogenesis. In seedlings which are three vieelis old or older the lamellae are mis-shape11 and bent in a n odd way (Figs. 9-11). Many of the grana profiles appear i n section as more or less semicircular or even circular. They are, howe\er, of normal size, in contrast to the aberrant, c u p s h a p e d grana of lethal mutants i n barley (see ~I'ILLES, 1963). T h e structurally ahnornlal chloroplasts are obserred in the anrea needles until they t u r n normally green. Old. dark-green needles v e r e found io h a ~ e normal chloroplasls (Figs. 12, 1 4 ) . A lew exceptional chloroplasts had features reininiscent of t h e sickle-shaped grana (Fig.

13) a n d might represent a transitional stage i n the normalisation process.

Discussion

The majority of t h e mutations affecting chloroplasts a r e recessi:e.

They interfere with t h e hiosynthesis of chloroplast constituents ( c f .

Fig. 1. Chloroplast in colyledon of 27-cia)--old dar1;- grow^ ~ ~ - i l d t y p e spruce. Fixed in glnlaralclel~yde-Os0,.

Fig. 2. Prolamellar b o d y i n cllloroplast of 16-day-old darlc-grovn v i l d t y p c spruce.

F i s c d i n I<i\InO,.

Fig. 3. Section t h r o u g h g r a a u m i n cllloroplast of 18-nlonth-old \viltl t y p e spruce. Fixed in g l u t a r a l d e l ~ y d e - I < A I ~ ~ O ~ ~

Fig. 4. C h l o r o p l a ~ t f r o m t h e sarnc needle a s illustrated i n Fig. 3. F i x e d i n glutaraldehyde -1<1\InO,.

Pig. 5. P l a s t i d in cot)-lcdon of 14-da~--olcl .cantha seedling. 12iscd i n glutaraldc11~-de

oso,.

Fig. 6. Plastid from Lhe s a m e cot?-ledon as illustrated i n fig. 3. Fixed in glutaraltlc- hytle-Os0,.

Fig. 5. Plastid in cotyledon oC 1G-day-old darli-gronm xnillha seedling. Fixed in ~ l u t a r - aldehyde-0~0,.

Fig. 8. Chloroplasi i n ycllo~\--green colylcdon of 24-clay-old darlc-gro\m oiresceils (cc~uecc) scedling. Fixed i n g l ~ ~ t a r a l d c l ~ y d e - O s 0 , .

Fig. 9. (:hloroplasl i n y e l l o w g r e e n cot?-lcdon of :3-71--eel;-old oirescprls seedling. F i x e d in gluLa~.aldch?-dc- OsO,.

Fig. 10. Chloroplast in ycllo\~--green cot!-ledon of -I-wceli-old rlirrscerls seedling. Fixed in I i l I n 0 4 .

Fig. 11. Dctail of chloroplait i n y e l l o w g r e e n colyledon of 3-\veek-old viresceris scetlliny.

Fixed in 1<3InO,.

Fig. 12. Scction t h r o u g h g r a n a in $reen 2 cnl long nrcdlc oi' IS-nionlli-old oi1,esceiis tree.

Ijixi.d i n glutaraldrliyde-lCN110~.

Fig. 13. C i ~ l o r o ~ ~ l a s t froin Lhe s a m e needle as i l l n s h a t c t l i n Fig. 1 2 . F i x e d i n glutaralcle hj-tie-I<llnO,.

l:ig. 14. Chloroplast f r o m Lhe s a m e ncedle as illustrated i n Fig. 1 2 . F i x e d in glularalde- hytle-I(i\InO,.

\ \ T ~ I , L L \ , 19Gi) a n d i n this may block the inorphogenesis or function of the chloroplast. Plantc ~ l h i c h are h e t e r o q g o u s for one of these reces- s i ~ e inutations delelop a normal phenotype, since one mild type allele is apparently sufficient for promoting synthesis of t h e choroplast con- stituent whicli is absent or reduced in ainount i n the m u t a n t homozy- gotc. For seini-doininant chloroplast mutations the biochemical m e c l ~ a n i s m has not j e t been established, but some kind of suppressing or inhibitory action of the mutated gene might be expected.

