of Chloroplast Development in Euglena · Euglena gracilis var. bacillaris Pringsheim was cultured...

STERN ET AL.-LIGHT INTENSITY IN PLASTID DEVELOPMENT

Light intensities of 400 to 700 ft-c inhibit develop-ment of photosynthetic capacity in Euglena.

Literature Cited

1. BEN-SHAUL, Y., J. A. SCHIFF, AND H. T. EPSTE]N.1964. Studies of chloroplast development in Eu-glena. VII. Fine structure of the developing plas-tid. Plant Physiol. 39: 231-40.

2. BERGERON, J. A. 1958. The bacterial chromato-phore. Brookhaven Symp. in Biol. 11: 118-31.

3. BROWN, J. S. 1959-1960. Factors influencing theproportion of the forms of chlorophyll in algae.Carnegie Inst. Wash. Ybk. 59: 330-33.

4. EILAM, Y. AND S. KLEIN. 1962. The effect oflight intensity and sucrose feeding on the finiestructure in chloroplasts and on the chlorophyllcontent of etiolated leaves. J. Cell Biol. 14: 169-82.

5. EPSTEIN, H. T., E. BoY DE LA TOUR, AND J. A.SCHIFF. 1960. Fluorescence studies of chloroplastdevelopment in Euglena. Nature 185: 825-26.

6. EPSTEIN, H. T. AND J. A. SCHIFF. 1961. Studiesof chloroplast development in Euglena. IV. Elec-tron and fluorescence microscopy of the proplastidand its development into a mature chloroplast. J.Protozool. 8: 427-32.

7. ERIKSSON, G., A. KAHN, B. WALLES, AND D. VONWETTSTEIN. 1961. Zur makromolekularen Phy-siologie der Chloroplasten. III. Deut. Bot. Gesell-sch. 74: 221-32.

8. LYMAN, H., H. T. EPSTEIN, AND J. A. SCHIFF. 1961.Studies of chloroplast development in Euglena.I. Inactivation of green colony formation by UVlight. Biochim. Biophys. Acta 50: 301-09.

9. NISHIMURA, M. AND H. HUZISIGE. 1959. Studieson the chlorophyll formation in Euglena graciliswith special reference to the action spectrum of theprocess. J. Biochem. 46: 225-34.

10. RABINOWITCH, E. I. 1945. Photosynthesis and Re-lated Processes. vol. I. Interscience, New York.p. 412-14, 423.

11. SCHIFF, J. A., H. LYMAN, AND H. T. EPSTEIN. 1961.Studies of chloroplast development in Euglena. II.Photoreversal of the UV inhibition of green colonyformation. Biochim. Biophys. Acta 5: 310-18.

12. SMITH, J. H. C. AND V. M. K. YOUNG. 1956.Chlorophyll formation and accumulation in plants.In: Radiation Biology. vol. III. A. Hollaender.McGraw-Hill, New York. p 412-15.

13. STERN, A. I., J. A. SCHIFF, AND H. T. EPSTEIN.1964. Studies of chloroplast development in Eu-glena. V. Pigment biosynthesis, photosynthetic02 evolution and CO2 fixation during chloroplastdevelopment. Plant Physiol. 39: 220-26.

Studies of Chloroplast Development in EuglenaVII. Fine Structure of the Developing Plastid 1, 2

Yehuda Ben-Shaul,3 Jerome A. Schiff, and H. T. EpsteinDepartment of Biology, Brandeis University, Waltham 54, Massachusetts

Dark-grown Euglena contain proplastids 1 ,u indiameter (1, 2) having double membranes and lackingany appreciable inner structure. When such cellsare exposed to the optimal intensity of light, a num-

ber of events occur leading to the formation of fullymature chloroplasts. The fine structure of thesefully mature chloroplasts has been described by Gibbs(4). We have previously inferred from both fluo-rescence and electron microscopy that the developmentof the proplastid into Oie mature chloroplast takesplace by internal forrmation of lamellae and fusioniof proplastids (1, 2). The sequence found by elec-tron microscopy indicated that the appearance oflamellae is linear vith time. It was also shown that

1 Received: July 15, 1963.2 Supported by research granit RG-6344 fromii the Na-

tional Institutes of Health.3 Yehuda Ben-Shaul wishes to acknowledge the granit

to him for travel expenses from the Fullbright Founda-tion.

the development of photosynthetic competence alsoexhibits linear kinetics. This paper describes thedevelopment of the ultrastructure of the chloroplastand provides further substantiation for several as-pects of our previous model (1, 2).

