Planta ([982) 154 : 259 265 Planta �9 Springer-Verlag 1982

Chloroplast development in low light-grown barley seedlings

A.R. Wellburn, D.C. Robinson, and F.A.M. Wellburn Department of Biological Sciences, University of Lancaster, Lancaster LA1 4YQ, U.K.

Abstract. Segments of 7-d low light-grown barley laminae cut at 0.5 cm intervals up from the intercalary meristem were examined ukrastructurally and bio- chemically. The different regions upwards showed the succession of plastid development in light-grown tis- sues of eoplasts, amyloplasts, amoeboid, immature and mature plastids as described by Whatley (1977). Semi-crystalline bodies were detected in all of them. The eoplast-amyloplast regions are characterised by a greater proportion of mitochondria and high levels of ATP and 3-phosphoglyceric acid, together with low levels of inorganic phosphate conducive to the activation of ADP glucose pyrophosphorylase. The amoeboid and immature plastid regions have higher levels of inhibitory phosphate and starch breakdown may be responsible for the release of metabolites and energy for development. Segments containing amoe- boid and immature plastids also have reduced levels of ATP (and 3-phosphoglyceric acid) as photosyn- thetic components are synthesised. Using ultrastruc- tural assessments of areas of thylakoids, first fi-caro- tene and violaxanthin, followed by chlorophyll a and lutein and, lastly, chlorophyll b are concentrated in the developing lamellar systems of the immature and mature chloroplasts. The formation of additional membraneous material which spreads these pigment systems over a greater thylakoid area within the plas- rids is the final stage of plastid morphogenesis in low light-grown seedlings.

Key words: Amyloplast - Chloroplast development - Hordeum - Starch.

Introduction

In studies of higher plant chloroplast development, well over 95% of literature in the past refers to the

Abbreviations: Chl = chlorophyll; 3-PGA : 3 phosphoglyceric acid

' greening' of etiolated tissue involving the conversion of etioplasts to mature plastids. There have been many reasons for this but principally this system en- abled a 'slowing-down' of parts of the developmental process so that they could be studied in detail. As a result much is now known about the sequential assembly of the photosynthetic apparatus. Further- more, the r61e of light quality particularly at the red end of the spectrum in controlling many aspects of development could only have been evaluated by ex- perimenting with dark-grown tissue (e.g. Kasemir 1979). Nevertheless, with the advent of more sensitive techniques, biochemical and physiological studies of plastid development in light-grown tissue grown under more normal conditions and similar to those in the field are possible provided that a sound and comprehensive understanding of the basic ultrastruc- ture of the system underpins each study.

Ultrastructural assessments have been made of di- cotyledonous and mono-cotyledonous light-grown plants. Whatley (1974, 1977) has shown that the plas- tids in all cells of the primary leaves of light-grown Phaseolus vulgaris undergo the same sequence of structural changes and has identified 7 basic and dis- tinct stages with optional diversions in several species. The first three proplastid stages she describes, namely those of the eoplast, amyloplast and amoeboid plas- tids, are quite unlike plastids in mature etiolated or greening tissue.

Monocotyledons differ developmentally from di- cotyledonous plants by virtue of having a basal inter- caIary meristem (Esau 1953). Leech and co-workers have undertaken a series of light-grown studies using maize (Zea mays) and lately wheat (Triticum aesti- rum). Leese et al. (1971) first showed that different populations of plastids could be effectively prepared from segments taken along the length of the maize seedling which correspond with the in situ appearance of the plastids. Using this system, Leech and co-

0032-0935/82/0154/0259/$01.40

260 A.R. Wellburn etal. : Light-grown plastid development

workers have examined lipid biosynthesis (Hawke etal. 1974), photochemical activities (Baker and Leech 1977) and changes in pool sizes of amino acids and amides (Chapman and Leech 1979). Previously Robertson and Laetsch (1974) had shown that the age of developing etiolated tissue has a considerable effect on the rate of chlorophyll formation during greening. Boffey etal. (1980), using light-grown wheat to avoid dimorphism, have extended this to show that the greening of mature etioplasts cannot be used as a model for the normal development of proplastids into mature chloroplasts, as not only exposure to light, but also chronological age of the plastid are important factors in development.