Some reports p r o ~ i d e information on h o n chloroplasts can be in- fluenced by seini-doininant arlrea mutations. I n soybean (Glycine m a d

\eedlings carrying the gene y,, t h e chlorophyll content, expressed as ,,g chloroplq11 per grain fresh \\eight, v a s found to be 1.51 i n yl1yI1 seedlings, 379 in YI1yll seedlings a n d 738 in Y,,T,, ( n i l d type1 seedlings (\Vor,r, 1963). I n all genotypes t h e amount of chlorophyll increase5 nit11 the age of the plants and the figures cited are t h e highest obtained. According to a n electron microscopical study by S u s (1963 1 , the bright yellow, lethal h o m o ~ y g o t e s (yl1yL1) can produce some t h j l a - Loids and e l en a fev g r a m i n their plastids. In some plastids the thyla- Loids are large a n d extend through nearly t h e whole plastid or are bent and assernbled into a coi~cenlric arrangement. T h e plastids contain also large ~ a c u o l e s a n d groups of plastoglobuli. Heteroaygous aurea plants i n tobacco ( B ~ R K a n d R I m s m , 1964) h a l e been analysed by SCHMID a n d G A ~ F I I ~ A (SCHMII), P R I C T and G ~ F F K O Y , 1966; SCHIIID and G ~ E ~ K O N , 1966, 1967). The plants are reported to h a ~ e a chlorophyll cnritent n h i c h is only one-eighth of t h a t of the wild type. The photo- synthetic capacity ot the a r ~ r e a plants at higher light intensities is superior to t h a t of t h e ~ \ i l d type, a n d photosynthesis i n the m u t a n t be- comes saturated at a considerably higher light intensity t h a n t h a t in green plants, The lamellar system in the arrrea cliloroplasts is much reduced. T h e g r a m consist of only t n o or three layers of thylaltoids, whereas 20-40 layers are found in grana of the mild type. T h e or- gallisation of t h e chloroplasts i n Lhis arrrea inutant seems similar to that of the chloroplasts in a liable uiridis ( c h l o r i n a ) m u t a n t i n drcibid- opsis thaliann (\VLHRMEIER a n d Kousrr.cn, 1965). This recessixe drabidopsis nlutant has a chlorophyll content of about t e n per cent of t h a t of the ~ ~ i l d type (ROEIEIELEN a n d \T'LHR\IEEER, 196.31.

I n the i n ~ e s t i g a t i o n presented here, a n aorea factor n a s found to affect the plaslid d e ~ e l o p m e n t i n two n a y s , u i z . by suppressing chloro- phyll synthesis a n d by interfering with t h e organisation of the thyla- Itoids. The s t ~ i d i e s on dark-gromn seedlings she\\ t h a t t h e decrease in amount of chlorophyll i n t h e mutant seedlings is not caused by bleach-

ing (1)11oto-ouitlatiol1) but must be due t o impeded biosynthesis. In t h e honiozjgous xunthu seedlings, no chlorophyll could be detected; the plastids a r e blocked in d e ~ e l o p m e n t prior t o tlie foriliation of pro- lamellar bodies. In tliis connection it is of interest to note t h a t the recessive chlorophyll-less m u t a n t x a n t h a - 1 0 i n barley can certainly form some discs but no prolamellar bodies i n its plastids ( Y O N \T7r,T1- s IEIK, 1 9 5 8 ; Y O N ISTETTSTTI~ and E n r ~ t s s o s , 1965). T h e kind of aberrant lamellar structure seen i11 tlie cliloropla~ts of yellom-green needles of the arlrea heterozygotes h a s so i a r not been reported for a n y other mutant. The needles are eventually normalised in regard to chlorophyll content and chloroplast structure. Spontaneous greening a n d nnrma- lisation of arrested plastids n as also obserl ed i n a r e c e s s i ~ e ~ r i r e s c e n s niutant of barley ( R I . ~ ~ I A ~ H I A N a n d ZALIR, 1963).