Materials and Methods

Euglena gracilis var. bacillaris Pringsheim wascultured in Hutner's pH 3.5 medium (6) or in restingmediuni as described previously (16). All detailsconcerned with the conditions for chloroplast de-velopment were the same as described previously(16, 17).

Samples were prepared for electron microscopyas follows. Cells were centrifuged lightly and thepellets fixe(d in 1.0% OsO in 0.28 Mr veronal acetatebuffer at pH 7.3 to 7.6 for 6 to 24 hours at 4°. Afterfixation, the cells were washed 2 to 3 times with bufferand were dehydrated by successive 15 minute ex-

231

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PLANT PHYSIOLOGY

posures to 25%, 50%, and 75% (v/v) acetone. Theywere then transferred successively to 96% (v/v)acetone for 30 minutes, 2 changes of 100% acetonefor 30 minutes each followed by 100% acetone towhich about 20% Vestopal (enough to cover thepellet) was added. This stood overnight at roomtemperature to permit evaporation of the acetone.The residue was placed in gelatin capsules which re-ceived more Vestopal until filled. The contents werehardened at 600 for 2 to 3 days before being sectionedby glass or diamond knives on a Porter-Blum micro-tome. The sections were transferred to coated grids

*. 45: B6: '~~~~~~1.

PP

by the usual procedures. In most cases, these gridsthen were stained with 1 to 2% KMnO4 for 10 min-utes according to Lawn's method (9). They werethen examined with an R.C.A. EMU-3C electronmicroscope.

Results and Discussion

Formation of Discs4 and Lamellae during theNormal Sequence of Chloroplast Development at100 ft-c in Nondividing Cells. In the following dis-cussion, dark-grown cells are taken as zero time ofdevelopment and all developmental times are measuredfrom the time at which these cells are placed in thelight.

In Figures 1 and 2 are shown the double-mem-

4 Throughout this paper we follow the nomenclatureof Gibbs (4). The basic internal chloroplast structureis the disc, a membrane 40 to 60 A in thickness. In themature chloroplast, each lamella consists of several discs(usually 4) which are fused together along their entirelength to form the elongated thickened lamella.

'I

3

N.... ..... . .Y. ... . i:,

DNM

V

FIG. 1. Nondividing cell at zero time of development(dark-grown cell), showing detail of proplastid. Abbre-viations used in figures: D, disc; E, endosome; G, golgiapparatus; L, lamella; M, mitochrondioii; MI\I, mem-brane; N, nucleus; P, p)yrenoid; Pmn, paramylum PP',proplastid; V, vacuole. Marker indicates I ,g.

FIG. 2. Nondividing cell at zero time of development(dark-grown cell). (Key to abbreviations in legend offigure 1.).

-------------------- 4Fic. 3. Nonldividing cell, plastic after 2 hours of de-

velopinent at 100 ft -(. (1l'e to albreiatioiis in legendof figure 1).

FIG. 4. Nondividing1 cell, plastid after 2 hours of (de-velopment at 100 ft-c. (Key to abbreviations in legendof figure 1).

- .- l..

232

A..........

,:. ', '.Ij


BEN-SHAUL ET AL.-FINE STRUCTURE OF DEVELOPING PLASTID

braned proplastids about 1 ,u in diameter which arefound in dark-grown cells. No prolamellar bodyas described for higher plants (7, 10, 13, 18) has beenobserved although on rare occasions small clumps ofparticles resmbling ribosomes or other small vesiclesare seen. The thickness of the double membrane isabout 100 to 150 A and the number of proplastidsper section was 2 to 6.