In the past we have endeavoured to examine the sources of energy for the development of plastids whilst effective photophosphorylation is absent and have shown that mitochondria are specifically syn- chronized within the greening process (Hampp and Wellburn 1980). Consequently it is of interest to know if this is also a feature of plastid development in light-grown tissues. This paper describes a series of experiments which, whilst not demonstrating mito- chondrial-plastid co-operation, reveal several features of light-grown plastid development that are not asso- ciated with etioplast-chloroplast transformations. The lower Ievels of light and temperature, by comparison to other studies, employed in this study were deliber- ately chosen not only to slow down the early stages of light-grown development but to simulate as close as possible the cultivation conditions around soil level for autumn-sown cereals in latitudes about 53 ~ N.

Material and methods

Pre-soaked (8 h) seeds of barley (Hordeum vulgare L. cv. Ark Royal) were germinated overnight on the surface of moist peat, lightly dusted the following morning with peat and then grown for the next six-full days in a light/dark (16 h/8 h) regime at 20 ~ C on a moist capillary bed. The light fluence rate was determined by the appropriate probes of a Li-Cor Quantum Photometer (Mod- el L1-185) to be 5 gmol m-2s-1 (250 lux). Under these conditions, at 7 days from soaking, the lengths of the laminae were 8 to 9 cm and the coleoptiles approximately 2.5 cm. By exerting a gentle but steady pull on the blades they usually break below the inter- calary meristem and the laminae slide out from the coleoptiles revealing white tissue immediately above the node of the meristem about 1 cm in length with a short distinct yellow to yellow-green region before merging into the full green of the remainder. After practice several hundred laminae can be harvested within a short period, placed with their meristems in line on a glass thin layer chromatography plate (with squared graph paper beneath) and the lengths of the arrayed laminae severed with a fresh and de- greased razor blade at 0.5 cm intervals. These different populations of segments were used for a variety of determinations. Usually one hundred sections were sufficient but occasionally 500 were required if sensitivity became a limitation. For electron microscopy 1 mm sections taken just above the meristem and from the middle of the different 0.5 cm sections were processed according to a

procedure described earlier (Wellburn and Wellburn 1979). Areas and distances on electron micrographs were measured using a Graphics Tablet attached to an Apple mini-computer. For each segment between 50 and 100 microgaphs were employed. The mean values of the top 10% of the size distribution range of organelles and starch grains were taken as those most likely to represent the centres of the structures and are referred to as maxi- mum areas. It was using such plastids that the lengths of the internal membrane (L) were measured. The total thylakoid area was calculated per plastid, per cell and per 0.5 cm segment by determining a notional radius from the circumference (L)/2~ and finding the value for 4~r 2. In addition longitudinal and transverse sections of the 0.5 cm segments from each region were examined by light microscopy. The number of cells in each dimension were counted and hence the total number per tissue volume calculated. Protoplasts were made from 100 of each of the sections by the procedure of Hampp and Ziegler (1980) and examined using a phase-contrast fluorescence microscope. By moving through the depth of field individual plastids in the protoplasts were counted and the number of plastids per cell for the different regions of the laminae obtained.

Levels of total ATP in extracts from the different segments were measured by bioluminescence using the method described by Wellburn et al. (1981) and energy charge ratios were calculated by the procedure adopted by Hampp and Wellburn (1980). Phos- phate was determined by the method of Taussky and Shorr (1953) and total protein by that of Lowry et al. (1951). Amounts of phospho-glycerate in different segments were estimated in triethan- olamine buffer (0.1 M, pH 7,6) using ATP in the presence of 3- phosphoglycerate kinase to form ADP and 1,3-diphosphoglycerate and following the disappearance of ATP using bioluminescence. Crude extracts from the different segments were prepared using the method of Ozbun et at. (1972) and e-amyIase and ]LamyIase activities were determined by the method of Abbott aud Matheson (1972). Starch synthetase was estimated in similar extracts by ad- ding pyruvate kinase and phospho-enol pyruvate to convert ADP from AD P-glucose to ATP which was measured by bioluminescence.

