MACROMOLECULAR PHYSIOLOGY OF PLASTIDS · dark-grown seedlings (Sisle &r Klein, 1963; Akoyunoglo &u Argyroudi-Akoyunoglou, 1969), the rate of chlorophyll synthesis on temperature and

jl. Cell Sci. i s , 31-55 (1974) 31Printed in Great Britain

MACROMOLECULAR PHYSIOLOGY

OF PLASTIDS

IX. DEVELOPMENT OF PLASTID MEMBRANES DURINGGREENING OF DARK-GROWN BARLEY SEEDLINGS

K. W. HENNINGSEN AND J. E. BOYNTON*Institute of Genetics, University of Copenhagen, (f>ster Farimagsgade 2.A,'353 Copenhagen, Denmark

SUMMARY

The development of plastid membranes was studied in relation to chlorophyll accumulationin dark-grown barley seedlings of various ages after transfer to light. Quantitative electronmicroscopy showed that the prolamellar body membranes are reorganized into primarylamellar layers which contain sufficient membranes to support grana formation during 24 hof greening. Structural reorganization of the plastid membranes is completed rapidly in youngseedlings, but is slow in older seedlings. Chlorophyll accumulates rapidly in young leaves aftera short lag. In older leaves there is a longer lag phase before the onset of chlorophyll synthesis,and the final rate of synthesis is lower.

Shortly after transferring to light, the crystalline prolamellar bodies in the etioplasts aretransformed and then dispersed into lamellar layers with numerous perforations and pro-tuberances. Before the phase of rapid chlorophyll synthesis, many small-diameter 2-disk granaare formed. When chlorophyll begins to accumulate, the perforations are rapidly eliminatedfrom the lamellar layers and a maximum number of 2-disk grana are formed. As greeningproceeds additional disks are added to these original 2-disk grana.

During the phase of rapid chlorophyll synthesis, pairing of the lamellae is positively corre-lated with the accumulation of chlorophyll. During greening less chlorophyll appears to beincorporated into the paired regions of the lamellae in young leaves as compared to old leaves.The results on the structural aspects of plastid development are discussed in relation to theformation of photosynthetic capacity.

INTRODUCTION

The formation of prolamellar bodies during the development of etioplasts in dark-grown seedlings of angiosperms has been studied in some detail (von Wettstein, 1958;Miihlethaler & Frey-Wyssling, 1959; von Wettstein & Kahn, i960; Gunning & Jagoe,1967; Henningsen & Boynton, 1969; Weier & Brown, 1970; Klein & Schiff, 1972;cf. also Kirk & Tilney-Bassett, 1967). The accumulation of protochlorophyll(ide) indark-grown leaves and the capacity for its resynthesis depends on the seedling age(Virgin, 1955; Akoyunoglou & Siegelman, 1968; Henningsen & Boynton, 1969). Theformation of prolamellar bodies is dependent on the synthesis of protochlorophyll(ide)(Boynton & Henningsen, 1967; von Wettstein, Henningsen, Boynton, Kannangara &Nielsen, 1971; Henningsen, Boynton, von Wettstein & Boardman, 1973).

* Present address: Department of Botany, Duke University, Durham, North Carolina 27706,U.S.A.

32 K. W. Henningsen and J. E. Boynton

Photoconversion of protochlorophyllide in dark-grown leaves (absorption maximum650 nm in vivo) initiates the loss of the crystalline configuration of the tubules in theprolamellar bodies in the etioplasts (von Wettstein & Kahn, i960; Virgin, Kahn &von Wettstein, 1963; Klein, Bryan & Bogorad, 1964; Kahn, 1968; Henningsen &Boynton, 1969; Klein & Schiff, 1972). The subsequent dispersal of the prolamellarbodies into primary lamellar layers is correlated with the spectral shift of the absorp-tion maximum of chlorophyllide from 684 to 672 nm in vivo (Butler & Briggs, 1966;Henningsen in von Wettstein, 1967; Henningsen & Boynton, 1969; Henningsen,1970). Both processes depend on seedling age and temperature. The esterification ofchlorophyllide to chlorophyll a accompanies or follows the spectral shift from 684 to672 nm (Sironval, Michel-Wolwertz & Madsen, 1965; Boardman, 1967; Akoyunoglou& Michalopoulos, 1971; K. W. Henningsen & S. W. Thorne, in preparation).

Light energy over a prolonged period is required for the formation of grana andfurther accumulation of chlorophyll (Eriksson, Kahn, Walles & von Wettstein, 1961).Continuous illumination of dark-grown seedlings with moderate to high intensity ofwhite light results in typical chloroplasts with a system of lamellae (i.e. thylakoids)differentiated into grana comprised of stacked or paired regions of the lamellae (i.e.disks), and unpaired stroma lamellae connecting two or several grana (cf. Kirk &Tilney-Bassett, 1967). Crystalline prolamellar bodies are formed in addition to granaand stroma lamellae in plastids of dark-grown seedlings greening in low intensity ofwhite light (Miihlethaler & Frey-Wyssling, 1959; Eilam & Klein, 1962; Wrischer,1966; Henningsen & Boynton, 1970) or in red light (Boardman, Anderson, Kahn,Thorne & Treffry, 1971). Irradiation of dark-grown bean leaves with 200-300 lightflashes separated by dark periods of 15 min (Sironval, Michel, Bronchart & Englert-Dujardin, 1969), or prolonged irradiation with far-red light (De Greef, Butler &Roth, 1971) results in long profiles of largely unpaired lamellae arranged in loosestacks.

The initial photoconversion of protochlorophyllide to chlorophyllide is oftenfollowed by a lag period before additional chlorophyll is accumulated (Virgin, 1955;Wolff & Price, 1957; Virgin, i960). The duration of the lag depends on the age of thedark-grown seedlings (Sisler & Klein, 1963; Akoyunoglou & Argyroudi-Akoyunoglou,1969), the rate of chlorophyll synthesis on temperature and light intensity (Smith &Koski, 1948; Virgin, 1955). Shortly after the onset of illumination a small amount ofchlorophyll b is detectable (Shlyk, Rudoi & Vezitskii, 1970; Thorne & Boardman,1971; Henningsen & Boardman, 1973).

During greening of dark-grown mutant cells of Chlamydomonas (Ohad, Siekevitz& Palade, 1967; Goldberg & Ohad, 1970) and Chlorella (Bryan, Zadylak & Ehret,1967), the formation of plastid lamellae parallels the increase in chlorophyll. Thedevelopment of photosynthetic activity of the cells correlates with the chlorophyllcontent, although pairing of the lamellae to form grana was delayed. Studies on thechanges in the content of plastid specific proteins and lipids suggest that functionalphotosynthetic membranes in green algae are assembled from single components in amulti-step process (Petrocellis, Siekevitz & Palade, 1970; Eytan & Ohad, 1970, 1972;Hoober, 1970; Bar-Nun, Wallach & Ohad, 1972).

Macromolecular physiology of plastids. IX 33

During greening of dark-grown angiosperm seedlings the photosynthetic capacitydevelops through a sequence of steps. Photosystem I activity can be detected earlierthan photosystem II, which precedes or coincides with the appearance of Hill activity(Gyldenholm & Whatley, 1968; Boardman et al. 1970; Thorne & Boardman, 1971;Oelze-Karow & Butler, 1971; Henningsen & Boardman, 1973). It appears that thephotosynthetic units are assembled at an early stage of greening, whereas accumulationof further chlorophyll molecules at later stages of greening serve to increase the sizeof the units (Henningsen & Boardman, 1973). It appears that during the developmentof chloroplasts in higher plants, the lamellae are completed by stepwise addition ofsingle components.