At 2 hours the developing plastids contain at least1 disc (figs 3, 4). One end of the developing disc isfrequently seen to be associated with a clump of ribo-some-like particles (fig 4).

After 4 to 6 hours, the plastids generally contain1 to 3 individual discs and 1 lamella usually con-taining 2 discs (fig 5). The plastids are furtherelongated with practically no change in width.

After 7 to 10 hours, the typical plastid has 2 to 3individual discs and 1 to 2 lamellae each containing2 discs (figs 6, 7). Sometimes the lamellae at thisstage contain 3 discs each, 40 to 60 A in thickness(fig 8). The plastids continue to elongate.

After 12 to 24 hours, there are miiany changes in

the developing plastids (fig 9). The number ofindividual discs has risen to about 6 and the numberof lamellae is 5 to 6. The number of discs per lamellahas increased to about 2 to 4. The lamellae contain-ing 4 fused discs appear as 2 thin outer membranesenclosing an inner membrane double in thickness asdescribed by Gibbs for mature chloroplasts (4). Atthis time, the pyrenoid appears as a region of densematerial containing lamellae. The length of theplastid is now about 2 to 3 times the length that itwas after 7 to 10 hours of development.

From 24 to 96 hours of development, the numberof lamellae increases to about 13, containing 4 to 6discs each, but the number of unfused discs decreases

V

6FIG. 5. Noindividiing cell, plastid after 6 hours of de-

velopliieilt at 100 ft-c. (Key to abbreviationis in legenidof figure 1).

FIG. 6. Nonidividinig cell, plastid after 8 hours of de-velopment at 100 ft-c. (Key to abbreviations in legendof figure 1).

FIG. 7. Nondividing cell, plastid after 10 hours of de-velopmieift at 100 ft-c. (Key to abbreviations in legendof figure 1).

FIG. 8. Nondividing cell, exceptional plastid after 18hours of development at 100 ft-c. (Key to abbreviationsin legend of figure 1).

233

Ak,

S.,

':.I .'.'t


PLANT PHYSIOLOGY

M

G....

FIG. 9. Nondividing cell, plastid after 24 hours of de-velopment at 100 ft-c. (Key to abbreviations in legendof figure 1).

to zero, eventually (figs 10, 11, 12, 13). There islittle change in the length of the plasti(d (luring thisperiod.

Parallel studIies following chloroplast develop-ment on cells kept continuously in the logarithmic

A.

*...X.t.-' %.~~~~~~..vo:

tA

M

FIG. 10. Nondividnig cell, p.astid after 36 bours of dc-

velopmienit at 100 ft-c. (Key to abbreviations in legend

of figure 1).

* ~~~~~~~~11


phase of growtlh showva similar seqluenice of de-velopnmental events to the ones (lescribecl above fornondividling cells. This is in agreement with ourpreviously published observations on chloroplast de-velopment in growing cells (1, 2).

Kinetics of Appearance of Discs and Larnellac(iuring the Normiial Sequence of Chloroplast De-velop-miient at 100 ft-c in, Nondividing Cel-ls. Figure 14summarizes the numlber of discs and lamellae at vari-ous levelopmental times. The number of free (liscsis at a mlaximumii after 12 tso 24 hours and subsequentlyleclines to zero. Lamella formation, however, con-tinues and is essentially linear from 14 hours onwardsuntil 12 to 13 lanmellae are fornmed characteristic ofthe mature chloroplast in this species under theseconditions. The simplest interpretation of these re-sults is that discs are fornmed up to a certain numberafter whiclh their formiiatioin slows (down and thenceases comlpletely. As (liscs are forming, however,they become fused into lamellae, eventually leadlingto thle coixSullIp)tioll of alll thle av.ailable (discs as th.eybecoinie In1corp)orated ilito coni)leted( laiiellae.

Another initer-est;ing inferenice \w1hich CanlI be (dla\nfroimi th1ese (lata concerns the (Irtamiatic in1creaIse inlnumbers of free discs and lanmellate betwveen 10 to 14

234

.............


BEN-SHAUL ET AL.-FINE STRUCTURE OF DEVELOPING PLASTID* : $F b'. F. o: . .^ ...................... S . .. Y .. , . .. s,..R" X.E. .',:ffi:' ...... b ;@o * ...................... :* . fi: ziE : :.:.< X . X ' t e e M.* ^.'.z., a ,;&<< ........... ^ - - F' ..... g'.F w l fr' x ^ ^.X r S | ^ i g i ire is .o*' 2 .. .;g, I | @S.:. .............. .d'g' ..:5: :'< :N { P.N, | :, @ .Za :1';. . .*vP... ' .@. ^' . ag i,. ,g aj, w1 w* . :t :: ,: : , ... ... X.§8:.A.^ . :: . 8. ,: .

12


hours of development. Our previous work (11) sug-gested that there are approximately 30 proplastids ina dark-grown cell which fuse at some time duringdevelopment in groups of 3 to form the approximately10 chloroplasts characteristic of light-grown cells.It is interesting, therefore, that the number of freediscs per plastid roughly triples between 8 and 14hours. If the factor of increase is assumed to be an

integer, it is best considered to be a tripling as canbe seen from the statistical analysis in table II.

In the case of the increase of lamellae between8 and 14 hours, quadrupling gives the best fit. Sincediscs undoubtedly continue to be formed even duringthe fusion process, one would expect the chance oflamella formation to be enhanced leading to a some-

what greater increment during fusion.Size of the Chloroplast during Development.

Figure 14 also shows that there is a tripling of thelength of the chloroplast at the same time as thetripling of the number of discs and this is again sub-stantiated statistically in table II. The width of thechloroplast remains relatively constant during de-velopment except for a statistically significant (tableII) transient change at about 10 hours. These data


for plastid length and numbers of lamellae and discsagain suggest the fusion of 3 developing proplastidsto make 1 chloroplast. This would lead to the con-clusion that fusion occurs linearly, i.e. the 3 pro-plastids become fused in a linear sequence whichincreases the length 3 times without markedly affect-ing the eventual width of the plastid. Occasionally,pictures of developing chloroplasts show a tripartitearrangement within the plastid (fig 15). Whetherthis is the direct result of the fusion process is notknown.

A Model for the Formiiation of Discs and Lamellae.Figure 16 diagrams our interpretation of the forma-tion of discs and lamellae inferred from the electronmicrographs presented above. We start with theproplastid (fig 16A and 1). The discs are invag-inated successively from the inner proplastid mem-brane (ref 2, fig 13). After the first disc is formed(fig 16B), the second disc begins to invaginate (fig16C) and disc formation continues in this manner.As more discs form, some begin to fuse (fig 16D, 7)to form lamellae. We do not know whether the fusionof discs is a random process or whether it proceedsin some orderly manner. This fusion would latercontinue until all the discs which had been formedare fused in groups of 2 to 6 to form completedlamellae.