Pigments were rapidly isolated from segments by maceration in 50% (v/v) methanol for 15 seconds using an Ultraturrax with a minature probe, adding triple the volume of chloroform and shaking vigorously. The phases were separated using centrifugation (100 g for 3 rain) and the lower chloroform layer, containing vir- tually all the colour, was evaporated under O2-free N2. Half was redissolved in high performance liquid chromatography (HPLC) eluting solvent (methanol: acetone : water, 67.5: 25 : 7.5% by vol.) and the remainder in di-ethyl ether for determination of total chlo- rophylls and carotenoids using the method of Gaudill6re (1974). Pigments were separated isocratically by reverse-phase (C18) HPLC (Spectra-Physics Model 3510) through a 250 mm column (4.6 mm internal diameter) packed with Partisil 10 ODS (Whatman 10 gm bead size) using the above de-gassed eluant at a flow rate of 15 mls rain- 1 and measuring the absorbance of the effluent at 440 urn. The amounts of individual pigments were calculated from integra- tor values (Minigrator) and from measured areas using the Apple Graphics Tablet. Correction values for the differential sensitivities of absorption of the different pigments at the single wavelength, previously calculated from HPLC of single components, were ap- plied.

Pigments, metabolites and levels of enzymic activity were deter- mined in triplicate from three separate experiments each. Where appropriate the error bars on the different figures represent the standard deviations of each set of determinations. Graphs have used ' length up laminae' as the developmental parameter; repre- senting that distance as measured from the node of the intercalary meristem upwards towards the tip of the monocotyledonous blade. The levels of components are expressed in terms of protein content rather than per segment. Of major interest in this study were the

A.R. Wellburn et al. : Light-grown plastid development 261

first 4 cm (i.e. all that tissue below 4 cm up fi'om the meristem) where the protein content does not vary significantly. Consequently there is little difference between the two types of expression. Over the next 3 cm (4-7 em) upwards the protein content increases and the corresponding values on a per segment basis are higher but retain the same relationship to each other.

Resuhs

After much testing of oats, wheat and barley under different growth conditions, low intensity light-grown barley seedlings were found to have the longest non- green regions above the intercalary meristem and to offer the best prospect of resolution of the very early stages of development. Electron microscopy estab- lished the presence of the first five basic stages of light-grown plastid development as described by Whatley (1977) at the following intervals up the la- minae: 0.05-0.15 cm, eoplasts; 0.154).75 cm, amylo- plasts ; 0.75-1.25 cm amoeboid plastids; 1.25-1.75 cm, immature plastids with perforated stroma lamellae and incipient grana; 1.75 cm and above, mature chloroplasts. Figures I A to D show examples of eoplasts and amyloplasts and the later stages. One difference from those plastids described by Whatley for Phaseolus tissue of different ages are the larger amounts of undifferentiated internal mem- brane and even semi-crystalline bodies to be found in barley eoplasts, amyloplasts and amoeboid plastids.

Another variation concerns the appearance of the so-called prolamellar bodies (PLBs). In light-grown barley they are not as large and do not have the tight crystallinity of light-grown maize (Rascio et al. 1976) or beans (Wrischer 1966) or even the open or loose structure of barley etioplasts (e.g. Robertson and Laetsch 1974) but an amorphous irregular ap- pearance, similar to structures we observed in green- ing Scenedesmus mutants (Wellburn et al. 1980). We believe both these structures differ from conventional PLBs and in this paper propose the name of prothy- lakoid bodies (PTBs) to distinguish them (see discus- sion). An alternative name, tubular connections has already been proposed by Platt-Aloia and Thomson (1977) but unfortunately this term has the possibility of confusion with additional artefactual tubular con- nections associated with detached PLBs or isolated etioplasts (see Wellburn et al. 1977). We have used this terminology before (Wellburn et al. 1980) but have now discontinued it because of these connota- tions.