In order to obtain a better understanding of the relationship between the structuraland biochemical aspects of plastid development during greening, we have examinedthe effects of seedling age and light intensity on the time courses of chlorophyllaccumulation, of reorganization of the prolamellar bodies into lamellae, and of theformation of grana. The dark-grown seedlings of various ages provided leaf tissueswith different capacities for chlorophyll synthesis and grana formation. This facilitatedthe distinction of steps in the development of plastid structures in relation to chloro-phyll accumulation.

MATERIALS AND METHODS

Plant material. Seedlings of barley (Hordeum vulgare L., cultivar Svalofs Bonus) were grownin darkness at 23 ± 2 °C and 70-80 % relative humidity. The seeds were planted in Vermiculiteand the developing seedlings watered with tap water. The seedlings were transferred to lighton the 5th, 7th, 9th and n t h day after planting. The illumination was either with 3200 or20 lux of white light from a bank of fluorescent tubes (Philips, 1 daylight + 2 warm white), atan air temperature of 25 ± 2 °C and a relative humidity of 60-70 %. For each treatment thesame 10 seedlings were sampled for electron microscopy and analysed for pigment content byin vivo spectrophotometry. The apical 10-mm segments of the primary leaves from 7-, 9- and11-day-old seedlings were discarded, and the adjacent 15-mm segments used for the investi-gation. From 5-day-old seedlings the coleoptiles were removed, the apical 5-mm segments ofthe primary leaves discarded, and the adjacent 15-mm segments studied.

Electron microscopy. A short piece from each of the 10 leaf segments was fixed in 4-2 %glutaraldehyde in 0-07 M phosphate buffer, pH 7-2. The leaf tissue was dissected in dim light,but kept dark during the 2-h fixation at o °C. All subsequent treatments were carried out innormal room light. After 4 changes of phosphate buffer (C07 M, pH 7'2) at 4 °C overnight, a2-h postfixation with 2 % OsO4 in 0-07 M phosphate buffer, pH 7-2, was carried out at roomtemperature. Dehydration in a graded ethanol series preceded embedding in epoxy resin(Spurr, 1969). Thin sections were cut with a diamond knife on a Cambridge ultramicrotome.The sections were mounted, unsupported, on grids and contrasted with uranyl acetate and leadcitrate. Micrographs of non-serial sections were obtained with a Siemens Elmiskop I or aZeiss EM 9 A electron microscope. Quantitative measurements of various parameters from theplastid sections were carried out on photoenlargements ( x 3) of micrographs taken at x 10 000.Measurements of areas were carried out with a planimeter (Haff, No. 315) and the length ofdistinct membrane profiles was determined with a precision map distance reader (Minerva).From every sample 2 or more leaf pieces were used to obtain measurements from 30 to 40plastid sections.

Pigment determinations. In vivo absorption spectra were obtained with a Zeiss RPQ-20Arecording spectrophotometer equipped with an integrating sphere attachment modified toallow correction for unspecific absorption. The correction was carried out at 750 nm. The10 leaf pieces were arranged parallel in a single layer between the glass windows of a metalframe which could be attached to the integrating sphere.

3 CEI 15


The chlorophyll content in greening seedlings of the different age groups was determined inextracts of leaf pieces from two or more separately grown batches of seedlings, which otherwisereceived the same treatments as the leaves used for electron microscopy. One-gramme of leafpieces from the 10-25 rnm band or in the case of 5-day-old seedlings from the 5-20 mm bandwas extracted with 80% acetone. Total chlorophyll (a + b) was determined by the method ofBruinsma (1961). For each sample of leaves used for chlorophyll extraction, the in vivo absorp-tion spectrum was recorded from two or three sets of 10 leaf segments.

The chlorophyll concentration in the leaf segments used for electron microscopy wasestimated from the in vivo absorbance.

RESULTS

Characteristics of the plant material. During development of barley seedlings in dark-ness the fresh weight of the shoots and the length of the primary leaves increased untilthe 9th day after planting, whereafter no significant increase in these two parameterswas observable (Henningsen & Boynton, 1969). The elongation of the primary leaveswas slowed down or ceased when the seedlings were transferred to light prior to the

Table 1. Growth parameters, ± s.E. of means, for barley seedlings developed in darkness(23 ± 2 °C) for 5, 7, 9 and 11 days and illuminated for 24 h with 3200 Ix of white light(25±2°C)

Seedlingage,days

579

1 1

Fresh weightof shoot,

mg

89 ±3n 6 ± 5170 + 5I 8 I ± 8

Length ofprimary leaf,

mm

88 + 2152 ± 1235 ±5247 ±6

Length ofsecondary leaf,

mm

2I±3

67 + 6193 + 7233 ±8

Length ofcoleoptile,

mm

59 ± 160 + 264 ± 167 ± 1

9th day. The growth characteristics of the seedlings used for the electron-microscopicanalysis of plastid development are shown in Table 1. The primary leaf of dark-grownbarley seedlings was tightly rolled up to the 7th day, and then the leaf started tounroll. Unrolling of the leaf took place after transfer to light. For the 9- and 11-day-oldleaves, unrolling was completed after about 6 h in the light (3200 lx), but took from8 to 16 h in leaves 7 and 5 days of age, respectively. With 7-day-old seedlings illumi-nated at 20 lx the leaves were not completely unrolled until after 12 h in the light.

The protochlorophyllide content of the primary leaves reached its maximum onthe 7th day after planting and then declined rapidly (Henningsen & Boynton, 1969).The distribution of protochlorophyllide and chlorophyll over the length of the primaryleaf was analysed for 7-day-old seedlings (Fig. 1). The apical 45 mm of the leaf con-tained about 80 % of the protochlorophyllide present in 7-day-old dark-grown leaves.After illumination for 4 h the highest amount of chlorophyll was accumulated in the15-45 m m leaf segment. After 24 h illumination the chlorophyll content was maximalin the 15-60 mm leaf segment and appeared to be evenly distributed in this regionof the leaf. Thus, the apical leaf segment of the 7-day-old seedlings greens moreslowly than the proximal 15-60 mm leaf segment, indicating that the cells in the apical


leaf segment are physiologically the oldest. From visual inspection of the seedlingsduring greening, the tips of the leaves of 9- and 11-day-old seedlings were also green-ing more slowly than the rest of the leaf, whereas the leaves of 5-6-day-old seedlingsappeared to green uniformly over the whole length of the leaf.

0-2

30 60 90

-o 06<

0-4

0-2

0 I—

1-4

1-2

1 0|

!>8 £

0-6 8

0-4

0-2

30 60 90 0 30

Distance from tip of leaf, mm

. . 1 .60 90

Fig. i. Distribution of protochlorophyllide and chlorophyll in the primary leaves of7-day-old barley seedlings. The in vivo absorbance at the red absorption maximum isused as a measure of the pigments in consecutive 15-mm segments of the leaves.A, in darkness; B, 4 h, 3200 lx; c, 24 h, 3200 lx.

To allow comparison with our previous studies of plastid development in dark-grown seedlings (Henningsen & Boynton, 1969, 1970), we chose to analyse the10-25 m m leaf segment of 7-, 9- and n-day-old seedlings and the 5-20 mm leafsegment of 5-day-old seedlings.