Development at Low Light Intensity (7 ft-c) inNondividing Cells. Cells which develop at 7 ft-c

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PLANT PHYSIOLOGY

(I)z0L.)i

02

2- ---y-5434+0879x

0.

UNFUSED DISCS/PLASTID

6-

z

051424 36 48 80

TIME IN HOURS

FIG. 14. Kinetics of plastid parameter during plastiddevelopment showing leng-th of plastid, width of plastid,number of lamellae anld number of unfused discs. Eachpoint is a mean of several observations, thle har represent-ing the 95%S confidence interval of the mean (see tableI). The broken line showrs the least squares fit to lamIel-lar data from 14 to 80 hours.

of light contain less chlorophlyll and have lowerphotosynthetic capacities ( 17) . It was of interest,therefore, to study the fine structure of plastids whlichhad developed at this low intensity.

Dark-grown cells, exposed to 7 ft-c of light, showinitially a developmental pattern similar to that de-scribed above for cells at 100 ft-c. After 10 to 15hours, hownever, abnormalities begin to appear. At15 hours (fig 17), the size of the plastid does not ex-ceed 3y compared with 5.5,u found for plastids de-veloping at 100 ft-c. The number of lamellae is lessthan normal (2-5) andl there are few unfused discs.Between 45 and 95 hours, the plastids are definitelyabnormal. Either the lamellar system is incomplete

(fig 18, plastid A) or nmany discs lhaxe not fused toform lamellae and are concentrated in the interior ofthe plastid (fig 18, plastid B ). Occasionally, theplastids seem normal in internal structure but are

Sunltnary

Time*

0 hours24-67-1012-1624364872-95

024-67-10

12-1624364872-95

0 hours24-67-10

12-1624364872-95

N024-67-10

12-1624364872-95

Table Iof Mleasurements of Developing Plastids

Range** N*** x; +SE-4-

Length of plastid (U)0.78-1.210.87-2.081.3-2.21.4-2.653.50-8.504.3-8.55.0-9.45.0-11.13.6-8.8

9 1.04 +- 0.260) 1.57 + 0.329

10-) 1.75±0.17810 2.21 ± 0.34211 5.45 + 0.893S 6.13 ± 1.11

6.85 ± 1.5319) 6.99 +- 1.2910 6.62 -H 1.27

Width of plastid (a)0.56-0.860.63-1.250.54-1.200.84-1.500.50-1.100.5-0.70.55-1.50.54-1.20.30-1.2

99

10)11

1(01 ()

0.74 -+ 0.040.96 -+ 0.180.88 -+ 0.151.35 --+ 0.220.70 -1- 0.120.64 -+ 0.100.82 -+ 0.300.83 -+ 0.160.84 ± 0.22

Number of Lamellae per plastid001-21-23-86-96-117-138-16

9 0-+09 0-HO10 1.3±0.2519 1.4-+-0.3711 5.5 -+ 1.018 7.8 -+ 1.01/ 8.1 ± 1.6

1() 10.2 -+ 1.410 13.3 ± 2.2

unmber of Unfused Discs per .iastid00-21-31-33-74-81-30-20-2

9 0+09J 1.0 +- 1.5

19( 1.5 ± 0.511 () 2.2 -+ 0.6611 .5-4- 0.698 5.9 ± 1.04/ 2.4 + 0.72

10 0.30 ± 0.4810 (0.50 ± 0.61

* Developmental time in hours (dark-grown cells takenat zero time; all times of development represenit timedark-grown cells have been in the light).

** Highest and lowest measuremtent.*** Number of measurements taketn.

t Mean of measurements (x) plus or miiinus the stan-dard error of the mean for 95% confidence level(SE95). SE95 was computed as follows: the stan-dard deviation of the measuremenits (s) was com-puted in the usual way. The standard error of themean (s,,) was computed from sx- = sN/' and wasmultiplied by t95 for the appropriate number of de-grees of freedom to yield SE,5.

236



Table IISumm,ary of Coniiputtation of the Magnitude of Change in Plastid ParamZeters betweent 7 to 10 anid 12 to 16 Hours

Plastid length (,u) Plastid width (,u) Number of Number oflamellae unfused discs

A) Observed(7-10 hrs) 2.21 1.35 1.4 2.2

B) Observed(12-16 hrs) 5.45 0.70 5.5 5.5

C) B/A ± SE95* 2.48 ± 0.523 0.522 ± 0.115 3.93 + 1.17 2.50 + 0.75

* The standard error of the ratio B/A for 95%o confidence was computed using Fieller's theorem.

rounded rather than elongated, and are lacking inpyrenoids. (fig 19).