Three other features are of note in the micro- graphs. In Fig. 1 A a particularly close appression of an eoplast with a mitochondrion is shown (arrowed). This type of association is quite common in the early protoplast segments. The pointer in Fig. 1 C indicates

a cluster of plastid tubules cut at right angles. These are similar to sections of protrusions of the inner plastid envelope recently described by Newcombe et al. (1981) and may indicate that the PTBs and the inner envelopes are interconnected in some manner. The micrograph of Fig. 1 D shows a close association of ribosomes with thylakoid membranes (arrowed) a feature shown by nearly all the plastids in these laminae (see also Figs, 1A and C) and common in other developmental systems (Wellburn et al. 1980).

Figure 2 shows that the percentage of plastids with PTBs is high even at the eoplast stage, highest in association with starch grains and then falls to a steady value of 10% for successive plastid types. These may be low estimates because the cross-sec- tional areas (and hence the plastid volumes) increase considerably (Fig. 2) thereby reducing the frequency of intersection with PTBs. The same figure also shows approximately a tripling (ca. 8 to 22) of the number of plastids per cell starting at the amoeboid phase. After vacuolation the number of cells per segment is reasonably constant over the first 4 cm (Fig. 3) but when the internal plastid membrane areas are calcu- lated it can be seen that significant increases in thylak- old membrane are a late phenomenon in chloroplast development (Fig. 3) and occur well after photosyn- thetic competence.

Figure 4 reveals an interesting bi-phasic relation- ship of starch with respect to plastids. When expressed either as an accumulated total ratios or as the largest cross-sectional area for each segment the amyloplast stage is clearly recognizable, with secondary starch concentrations occurring in the 2.25 cm segments. Plastids in Figs. 1 B and D are representative of each.

The mitochondrial relationships (Fig. 5) are also interesting. Whilst the maximum cross-sectional areas do not change noticeably along the laminae the accu- mulated total mitochondrial area, as compared to that of plastids and expressed as a ratio, is extremely high at the eoplast stage and thereafter drops to a steady value.

Pigment distributions are shown in Fig. 6-8. When pigment concentrations are expressed simply on the basis of amounts per section, logarithmic curves similar to those shown for the chlorophylls (Fig. 6) are found. Stow initial increases for the first segments are followed by exponential phases with le- velling off towards the tip. This belies the internal concentrations revealed by expression of pigments on a membrane area basis. When this is done (Fig. 6) a peak concentration of chlorophyll a within thylak- oid membranes occurs in segments centred at 2.75 cm with accumulation of chlorophyll b slightly higher up. Although pigments are difficult to determine in the lower segments, the chlorophyll a/b ratio (Fig. 7)

Figs, 1A-D. Representative m/crographs o f plastids f rom different regions of light-grown 7-day barley laminae, A Eoplasts from close to the meris tem with a close appression of a mitochondrion arrowed. B Large starch grains (S) and a prothylakoid body (PTB) within an amyloplast taken from a section 0.25 cm up the leaf blade. C An immature plastid from the 1.25 cm region containing a PTB and an array of microtubules (arrowed). D Material from 2.75 cm up a lamina showing a mature plastid with starch grain (S), small PTB and distinctive arrays of r ibosomes associated with the thylakoids (arrowed). Bars = 1 /am

A.R. Wellbarn et al. : Light-grown plastid development

s

~ 3 ' ~

~0 o

2 s L r~ 03 ~ . 4 - r

~ . 3 . o

J:::

~.2

E_

o 5

" " , I Plastid no s" p O ~ ~ 1 2 ~

: ~,/ .--" ~l~=

/ ', ...-.,"'4~tid~rea ~ ~0 / ",... . . . . ~10"1 -

~ _.."'L ~ / ~ - ~4 w ~ . . . ~ %+PTBs .I-10 - -

. . . " ~ - . ~ _ . _ ~ - . - . o - - , - - 0 5 1 - ' o

a t ,o 1 2 3 4 Length up laminae(cm.) 3

�9 . t . . � 9 . t - ~ r M i t . A r e Z | . - - -E~ �9 - ~

~".~. Mit/PI .ratio

I I I 1 2 3

Length up laminae (cnn.)