Chlorophyll formation. Photoconversion of protochlorophyllide in dark-grown barleyseedlings was completed after less than 5 min illumination with white light at either20 or 3200 lx. The spectral shift of the in vivo absorption maximum from 684 to672 nm was completed in less than 10 min in 5- and 7-day-old leaves, but took20-40 min with leaves of age 9 and 11 days. The shift of the maximum from 672 to678 nm also depended on seedling age. In 5- and 7-day-old seedlings this red shiftstarted after 2 h in the light and was completed at 4-6 h. With 9-11-day-old seedlings,a 16-24 h illumination was required before the absorption maximum reached 678 nm.The red shift to 678 nm also depended on the light intensity. In 7-day-old seedlingsthe absorption maximum reached 678 nm after 4-6 h at 3200 k, but 8-16 h wererequired at 20 k. In summary, a considerable amount of chlorophyll (150-200/^per gram fresh weight) was present in the plastids, before the in vivo absorption at678 nm dominated.

The accumulation of chlorophyll in the primary leaves of 5- and 7-day-old seedlingshad an initial lag of 30 min. Chlorophyll accumulated slowly during 30 min to 2 h inthe light (Fig. 2), whereupon the rapid phase of chlorophyll synthesis began. In

3-2

36 K.W. Henningsen andj. E. Boynton

7-day-old seedlings the rate of chlorophyll accumulation was reduced after 12 hwhereas in the 5-day-old seedlings the high rate of chlorophyll synthesis continuedthroughout the 24-h period studied. As is also apparent from Fig. 2, the accumulatienof chlorophyll in 7-day-old seedlings kept at 20 lx is slower than in those given3200 lx. With seedlings of age 9 and 11 days, the lags were 1 and 4-6 h, respectively.Chlorophyll accumulated thereafter at lower rates than in the young seedlings.Increasing age of the dark-grown seedlings results in a longer lag phase and a decreasein the rate of chlorophyll synthesis.

10 12 14 16 18 20 22 24Illumination, h

Fig. 2. Accumulation of chlorophyll (a + b, /*g/g fresh weight) upon illumination ofdark-grown barley seedlings of various ages. A> 5 days, 3200 lx; # , 7 days, 3200 lx;O, 7 days, 20 lx; A, 9 days, 3200 lx; • , 11 days, 3200 lx.

The amount of chlorophyll in the leaf segments used for the electron-microscopicanalysis of the plastid membranes was judged by measuring the in vivo absorbance(Fig. 3). The chlorophyll accumulation curves obtained revealed similar trends tothose obtained when the chlorophyll was extracted (Fig. 2). Exceptions noted werethat the 9- and n-day-old seedlings had longer lag periods and were greening moreslowly, suggesting that the material used for the actual electron microscopy werephysiologically older than the average 9- and 11-day-old seedlings. Thus, the materialused for the ultrastructural analysis accentuates the differences between the 4 agegroups, an advantage for the present investigation.

Plastid size and shape. The etioplasts in the primary leaf of barley seedlings develop-ing in darkness increase in volume up to the 7th day, and then the volume decreasesagain (Henningsen & Boynton, 1969; and Table 2). The average area of the plastidsections in the primary leaves was determined during greening and the significantchanges found have been presented in Table 2. Changes in the area of plastid sectionsrepresent changes in volume as well as in shape of the plastids. Thus, 5- and 7-day-old


0 2 4 6 8 10 12 14 16 18 20 22 24

Illumination, h

Fig. 3. Changes in the in vivo absorbance at the chlorophyll absorption maximum uponillumination (3200 lx) of dark-grown barley seedlings of various ages. Results for7-day-old seedlings illuminated with 20 lx are also shown (CO- The data were takenon the leaf segments which were used for electron-microscopical analysis. The averageof the measurements from 14 separate plantings of 7-day-old seedlings illuminated for4 and 24 h, respectively, are indicated with X. A, 5 days old; # , 7 days; A, 9 days;D, 11 days.

Table 2. Changes in the size of plastids in primary leaves of barley seedlings grown indarkness and then illuminated with white light

Age of seedlings,days

5

7

9

1 1

Condition

darkness0-5-12 h16-24 hdarkness0-5-24 h0-5 h1-24 h

darkness0-5 h1-24 hdarkness0-5-24 h

3200 lx3200 lx

3200 lx20 lx20 lx

3200 lx3200 lx

3200 lx

Average area of plastidsections, /tm2 ± s.E.

4-2 ±0-24-6 + 0-28-2 ±0-36-510-35-8±o-36-8 + 0-87-0 + 0-34-5 + 0-24-4 + 0-46-7 ±0-43-1 ± 0 1

45 ±0-5


etioplasts were elongated ellipsoids, whereas 11-day-old etioplasts are almost spherical(cf. Fig. 13). During greening of 5-day-old seedlings the plastid volume remainedconstant up to 12 h in the light, and then doubled between 12 and 16 h. No changesin plastid size were observed during greening of 7-day-old leaves at 3200 lx; however,at 20 lx a significant increase of plastid size was observed. In 9-day-old leaves illumi-nated at 3200 lx, the average plastid section area increased between 30 min and 1 h,and then it remained constant. The increase appeared to represent a change from aspherical to an ellipsoidal shape (cf. Fig. 14). This change in shape was also observedfor plastids of the n-day-old leaves (cf. Figs. 13 and 18).

0-3

2 02

•a. 0-1

"E

-o 0« 0-2

0-1

0 4 8 12 16 20 24 26

Illumination, h

>----/!]

0 4 8 12 16 20 24

Illumination, h

Fig. 4. Dispersal of the prolamellar bodies upon illumination of dark-grown barleyseedlings of various ages. The percentage of plastid sections with prolamellar bodiesand the average area of prolamellar bodies per area plastid section are plotted againsttime of illumination. A, 5 days old, 3200 lx; # , 7 days, 3200 lx; O, 7 days, 20 lx,transferred to 3200 lx at arrow; A, 9 days, 3200 lx; • , 11 days, 3200 lx.

Reorganization of prolamellar body membranes. In leaves of all ages, structural trans-formation of the prolamellar bodies (cf. Fig. 13) was completed in less than 30 minafter transfer to light. With respect to the older leaves (9 and 11 days), tube trans-formation was completed faster in continuous illumination at 3200 lx, than in darknessafter a brief illumination at a high light intensity (Henningsen & Boynton, 1969).Dispersal of the prolamellar bodies was measured by the number of plastid sectionscontaining a prolamellar body, and by the area of the prolamellar bodies per areaplastid section (Fig. 4). In 7-day-old leaves illuminated for 30 min at 3200 lx (cf.Fig. 12), the number of plastid sections with a prolamellar body was reduced to athird of the dark value. In leaves of age 5, 9 (Fig. 14) and 11 days (Fig. 13), a significantdecrease in the number of prolamellar bodies was not detected until after 1 h of


illumination. The size of the prolamellar bodies remaining at 1 h was drasticallyreduced. In leaves illuminated at 3200 lx the remaining prolamellar bodies were slowlydispersed, particularly in the 11-day-old leaves where prolamellar bodies weredetected for up to 12 h in the light. In general, the dispersal of the prolamellar bodieswas slower in leaves continuously illuminated at 3200 lx than in darkness following abrief illumination at high light intensity. In the 7-day-old leaves with a maximumcontent of protochlorophyllide, dispersal of the prolamellar bodies is faster than inleaves of the other age groups. The shift of the chlorophyllide absorption maximumin vivo from 684 to 672 nm precedes or accompanies the dispersal of the major portionsof the prolamellar bodies.