The conspicuous differences between developmentat 7 ft-c and normal intensities are: the plastidsnever exceed about 3 , in length compared with about5.5 ,u for normal plastids; and disc formation appearsunaffected, but the fusion to form normal lamellaedoes not proceed in most cases. This would suggestthat the process which is prevented at low lightintensities is the fusion of all 3 proplastids to yieldone chloroplast as well as the normal, orderly fusionof discs to make lamellae. Indeed, the 2 fusion proc-esses may be closely interrelated and plastid fusionmight precede and be the initiator of the fusion ofdiscs into lamellae. It is very likely that the plastidsdescribed by Aloriber et al. (12), from cells grownon agar, actually developed at low light intensity,since their pictures resemble ours for chloroplastsdeveloped at 7 ft-c. This is particularly likely sincelight would not penetrate readily to the innermostcells due to self-absorption.

When cells which have undergone chloroplastdevelopment at 7 ft-c are exposed to normal intensities(100 ft-c), pigments and photosynthetic abilities in-

FIG. 15. Nondividing cell, plastid after 24 hours of de-velopment at 100 ft-c showing exceptional tripartite inter-nml structure. (Key to abbreviations in legend of figure1).

a- MbMb

BA

DCFIG. 16. Model for the early developmenit of the Eu-

glena chloroplast. A) Diagram of proplastid showingdouble membrane. B) Invaginiationi of first disc frominner membrane. C) Formation of more discs in thesame manner. D) Fusion of 2 discs to form a lamella.(Key to abbreviations in legend of figure 1).


237


PLANT PHYSIOLOGY

.. L

p

..4.

FIG. 18. Nondividinig cell, plastid after 72 hours of de-velopment at 7 ft-c. (Key to abbreviations in legendof figure 1).

Fi(;. 19. Nondividinig cell, plastid after 95 hours of de-velopmenit at 7 ft-c. (Key to abbreviationis in legendof figure 1).

crease ancl eventually exceed those of cells which havedeveloped entirely at 100 ft-c (17). These hyper-dleveloped cells exhibit approximately double thenumber of lamellae per chloroplast when comparedwith normally developed cells (figs 20, 21). Thelamellae appear to have a normlal composition of discs.

Relation of Strufctutre and Functiont ditring Chioro-plast Developmnent. The normal sequence of de-velopment described above can be correlated with thedata on pigment formation and photosynthetic abilitiesobtainedl under identical conditions (16). Duringthe initial formation of discs, before any lamellae iscompleted, (1-4 hours of development) there is a

small increase in chlorophyll, but carotenoids remainrelatively constant. Net photosynthetic 02 evolu-tion commences at about 4 hours of development butmay be present to some extent prior to this time (15).Thus photosynthetic 02 evolution nmight be mostefficient in completed lamellae but mighlt be associatedto somle extent with free discs.

220

FIG. 20. Nondividing cell, plastid showing hlyper-development after 95 hours at 7 ft-c followed by 95 hoursat 100 ft-c. (Key to abbreviations in legenid of figure 1).

FIG. 21. Nondividing cell, plastid shiowinig hlyper-developmenit after 95 hours at 7 ft-c followed by 160 hoursat 100 ft-c. (Key to abbreviations in legeend of figure 1).

Photosynthetic CO, fixation is first clearly seenat 6 hours of development when there is at least 1complete(d lamella per plastid. The rate of photo-synthetic °2 evolution, CO2 fixation, and pigmentfornmation increases dramatically at about 10 hours ofdevelopment and thereafter the increase in these para-meters is linear with time until they achieve constantvalues after about 72 hours of development. Fromthe developmental micrographs presented above, thisdramatic inception of linear kinetics in photosyntheticparameters correlates well with the linear rates oflamellar comlpletion following the jumlp at 12 to 16hours.

The reduced pigment contents anid photosyntheticabilities of cells developing at 7 ft-c of light (17)correlate wvith the abnormal structure of the plastidsunder these conditions.

The hyperdevelopment in all photosynthetic para-meters (17) which occurs when cells which haveundergone clhloroplast developnment at 7 ft-c of lightare subsequently exposed to normal intensities (100ft-c) can be correlated with the electron micrographsshown in figures 20 and 21. As noted above, thesehyperdeveloped chloroplasts appear normal in basic

238

.........



structure but have almost double the number oflamellae when compared with normal chloroplasts.