Acknowledgements

The seed malerial for tliis inrestigation \\as kindly supplied by Proiessor 11. L ~ G N E K , Schmalenbecli, Germany. 1 should like to aclinonledge the s ~ i p p o r t gilcn by Professor 3. G r ~ s ~ ~ r s s o s and Pro- fessor D. T O N \ T ' E T T S T ~ I ~ . I all1 indebted to H. K. J r - Y G for c o n t i l i ~ ~ o u s mgenious maintenance a n d improrement of the instrumental equip- ment arid to RIrs B. O\c i ~ s b o \ , hrrs E. h I . i ~ ~ s s o h a n d hIr F. ~ ' . ~ D E R

tor tcclinical assistance.

Financial support has been obtained from "Fonden for sltoglig forsk- ning", tlie United States Public Health S e n i c e (National Institutes of Health, GAI 0 8 8 7 7 ) a n d "Knut och Alice Tf'allenbergs stiftelse".

Summary

A11 electron niicroscopical investigation has been made of plastid d e ~ e l o p n i e n t i n spruce plant\ ~ ~ l i i c h are llornozygous or lieterozygous for a semi-dominant alzrea mutation. T h e cotyledons of normal spruce seedlings sgnthesise c h l o r o p l ~ ~ l l a n d delelop chloroplasts both i n the d a r k and i n t h e light. The chloroplasts i n d a r k - g r o r ~ n seedlings have grnna a n d se\ era1 prolamellar bodies. So chlorophyll n as detected i n

acetone extracts of light-grown or dark-grown seedlings which \yere lmoii~ozygous for the arrrcJa factor. I n these yellow lethal seedlings, plastid morphogenesis is blocked prior to t h e formation of prolamellar b3dies. I n the yellon-green heterozygous azzrea seedlings, c11l )rophyll synthesis is reduced; when grown i n the d a r k they h a l e a cl~lorophyll content t h a t is only 50 per cent of that i n correspondiilg mild type i n d i ~ i d u a l s . T h e chloroplasts of these dark-gromn azrrea seedlings pJssess several prolamellar bodies and a poorly differentiated lainellnr system, i n n h i c h t h e grana usually contain only t w o or three discs.

d urea plants de\ elop i n the light a n abnormal lamellar systein with grana which appear sickle-shaped in cross-section. The yellon-green needles possess the ability of spontaneous greening. The chloroplasls in oldcr green needles of the m u t a n t are normalised a n d indistinguish- able from n i l d type chloroplasts.

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De homozygota och heterozygota effekterna p& plastidutvecklingen av en aurea-mutation hos gran (Picea abiss (L.) Karst.)

E n elelitro~l~l~ilrrosliopislc undersokning h a r utforts over plastidutvccli- lingen 110s granplantor, som iir homozygota eller heterozygota for en semi- dominant a17rea-mutation. Hos normala groddplantor as7 gran bildar lljiirt- bladen klorofyll och kloroplaster siviil i ~ n i j r k e r soin i Ijus. I mijrkerodlade groddplantor inneliRller kloroplasterna prolamellarkroppar sarnt s. k. grana, Grana bestar av skiulika bildningar (thylakoider), staplade pa varanclra.

Klorofyll kunrie intc p8visas spektrofoton~etriskt i acetonextrakt av grodd- plantor, son1 var homozj-gota fijr a~zrea-faktorn, oavsett o m dessa hacle vusit i morlrer eller i ljus. I dessa gula och letala plantor iir plastidutveckli~lgen bloclreracl p & ett tidigt stadium fore bildningen au prolarnellarkroppar. Hos de gulgrona heterozygota aurea-plantorna 51- klorofyllbildni~lgen hiiinnlad;

11% groddplantstadiet Br lilorofyllhalten i inorkerodlade arzrea-plantor endast 50 7; aav klorofj-llhalten i motsvarande vildtypsindivider. Kloroplasterna hos nlorkerodlade anrea-plantor inneh8ller ett flertal prolamellarkroppar och ett fijga utvecklat laniellsystenl nled grana, vilka vanligen best2r av endast tvi-tre thylakoider. I ljuset utveclrlar arrrea-plantorna ett defelrt lamell- system nied grana, sorn i tvlrsnitt ar bBgforrnigt bojda. De gulgrona barren ltan rned tiden bli grona. Kloroplasterna i sjclana grona b a r r a r normalise- rade och lilrnar kloroplasterna hos normal gran.

Electronic version

0 Studia Forestalia Suecica 2002 Edited by J.G.K.Flower-Ellis