80

o6

10

8

6

4

2

n

-

-

-I A£0 \

~ / ' \A,' 0

-

i i i

/

-

\ t 1 1 1 1 1 1 1 f

0 4 8 12 16 20Illumination, h

4 8 12 16 20 24 26

Illumination, h

Fig. 5. Changes in total profile length of lamellae (thylakoids) upon illumination of5- to 11-day-old dark-grown barley seedlings. The average length of total lamellarprofiles per plastid section and per plastid section area are plotted against time ofillumination. A, 5 days old, 3200 lx; %, 7 days, 3200 lx; O, 7 days, 20 lx, transferredto 3200 lx at arrow; A, 9 days, 3200 lx; • , 11 days, 3200 lx.

Crystalline prolamellar bodies were reformed in the plastids of 7-day-old leavesilluminated at 20 lx (Figs. 4, 20). Under this condition the dispersal of the prolamellarbodies appeared inhibited in comparison to leaves illuminated at 3200 lx. The refor-mation of crystalline prolamellar bodies and the reaccumulation of protochlorophyllidein leaves of barley seedlings illuminated at 20 lx have been described in detail else-where (Henningsen & Boynton, 1970). Small crystalline prolamellar bodies wereoccasionally observed in 5-day-old leaves illuminated for 6-12 h with 3200 lx. It isconceivable that portions of the tightly rolled leaves received a lower intensity of lightand this might lead to accumulation of some protochlorophyllide during the rapidphase of chlorophyll synthesis.

Dispersal of the prolamellar bodies resulted in the formation of primary lamellarlayers. This involved a restructuring of membraneous tubules into membrane sheets.


The newly formed lamellar layers had numerous perforations and protuberances(Figs. 12, 14). The total amount of lamellae (thylakoids) present at various stages ofgreening was estimated by determining the length of distinct lamellar profiles perplastid section (Fig. 5). The total length of the lamellae increased simultaneously withthe decrease in size of the prolamellar bodies. Following the initial increase in lengthof the lamellar profiles due to dispersal of the prolamellar bodies, a small decrease wasobserved. This decrease is presumably related to the elimination of perforations fromthe primary lamellar layers, and would imply that the perforations are eliminated bycontraction of the membrane material. The total amount of lamellae was also calcu-lated as profile length of lamellae per plastid section area (Fig. 5). In 5-day-old leavesthe total length of lamellar profiles per plastid section gradually increased up to 12 hin the light, and then remained about constant over the next 12-h period. The mem-branes participating in the differentiation of the lamellar system in the etioplastsduring greening of 7-, 9- and 11-day-old leaves (during the first 24 h) were largelyderived from the prolamellar body membranes. This was also true for the membranesparticipating in the formation of grana. Additional membranes were synthesized onlyat later stages of greening. In the etioplasts of 5-day-old leaves the lamellar systemwas formed from reorganized prolamellar body membranes as well as additionalmembranes synthesized during greening.

The perforations in the lamellar layers of the etioplasts and those formed by dis-persal were eliminated during greening. The number of perforations in profiles of thelamellar layers were quantitated in 7-day-old leaves illuminated with 3200 or 20 lx(Fig. 6). The number of perforations reached a maximum after 2 h in the light. Duringthe following 6 h, the number decreased to a low value that remains constant. Leavesilluminated 24 h at 20 lx contained both grana, prolamellar bodies and lamellar layerswith few perforations. Transfer of the seedlings from low (20 lx) into high light(3200 lx) resulted in the dispersal of the prolamellar bodies which accumulated in dimlight, and resulted in an increase in the number of perforations observed (Fig. 6). Inthe 5-day-old leaves the elimination of the perforations from the lamellar layers wasfaster than in 7-day-old leaves illuminated at 3200 lx. Most of the perforations hadalready been eliminated after 4 h in the light. In 9-day-old leaves the perforationswere eliminated at about the same time as in 7-day-old leaves, but in the 11-day-oldleaves many perforations remained in the lamellar layers for up to 12 h.

The occurrence of fine tubules. Fine tubules (cf. Figs. 12, 14) were found in the stromaof plastids in dark-grown barley seedlings (Henningsen & Boynton, 1969). The finetubules occurred in bundles of 10-42 parallel tubules, and reached a maximumlength of 2 fim. The fine tubules could be found in the plastids at any stage of greening.Fine tubules occurred with particular frequency in plastids of 5- and 7-day-old leavesilluminated with 3200 lx for 30 min to 2 h, as well as in 7-day-old leaves illuminated2-4 h at 20 lx. Fine tubules were also frequent in 5-day-old leaves illuminated for6 h at 3200 lx and in 7-day-old leaves after 8 h at 20 lx.

Grana formation. A granum was considered to consist of two or more closely paired,appressed lamellae (cf. Figs. 15, 17). The formation of grana was followed by deter-mining the number of grana per. plastid section area (Fig. 7), the number of disks per


granum (Figs. 8, 9) and the total length of paired lamellar profiles per plastid sectionarea (Fig. 10).

In etioplasts of all ages, between 0-2 and 1 cross-sectioned granum was found per/tm2 plastid section area (Fig. 7). It remains to be investigated whether these granaare homologous to those developing during greening. After 24 h of greening, seedlings

12 16Illumination, h

Fig. 6

12 16 20 24

Illumination, h

Fig. 7

Fig. 6. Number of perforations in profiles of the lamellae in plastids of 7-day-olddark-grown barley seedlings illuminated from o to 24 h with 3200 or 20 lx ( • and O,respectively). The 20-lx specimen was transferred to 3200 lx at arrow.

Fig. 7. Number of grana per plastid section area during illumination of dark-grownbarley seedlings of various ages. A, 5 days, 3200 lx; # , 7 days, 3200 lx; O, 7 days,20 lx; A, 9 days, 3200 lx; • , 11 days, 3200 lx.

of all ages contained about 3 sectioned grana per/«n2 plastid section area. In 5-day-oldleaves the number of grana increased rapidly, reaching a maximum of 4-5 between6 and 12 h of illumination, and then declined thereafter to the level reached by allplastids at 24 h.

During greening of the 5- and 7-day-old leaves the average number of disks per


granum remained close to 2 during the first 4-h (Figs. 8, 15). Few grana containing3-6 disks were observed during the first 4 h (Fig. 9) of greening, although successivelymore grana with 3 or more disks appeared thereafter. Figs. 8 and 9 demonstrate that themore rapidly greening 5-day-old leaves contained grana with more disks earlier thanin the 7-day-old leaves. The increase in the number of disks per granum occurred ina similar manner in 7-day-old leaves greening at 20 lx and at 3200 lx. In the 9-and

0 s -

-aai_o

Illumination, h

Fig. 8. Average number of disks per granum as a function of time of illumination of5- to 11-day-old dark-grown barley seedlings (in upper diagram). The percentage ofthe grana with more than 2 disks as found in dark-grown barley seedlings duringgreening in 3200 lx of white light (in lower diagram). A, 5 days old; O, 7 days;T , 9 days; D, " days.