Comitparison of Chloroplast Development in Eu-glena, Other Algae, and Higher Plants. Many algaeare able to form chloroplasts in the dark and, there-fore, considerations of chloroplast development donot apply. In a few cases, such as Ochromonas (5)and a mutant of Chlamydemonas (8, 17) attrition ofthe chloroplast occurs in the dark. The details forOchromonas suggest that the developmental processis different from that in Euglena.

In higher plants (7, 10, 13, 18) however, the usualsituation is the appearance of tubules or vesicles ap-parently invaginating from the inner membrane ofthe proplastid on exposure to light. These vesiclesarrange themselves in layers and fuse to form discs.These discs eventually organize into grana andstroma lamellae.

It would appear that Euglena shows a very distinc-tive type of development unlike these other systems,since discs appear to form directly from the innerplastid membrane and grow steadily without neces-sitating the fusion of tubules. In addition, discfusion and perhaps plastid fusion appear to play im-portant parts. Finally, the sequential nature ofplastid development in Euglena permits a quantita-tion which has not been possible in other systenms.

SummaryRepresentative electron micrographs of Euglena

cells undergoing chloroplast development are pre-sented. Dark-grown cells contain proplastids 1 u indiameter which have double membranes but littleinternal structure. The prolamellar body reportedfor plastids of higher plants appears to be absent.When nondividing dark-grown cells are exposed tooptimal light intensities (100 ft-c), chloroplast de-velopment begins. During the first 10 hours, num-erous discs are produced by invagination of the innerproplastid membrane. After a few discs are formed,some of them fuse into lamellae and this fusion be-comes a continuing feature of development. A modelfor this process is presented. At about 10 hours ofdevelopment, the number of discs and the length ofthe plastid triples while the width of the plastid, asidefrom a small transient change at about this time, re-mains essentially constant throughout development.These observations give further support to our pre-viously presented model of proplastids fusing bythrees to form the basis of the mature chloroplast.The pyrenoid becomes evident in the plastid afterabout 18 to 20 hours of development. After about 14hours of development, the formation of lamellae islinear with time but disc formation begins to de-crease in rate and eventually ceases. By 72 hours,development is complete and the plastids containabout 13 lamellae and no unfused discs.

Observations of chloroplast development in non-dividing cells of Euglena at 7 ft-c reveal a patternsimilar to that found at 100 ft-c up to approximately10 hours of development. After this time, severalabnormalities appear. The plastids never triple in

length as do their normal counterparts. Discs appearto be formed at a normal rate but often fail to fuse intolamellae or else are observed to form lamellae of ab-normal shape and orientation. This suggests thatlow light intensities may not allow the fusion ofproplastids inferred above and also interfere withthe normal fusion of discs to form lamellae. Nopyrenoids have been observed in plastids developingat 7 ft-c.

The normal developmental sequence at 100 ft-ccorrelates well with physiological parameters suchas chlorophyll and carotenoid formation, photosynthe-tic 02 evolution and CO2 fixation reported previously.The inception of photosynthetic 02 evolution and CO2fixation occur at approximately the same time as theformation of the first lamella (4-5 hours). Allparameters become linear between 14 and 72 hoursof development as does lamella formation. The ab-normal structure of the plastid at 7 ft-c also correlateswith the impaired photosynthetic abilities observedfor these cells previously. Hyperdevelopment, whencells which have developed at 7 ft-c are exposed to100 ft-c, is also reflected in changes in chloroplaststructure.

Chloroplast development in Euglena appears todiffer from that described for higher plants sincediscs, rather than vesicles, are invaginated from theinner proplastid membrane. Proplastid fusion seemsto have no counterpart in higher plants. The se-