11-day-old leaves grana containing 2 disks remained dominating during the 24 hstudied (Figs. 8, 9, 18). The analysis reveals that the differentiation of the lamellarsystem in etioplasts occurred in the following way: first, a maximum number of2-disk grana were produced, and then more disks were added to these original granaas greening proceeded. Eventually two or more grana may be incorporated intoa single granum consisting of a very large number of disks which in some casestraverses the entire chloroplast (e.g. Weier, Stocking & Shumway, 1967).

Loose stacks of several lamellar layers are common in plastid sections from 9- and11-day-old leaves in darkness as well as after prolonged exposure to light (Figs. 13,


16, 18). Pairing between 2 of the lamellar layers in a loose stack is observed (Fig. 16).This type of membrane arrangement was rather frequent during the first 4-8 hin the light and might be similar to the regions of grana-like pairing between thelamellar layers found in unilluminated leaves.

Finally, we attempted to analyse whether the total profile length of paired regionsin the grana of a plastid was correlated with its chlorophyll content during the variousstages of greening. The increase in profile length of the paired regions of lamellae perarea of the plastid sections during illumination of the seedlings is shown in Fig. 10.The chlorophyll content of the analogous leaf segments is given for comparison. It

Number of disks per granum

Fig. 9. Histograms illustrating the distribution of the number of disks per granum inplastids of 7-day-old seedlings after various times of greening in 3200 lx of whitelight: B, c, D, E, 4, 8, 16, 24 h, respectively. For comparison the distribution in 5-,9- and 11-day-old seedlings (A, F, G) after 24 h of greening is given.

can be seen that there was a reasonably parallel increase in the 2 parameters duringthe greening of 7-day-old seedlings at 3200 lx. The detailed correlation between thetwo, however, breaks down. While the chlorophyll content increased smoothly after2 h, the length of the paired regions rose in a distinct step between 4 and 6 h and onlyafter 12 h did it begin to rise smoothly. In the 7-day-old leaves illuminated with 20 lx,the lag in chlorophyll synthesis was paralleled by a lag in the formation of pairedregions. Again, a distinct step in the formation of the paired regions occurred between4 and 6 h, and again between 16 and 24 h. In the 9- and n-day-old seedlings


(cf. Fig. 3) there was a slow greening paralleled by a slow increase in the length of thepaired profiles. The best correlation between increase in length of paired lamellarprofiles and chlorophyll was obtained in the 5-day-old seedlings (Fig. 10). Duringthe first 1-2 h of illumination the paired regions per plastid section area increasedsignificantly, although this initial increase was not accompanied by a comparable

a.ono

0 4 8 12 16 20 24

8 12 16 20Illumination, h

240 tfin-y r-«—0 4 8 12 16 20

Illumination, h

24

Fig. 10. Average profile length of paired regions of lamellae per plastid section areaduring greening of dark-grown barley seedlings of various ages (A)- The values arecompared to the content of total chlorophyll {a + b) per g fresh weight in similar leafsegments (O). A, 7 days old, 3200 lx; B, 7 days, 20 lx, transferred to 3200 lx at arrow;C, 5 days, 3200 lx; D, 9 days, 3200 lx; E, 11 days, 3200 lx.

increase in the chlorophyll content of the leaves. It can be concluded that the lengthof paired lamellae per /im2 plastid section area is the parameter that gives the bestcorrelation with the amount of chlorophyll in the developing plastids. On the otherhand, it is equally clear that the paired grana membranes can incorporate differentamounts of chlorophyll per unit of membrane area depending on the stage of greening


and the age of the seedlings. In the initial phase of greening, particularly in youngleaves, pairing of the lamellae precedes chlorophyll accumulation. At any stage ofgreening a small amount of chlorophyll can possibly be present in unpaired regionsof the lamellae.

The general relation between the length of paired lamellar profiles and the chloro-phyll content of the leaves during greening for each seedling age and light conditionis given in Fig. 11. The length of paired regions of the lamellae is positively correlatedwith the chlorophyll content of the leaf segments. The curves of the relation betweenpaired regions of the lamellae and the chlorophyll content have different slopes for

0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 600 700 800 900 1000

Hg total chlorophyll

Fig. ir. Diagrams illustrating the relation between the content of total chlorophyll{a + b) per g fresh weight in leaf segments and the profile length of paired regions ofplastid lamellae during greening of barley seedlings developed in darkness for 5, 7,9 and 11 days, A: # , 7 days old at 3200 lx; O, 7 days at 20 lx. B: # , A, M, 5, 9 and11 days old, respectively, at 3200 lx.

the different seedling ages (Fig. 11). Assuming that the increase in profile length isproportional to an increase in area of the paired regions of lamellae, the results suggestthat less chlorophyll is incorporated per area of paired lamellae in young leaves thanin older leaves. In 5-day-old seedlings 100 /tg chlorophyll per g fresh weight cor-respond to 1 /iva profile of paired lamellae per /mi2 of plastid section. In 9-day-oldseedlings this figure is about 500 fig chlorophyll per g fresh weight. Thus, about5 times as much chlorophyll is associated with the paired membranes in the greeningetioplasts of 9-day-old seedlings as in those of 5-day-old seedlings. At later stagesin the greening of young seedlings, increasing amounts of chlorophyll are incorporatedinto the already formed membranes of the grana (cf. 5-day-old seedlings, Fig. n ) .The 7-day-old seedlings illuminated with 3200 lx display a slope intermediate betweenthe 5- and 9-day-old seedlings. Probably both in young and old seedlings the chloro-phyll content per membrane area in the grana eventually will reach the same value.

46 K. W. Henningsen and jf. E. Boynton

DISCUSSION

In response to light an increase in size and a change in shape of the plastids wereobserved. The increase in plastid volume in greening barley leaves occurred earlierthan reported for plastids isolated from greening bean leaves (Mego & Jagendorf,1961). In darkness following a brief illumination the plastid volume increases rapidly,and then returns to the same volume as before illumination (Henningsen & Boynton,1969). It is conceivable that the changes in size and conformation of the plastids reflectchanges in the metabolic activity of the plastids.

If the rate of chlorophyll accumulation is used as a measure, then the 5-day-olddark-grown barley seedlings used in this study concerning the structural aspects ofchloroplast development are comparable to the 6-day-old dark-grown seedlings usedfor earlier studies about the development of the photochemical activities (Henningsen& Boardman, 1973). Furthermore, they are comparable to the seedlings used foranalysis of esterification of chlorophyllide (K. W. Henningsen & S. W. Thorne, inpreparation) and for changes in the properties of chlorophyll(ide) holochromes at theinitial stage of greening (K. W. Henningsen, S. W. Thorne & N. K. Boardman, inpreparation).

The spectral shift from 682-684 to 672 nm and esterification of newly formedchlorophyllide have similar time courses and both processes are temperature depen-dent in a similar manner (Sironval et al. 1965; K. W. Henningsen & S. W. Thorne,in preparation). Also the dispersal of the prolamellar bodies into primary lamellarlayers has been correlated with the spectral shift of chlorophyllide from 682-684 to672 nm (Henningsen & Boynton, 1969; Henningsen, 1970). In leaves of mutants inbarley where the spectral shift from 682 to 672 nm of newly formed chlorophyllide islacking, esterification and subsequent processes are also blocked (K. W. Henningsen& S. W. Thorne, in preparation). These mutants also fail to disperse the prolamellarbody membranes (von Wettstein et al. 1971; Henningsen et al. 1973). Thus, the682-684 to 672 nm spectral shift, the esterification of chlorophyllide and the dispersalof the prolamellar body appear interconnected.

Ultracentrifugation (Bogorad, Laber & Gassman, 1968), gel chromatography(Henningsen & Kahn, 1971; K. W. Henningsen, S. W. Thorne & N. K. Boardman,in preparation) and fluorescence spectroscopy (Thorne, 1971), give results consistentwith the translocation of the newly formed chlorophyll a protein complex in the plastidmembranes. The time course for the increase in fluorescence efficiency of newly formedchlorophyll a in barley leaves (K. W. Henningsen & S. W. Thorne, in preparation),correlates well with the appearance of the first photochemical activities (Henningsen &Boardman, 1973). Appreciable photosystem I activity is detected with plastids frombarley leaves illuminated for 15 min. Chlorophyll holochrome extracted with saponinfrom such leaves has photosystem I activity and appears to contain associated cyto-chromes (K. W. Henningsen, S. W. Thorne & N. K. Boardman, in preparation).Since cytochromes are not associated with protochlorophyllide holochrome extractedwith saponin from dark-grown leaves (Henningsen & Kahn, 1971), the associationbetween chlorophyll holochrome and cytochromes appears to be formed during the


light period. Thus, it appears that the newly formed chlorophyll-protein complex istranslocated from the original site in the prolamellar body membranes and incor-porated into reaction centre sites in the primary lamellar layers where it becomesassociated with components of the electron-transport system.

Photosynthetic oxygen evolution from leaves can be detected as soon as 30 minafter the onset of illumination. On a chlorophyll basis, the rate of oxygen evolutionfrom both leaves and isolated plastids reaches a maximum after 1-5-2 h of greening(Henningsen & Boardman, 1973). In young dark-grown barley seedlings, photo-synthetic CO2 fixation could also be detected after 3 h illumination (Rhodes & Yemm,1966). These results when considered in light of the observations reported above showthat both photosystem I and II are functional at a stage when the lamellae are eithersingle or paired into small diameter 2-disk grana. The early appearance of 2-disk granaand capacity for photosynthetic oxygen evolution in greening barley leaves agree withthe studies on lamellar structure and photosynthetic reactions in mutants of Nicotianatabacum by Homann & Schmid (1967). They concluded that photosystem I activitycan occur with single lamellae, but oxygen evolution (photosystem II) requires pairingbetween at least 2 lamellae. Oxygen evolution and photophosphorylation weredetected in dark-grown bean leaves irradiated with far-red light (De Greef et al. 1971).Under this condition most of the lamellae are unpaired; however regions of pairingbetween 2 or 3 lamellae are evident.

In contrast to the higher plants, the ac-31 mutant in Chlamydomonas has a func-tional photosystem II, but all the lamellae are unpaired (Goodenough, Armstrong &Levine, 1969). During greening of dark-grown cells of the mutant y-i in Chlamydo-monas, the developing lamellae are unpaired at the time when oxygen evolution isfirst detected (Ohad et al. 1967).

While the photosynthetic membranes in green algae appear to be homogenous(Eytan & Ohad, 1972), the lamellar system in chloroplasts of higher plants is differen-tiated into grana and stroma regions with distinctly different functions and com-position (Sane, Goodchild & Park, 1970; Allen, Good, Trosper & Park, 1972). Thisdistinction between the lamellar system in algae and higher plants could explain whypairing can be lacking in chloroplasts of algae, while some paired regions between atleast two lamellae occur in chloroplasts of higher plants under conditions wherephotosystem II is functional.

During the early stages of greening high light intensity is required to saturate thephotosystems (Kirk & Goodchild, 1972; Bar-Nun et al. 1972; Henningsen & Board-man, 1973). During this period the chlorophyll content is low and the plastid mem-branes consist of unpaired lamellae and small-diameter 2-disk grana. At later stagesof greening when grana with 3 or more disks are formed and more chlorophyllaccumulates the photochemical reactions are saturated at a lower light intensity. Itappears that the accumulation of chlorophyll in the light harvesting system and alsothe appearance of the high potential form of cytochrome 6-559 parallels the increasein paired regions with 3 or more lamellae.

The development of chloroplasts in 7-, 9- and 11-day-old barley seedlings followsthe same sequence as described for the 5- to 6-day-old seedlings. Chloroplast develop-


merit is slower in the older seedlings and it is evident that during greening for up to24 h, the lamellar membranes are all derived from membrane material present in theetioplast. However, the newly dispersed lamellar layers may function as a frameworkfor the insertion of further membrane components required for the formation of grana.

This work was supported by grants from the National Institute of Health, U.S. PublicHealth Service (GM 10819), the Carlsberg Foundation, and the Danish Natural ScienceResearch Council to Professor D. von Wettstein. The authors thank Professor Diter vonWettstein, Dr A. Kahn and Dr Niels Nielsen for valuable discussions. The technical assistanceof Miss K. B. Pauli and Mr P. Eriksen is gratefully acknowledged.

REFERENCES

AKOYUNOGLOU, G. & ARGYROUDI-AKOYUNOGLOU, J. H. (1969). Effects of intermittent andcontinuous light on the chlorophyll formation in etiolated plants of various ages. Physiol. PL22, 288-295.

AKOYUNOGLOU, G. & MICHALOPOULOS, G. (1971). The relation between the phytylation andthe 682 -> 672 nm shift in vivo of chlorophyll a. Physiol. PL 25, 324-329.

AKOYUNOGLOU, G. A. & SIEGELMAN, H. E. (1968). Protochlorophyllide resynthesis in dark-grown bean leaves. Plant Physiol., Lancaster 43, 66-68.

ALLEN, C. F., GOOD, P., TROSPER, T. & PARK, R. B. (1972). Chlorophyll, glycerolipid andprotein ratios in spinach chloroplast grana and stroma lamellae. Biochem. biophys. Res.Cotntnun. 48, 907-913.

BAR-NUN, S., WALLACH, D. & OHAD, I. (1972). Biogenesis of chloroplast membranes. X.Changes in the photosynthetic specific activity and the relationship between the harvestingsystem and photosynthetic electron transfer chain during greening of Chlamydomonasreinhardi y — 1 cells. Biochim. biophys. Acta 264, 138-148.

BOARDMAN, N. K. (1967). Chloroplast structure and development. In Harvesting the Sun,Photosynthesis in Plant Life (ed. A. San Pietro, F. A. Greer & T. J. Army), pp. 211-230.New York and London: Academic Press.

BOARDMAN, N. K., ANDERSON, J. M., KAHN, A., THORNE, S. W. & TREFFRY, T. E. (1971).

Formation of photosynthetic membranes during chloroplast development. In Autonomy andBiogenesis of Mitochondria and Chloroplasts (ed. N. K. Boardman, A. W. Linnane & R. M.Smillie), pp. 70-84. Amsterdam: North-Holland.

BOGORAD, L., LABER, L. & GASSMAN, M. (1968). Aspects of chloroplast development: transitorypigment-protein complexes and protochlorophyllide regeneration. In Comparative Bio-chemistry and Biophysics of Photosynthesis (ed. K. Shibata, A. Takamiya, A. T. Jagendorf &R. C. Fuller), pp. 299-312. Tokyo: University of Tokyo Press.