quential nature of development found here appearsto be unique to Euglena and permits a quantitation ofphysiological and developmental events not possiblebefore.

AcknowledgmentsThe expert technical assistance of 'Miss Nancy

O'Donoghue is gratefully acknowledged.

Literature Cited1. EPSTEIN, H. T., E. BoY DE LA TOUR, J. A. SCHIFF.

1960. Fluorescence studies of chloroplast develop-ment in Euglena. Nature 185: 825-26.

2. EPSTEIN, H. T. AND J. A. SCHIFF. 1961. Studiesof chloroplast development in Euglena. IV. Elec-tron and fluorescence microscopy of the plastid andits development into a mature chloroplast. J. Pro-tozool. 8: 427-32.

3. ERIKSSON, G., A. KAHN, B. WALLES, AND D. V'ONWETTSTEIN. 1961. Zur makro:nolekularen phy-siology der chloroplasten. III. Deut. Bot. Ge-sellsch 74: 221-32.

4. GIBBS, SARAH P. 1960. The finie structure of Ell-glenza gracilis with special reference to the chloro-plasts and pyrenoids. J. Ultrastruct. Res. 4: 127-48.

5. GIBBS, SARAH P. 1962. Chloroplast developmentin Ochromontas dantica. J. Cell Biol. 15(2): 343-61.

6. GREENBLArr, C. L. AND J. A. SCHIFF. 1959. Apheophytin-like pigment in dark-adapted Euiglenagracilis. J. Protozool. 6: 23-28.

7. HODGE, A. J., J. D. McLEAN, AND F. V. MERCER.1955. Ultrastructure of the lamellae and grana inthe chloroplasts of Zea mays. J. Biophys. Biochem.Cytol. 1: 605-13.

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8. HUDOK, G. 1963. Arginine biosynithesis and en-

zyme repression in chlamlydomnonas reinhardii.Thesis, Harvard University.

9. LAWN, A. MI. 1960. The use of potassium perman-

ganate as ani electron-dense stain for sections oftissue embedded in epoxy resin. J. Biophysic.Biochem. Cytol. 7: 197-98.

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Water Uptake and Heat Evolution by Germinating Cotton SeedJ. Dewez

Laboratory of Tropical Agronomy, University of Louvain, Louvain, Belgium

If the rate at whiicl water is taken up during theearly stages of germination has been studied for thesee(ls of many plants, the information on the cottonseedl is not very abundlant in that respect. On theother hand, no parallel studies on the kinetics of

water uptake and heat evolution have been made.The purpose of this investigation was to gather in-formation about the kiinetics of both processes duringthe first day of gerimination of the cotton seedl.

Materials and Methods

Cotton seeds (Gossypium hirsufituw, L. var. 1021-849) from the 1961 harvest at the Lubarika Agricul-tural Station of the Ruzizi Valley (Congo) were used.They were (lelilnted by treatmiient with concentratedsulfuric acid for about 5 minutes followed by rinsingwith water. During rinsing, the floating seecls were

eliminated. Atter wiping off excess moisture, they

were storedl at 250 and 40% relative humidity.Water uptake was miia(le to occur in a cylindricalglass vessel with a frittedI glass bottonm plate throughwhich a streami of water-saturated air was passed.

This vessel -vith a ,hallow layer of (listille(d water

1 Received July 15, 1963.

as kept in a w-ater thermiiostat, the temiiperatutre ofwhich wsas kept constant wsithin 0.10 from 200 to 47.50.Water uptake wsras imleasure(d on a 10 seed lot, afterwiping wvith blotting paper, by weighing after a given

perio(l of tinme. The et weiglht (letermiiinationi wrasfollowevd by oven drying at 1050.

Heat evolutionl was measure(l in a Calvet Micro-calorimeter (2, 3) on groups of 5 seed (about 0.4 gdry matter) to which 0.5 nml wvater was added. Theobservation of the heat evolution was made duringperiods of timle ranging froml 3 to 24 hours and( thewater uptake was determined at the completion ofeach experiment. The initial moisture content ofthe seeds which had been kept un(ler the conditionsindicated above namiiely 250 and 40% relative humlid-ity varied froml 7 to 10%/c aln(d their viability was

about 90%'c.Results

\When the water uptake during the first 12 hours

is plotted against time, fairly smooth sigmoid curves

are obtained (fig 1). It should be observed that thevariability of the results is fairly low even thouglheach experimental point corresponds to a different10 see(l lot. The sigmoicl aspect of the curves sug-

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