BOYNTON, J. E. & HENNINGSEN, K. W. (1967). The physiology and chloroplast structure ofmutants at loci controlling chlorophyll synthesis in barley. Studia biophys. 5, 85-88.

BRUINSMA, J. (1961). A comment on the spectrophotometric determination of chlorophyll.Biochim. biophys. Acta 52, 576-578.

BRYAN, G. W., ZADYLAK, A. H. & EHRET, C. F. (1967). Photoinduction of plastids and ofchlorophyll in a Chlorella mutant. J. Cell Biol. 2, 513-528.

BUTLER, W. L. & BRIGGS, W. R. (1966). The relation between structure and pigments duringthe first stages of proplastid greening. Biochim. biophys. Acta 112, 45-53.

D E GREEF, J., BUTLER, W. L. & ROTH, T. F. (1971). Greening of etiolated bean leaves infar-red light. Plant Physiol., Lancaster 47, 457-464.

EILAM, Y. & KLEIN, S. (1962). The effect of light intensity and sucrose feeding on the finestructure in chloroplasts and on the chlorophyll content of etiolated leaves. J. Cell Biol. 14,169-182.

ERIKSSON, G., KAHN, A., WALLES, B. & VON WETTSTEIN, D. (1961). Zur macromolecularenPhysiologie der Chloroplasten. III. Ber. dt. bot. Ges. 74, 222-232.

EYTAN, G. & OHAD, I. (1970). Biogenesis of chloroplast membranes. VI. Cooperation betweencytoplasmic and chloroplast ribosomes in the synthesis of photosynthetic lamellar proteinsduring the greening process in a mutant of Chlamydomonas reinhardi y—i.J. biol. Chetn. 245,4297-4307.


EYTAN, G. & OHAD, I. (1972). Biogenesis of chloroplast membranes. VII. The preservation ofmembrane homogeneity during development of the photosynthetic lamellar system in analgal mutant (Chlamydomonas reinhardi y — 1). J. biol. Chem. 247, 112-121.

GOLDBERG, I. & OHAD, I. (1970). Biogenesis of chloroplast membranes. V. A radioautographicstudy of membrane growth in a mutant of Chlamydomonas reinhardi y—i.jf. Cell Biol. 44,572-591-

GOODENOUGH, U. W., ARMSTRONG, J. J. & LEVINE, R. P. (1969). Photosynthetic properties ofac-31, a mutant strain of Chlamydomonas reinhardi devoid of chloroplast membrane stacking.Plant PhysioL, Lancaster 44, 1001-1012.

GUNNING, B. E. S. & JAGOE, M. P. (1967). The prolamellar body. In Biochemistry of Chloro-plasts, vol. 2 (ed. T. W. Goodwin), pp. 655-676. New York and London: Academic Press.

GYLDENHOLM, A. O. & WHATLEY, F. R. (1968). The onset of photophosphorylation in chloro-plasts isolated from developing bean leaves. Nezv Phytol. 67, 461-468.

HENNINGSEN, K. W. (1970). Macromolecular physiology of plastids. VI. Changes in membranestructure associated with shifts in the absorption maxima of the chlorophyll pigments.J. Cell Sci. 7, 587-621.

HENNINGSEN, K. W. & BOARDMAN, N. K. (1973). Development of photochemical activity andthe appearance of the high potential form of cytochrome 6-559 in greening barley. PlantPhysioL, Lancaster 51, 1117-1126.

HENNINGSEN, K. W. & BOYNTON, J. E. (1969). Macromolecular physiology of plastids. VII .The effect of a brief illumination on plastids of dark-grown barley leaves. J. Cell Sci. 5,757-793-

HENNINGSEN, K. W. & BOYNTON, J. E. (1970). Macromolecular physiology of plastids. VII I .Pigment and membrane formation in plastids of barley greening under low light intensity.J. Cell Biol. 44, 290-304.

HENNINGSEN, K. W., BOYNTON, J. E., VON WETTSTEIN, D. & BOARDMAN, N. K. (1973). Nuclear

genes controlling chloroplast development in barley. In Biochemistry of Gene Expression inHigher Organisms (ed. J. K. Pollak & J. Wilson Lee), pp. 457-478. Sydney: Australia andNew Zealand Book Co.

HENNINGSEN, K. W. & KHAN, A. (1971). Photoactive subunits of protochlorophyll(ide)holochrome. Plant. PhysioL, Lancaster, 47, 685-690.

HOMANN, P. H. & SCHMID, G. H. (1967). Photosynthetic reactions of chloroplasts with unusualstructures. Plant PhysioL, Lancaster 42, 1619-1632.

HOOBER, J. K. (1970). Sites of synthesis of chloroplast membrane polypeptides in Chlamydo-monas reinhardi y—i.J. biol. Chem. 245, 4327-4334.

KAHN, A. (1968). Developmental physiology of bean leaf plastids. I I I . Tube transformationand protochlorophyll(-ide) photoconversion by a flash irradiation. Plant PhysioL, Lancaster43, 1781-1785.

KIRK, J. T. O. & GOODCHILD, D . J . (1972). Relationship of photosynthetic effectiveness ofdifferent kinds of light to chlorophyll content and chloroplast structure in greening wheatand in ivy leaves. Aust.J. biol. Sci. 25, 215-241.

KIRK, J. T. O. & TILNEY-BASSETT, R. A. E. (1967). The Plastids. London: Freeman andCompany.

KLEIN, S., BRYAN, G. & BOGORAD, L. (1964). Early stages in the development of plastid finestructure in red and far-red light. J. Cell Biol. 22, 433-442.

KLEIN, S. & SCHIFF, J. A. (1972). The correlated appearance of prolamellar bodies, proto-chlorophyll(ide) species, and the Shibata shift during development of bean etioplasts in thedark. Plant PhysioL, Lancaster 49, 619-626.

MEGO, J. L. & JAGENDORF, A. T. (1961). Effect of light on growth of Black Valentine beanplastids. Biochim. biophys. Ada 53, 237-254.

MUHLETHALER, K. & FREY-WYSSLING, A. (1959). Entwicklung und Struktur der Proplastiden.J. biophys. biochem. Cytol. 6, 507-512.

OELZE-KAROW, H. & BUTLER, W. L. (1971). The development of photophosphorylation andphotosynthesis in greening bean leaves. Plant PhysioL, Lancaster 48, 621—625.

OHAD, I., SIEKEVITZ, P. & PALADE, G. E. (1967). Biogenesis of chloroplast membranes.II. Plastid differentiation during greening of a dark-grown algal mutant {Chlamydomonasreinhardi). J Cell Biol. 35, 553-584.

4 CEL I j


PETROCELLIS, B. DE, SIEKEVITZ, P. & PALADE, G. E. (1970). Changes in chemical compositionof thylakoid membranes during greening of the y—i mutant of Chlamydomonas reinhardi.J. Cell Bid. 44, 618-634.

RHODES, M. J. C. & YEMM, E. W. (1966). The development of chloroplasts and photosyntheticactivities in young barley leaves. New Phytologist 65, 331-342.

SANE, P. V., GOODCHILD, D. J. & PARK, R. B. (1970). Characterization of chloroplast photo-systems 1 and 2 separated by a non-detergent method. Biochim. biophys. Acta 216, 162-178.

SHLYK, A. A., RUDOI, A. B. & VEZITSKII, A. Yu. (1970). Immediate appearance and accumula-tion of chlorophyll b after a short illumination of etiolated maize seedlings. Photosynthetica 4,68-77.

SIRONVAL, C , MICHEL, J. M., BRONCHART, R. & ENGLERT-DUJARDIN, E. (1969). On the

' primary' thylakoids of chloroplasts grown under a flash regime. In Progress in PhotosynthesisResearch, vol. 1 (ed. H. Metzner), pp. 47—54. Tubingen: International Union of BiologicalSciences.

SIRONVAL, C , MICHEL-WOLWERTZ, M. R. & MADSEN, A. (1965). On the nature and possiblefunctions of the 673- and 684-m/t forms in vivo of chlorophyll. Biochim. biophys. Acta 94,344-354-

SISLER, E. C. & KLEIN, W. H. (1963). The effect of age and various chemicals on the lag phaseof chlorophyll synthesis in dark grown bean seedlings. Physiol. PI. 16, 315-322.

SMITH, J. H. C. & KOSKI, V. M. (1948). Chlorophyll formation. Carnegie Instn Wash. Yearbook47. 93-96.

SPURR, A. R. (1969). A low-viscosity resin embedding medium for electron microscopy.J. Ultrastruct. Res. 26, 31-43.

THORNE, S. W. (1971). The greening of etiolated bean leaves. I. The initial photoconversionprocess. Biochim. biophys. Acta 226, 113-127.

THORNE, S. W. & BOARDMAN, N. K. (1971). Formation of chlorophyll b, and the fluorescenceproperties and photochemical activities of isolated plastids from greening pea seedlings.Plant Physiol., Lancaster 47, 252-261.

VIRGIN, H. (1955). Protochlorophyll formation and greening in etiolated barley leaves. Physiol.PL 8, 630-643.

VIRGIN, H. (1960). Pigment transformation in leaves of wheat after irradiation. Physiol. PL 13,155-164.

VIRGIN, H., KAHN, A. & VON WETTSTEIN, D. (1963). The physiology of chlorophyll formation inrelation to structural changes in chloroplasts. Photochem. Photobiol. 2, 83-91.

VON WETTSTEIN, D. (1958). The formation of plastid structures. Brookhaven Syrnp. Biol. 11,138-159. New York: Upton.

VON WETTSTEIN, D. (1967). Chloroplast structure and genetics. In Harvesting the Sun, Photo-synthesis in Plant Life (ed. A San Pietro, F. A. Greer & T. J. Army), pp. 153-190. New Yorkand London: Academic Press.

VON WETTSTEIN, D., HENNINGSEN, K. W., BOYNTON, J. E., KANNANGARA, G. C. & NIELSEN,

O. F. (1971). The genie control of chloroplasts development in barley. In Autonomy andBiogenesis of Mitochondria and Chloroplasts (ed. N. K. Boardman, A. W. Linnane & R. M.Smillie), pp. 205-223. Amsterdam: North-Holland.

VON WETTSTEIN, D. & KAHN, A. (i960). Macromolecular physiology of plastids. Proc. Eur.Reg. Conf. Electron Microsc. Delft, vol. 2 (ed. A. L. Houwink & B. J. Spit), pp. 1051—1053.Delft: De Netherlandse Vereiniging Voor Electronmicroscopie.

WEIER, T . E. & BROWN, D. L. (1970). Formation of the prolamellar body in eight day, dark-grown seedlings. Am. J. Bot. 57, 267-275.

WEIER, T. E., STOCKING, C. R. & SHUMWAY, L. K. (1967). The photosynthetic apparatus inchloroplasts of higher plants. Brookhaven Syrnp. Biol. 19, 353-374. New York: Upton.

WOLFF, J. B. & PRICE, L. (1957). Terminal steps of chlorophyll a biosynthesis in higher plants.Archs Biochem. Biophys. 72, 293-301.

WRISCHER, M. (1966). Neubildung von Prolamellarkorpern in Chloroplasten. Z. Pflanzen-physiol. 55, 296-299.

{Received 29 August 1973)

Macromolecular physiology of plastids. IX

0-5

Fig. 12. Plastid section from the primary leaf of a 5-day-old dark-grown barleyseedling illuminated for 30 min with 3200 lx of white light. Remnants of a transformedprolamellar body (pb) connected to primary lamellar layers with numerous perforations(arrows) and protuberances (tr). Groups of fine tubules (/) in the stroma. x 37000.Fig. 13. Plastid section from the primary leaf of an 11-day-old dark-grown barleyseedling illuminated for 30 min with 3200 lx of white light. The plastid membranesconsist of a transformed prolamellar body and a stack of long parallel lamellar layerslargely without perforations, x 37000.

4-2

K. W. Henningsen and J. E. Boynton

P

P0-2 am

Fig. 14. Section through a plastid in the primary leaf of a 9-day-old dark-grownbarley seedling illuminated for 1 h with 3200 lx of white light. Primary lamellar layersare radiating from the periphery of a transformed prolamellar body. The lamellarsheets have numerous perforations (p) and protuberances (arrows and insets). A bundleof fine tubules (/) is present in the stroma. x 61000. Insets: Protuberances from theprimary lamellar layers, in cross-section (at left) and in surface view (at right),x 108000; scale line represents 0-2/tm.

Macromolecidar physiology of plastids. IX 53

gg

0-5 /mi

Fig. 15. Plastid section from the primary leaf of a 5-day-old dark-grown barley seed-ling illuminated for 4 h with 3200 lx of white light. Most of the perforations have beeneliminated from the lamellar layers and grana (g) are forming. Bundles of fine tubulesin the stroma are seen in longitudinal and cross-section (arrows), x 51000.Fig. 16. Plastid section from the primary leaf of a 9-day-old dark-grown barley seed-ling illuminated for 4 h with 3200 lx of white light. Many perforations are still presentin the lamellar layers. Regions of the lamellar layers are arranged in loose stacks andsome of the lamellae participate in grana-like pairing, x 30000.


0-5• 0-5 //m

»*

Fig. 17. Plastid section from the primary leaf of a 5-day-old barley seedling illumi-nated for 16 h with 3200 lx of white light. The lamellar system is well differentiatedinto grana consisting of several paired disks and inter-grana lamellae which connectadjacent grana. x 33 000.Fig. 18. Plastid section from the primary leaf of an 11-day-old dark-grown barleyseedling illuminated for 24 h with 3200 lx of white light. Several lamellar layers arearranged in a loose stack. Some short regions of the lamellar layers are paired (arrows),x 41000.

Macromolecular physiology of plastids. IX• - 1 * . - . » • . .

0-5

|.-:f :-f #l..fl;

Figs. 19, 20. Plastid sections from the primary leaves of 7-day-old dark-grown barleyseedlings illuminated for 8 h with white light at 3200 lx (Fig. 19) and at 20 lx (Fig. 20).Many grana, with several disks in each, are formed in the plastids under both condi-tions. The plastid from a leaf illuminated at 20 lx (Fig. 20) contains in additioncrystalline prolamellar bodies. The small prolamellar bodies are connected to severaladjacent lamellar layers. Fig. 19, x 48000; Fig. 20, x 41 000.

Documents

MACROMOLECULAR PHYSIOLOGY OF PLASTIDS · dark-grown seedlings (Sisle &r Klein, 1963; Akoyunoglo &u Argyroudi-Akoyunoglou, 1969), the rate of chlorophyll synthesis on temperature and