.4 ~10-

~ ~J .3 ~q

o

2 ~, 4.

~.1 2-

0 0

6

Mere..: area :"

.4..Cell no. r." , - . ,} . . . . . . 1 ' - -~ ~ - { - ..t,-"

..... ~..:.+---F-j" 1 2 3

Length up laminae (cm.)

O h , ~ f ..,..-)L,

" , /.- ,.' ',.

hi a tmem. *" .4 \

/ " . i / ChlWrnern. ~, ~.,, .... / . j ' - - .

I I , - , - , ,~,-- L ~ ' ~ I I

I 2 3 Length up laminae (cm,)

-120~

c l 8 0

03

-40 "6

E

3 ~

1 5.

263

'E~_ I ,Starch "-~ 4 [ !~Area ~,T!~ = | t i .~ 3-t- I ': 03" " �9

~ ~ 2 "

E

N 0 / 0

4

"~176 ~ " ' . ,, St./Pla.ratio

"% . , o . . * " l [ - ~ 1 7 6 i " " L , - ~ --~ .... "T ' ~ , :'~'*~.-,,

1 2 3

Length up laminae (crn)

-.3 .o

.,2~r

o

~ 4 .

~+3.

6 2,

at2 o

01

7

�9 0 ""-0 ~ , o ~ ~ - - o �9 "-~tChl a/b / Chl a/8-ca~:

%% /

I

/ , ~ ~ "" , .~" , ~ / 'Chl /Total Ca~

/ . ~ ,-..~ .1

o ~ .o - . . - o / /

@~ -4l-- "-~

I ~ ~ 1 2 3 4

Length up laminae (cm.)

.o

-12~

s

4~ 6

x3 .15 E

c~ ,10. 'E E

.05"

~-0 o

8

Lutein/ \,, / \

II / \

/ / @ \ 7 / " "*.Vi~

/.:" aid".. ', ,,,

~=.~_z.." ~d-, .eo a~,,: ----~ 1 2 3

Length up laminae (cm,)

c i

�9 7 03 -

rv

,~ 02-

o

Eo .01

ATP ,~'" .~ ".�9 {

" , ,~ . . " Energy Charge �9 : . t ~ . ~ � 9

, . .- �9 ~ ' ~ I . ~ ~ ~ ' ~ t l . . . . . �9

C3. �9 .15 ,7

~'4 .

.lO ~ 3

-.05 o ~ 1

&0

o

-'8~c

.6 7

9

}~ .~" ~)~"~"- g-Am,,lase �9 , , : ....... "r

Staroh''., A synthetase....|../ -

I I I I I 2 3 4 5 6

Length up laminae (cm)

~12.

?=.8- E

n ~ . 4 -

0 0

11

, ~ - . . ~,~o4 ~- -~ # X ")~

2 .......... fs':

1 0 Length up laminae (cm.) Length up laminae (era.)

c

..15

-.13'~ -?

~_c

".11 s <

- . 09~ o 0 -'

-.3 a_ 'I21 E

..2~ d~

-.1 -~

Fig. 2. Maximum plastid areas (top 10%), numbers of plastids and percentage of plastids containing PTBs along the laminae of 7-day light-grown barley seedlings

Fig. 3. Total number of cells per 0.5 cm segment and the areas of internal plastid membranes along similar leaf blades

Figs. 4 and 5. Maximum starch (Fig. 4) and mitochondrial (Fig. 5) areas (top 10%) and the accumulated starch (Fig. 4) or mitochondrial areas (Fig. 5) to total plastid area ratios along the laminae of 7-day light-grown barley seedlings

Fig. 6. Amounts of chlorophylls a and b expressed as logarithmic amounts per segment and on a thylakoid area basis along the laminae of 7-day light-grown barley seedlings

Fig. 7. Chlorophyll a/b, chlorophyll a//3 carotene and chlorophyll a + b/total carotenoid ratios along similar leaf blades

Fig. 8. Amounts of different carotenoids expressed on a thyiakoid area basis along the leaf seedlings

Fig. 9. Levels of activity of starch synthetase and cz- and fl-amylases along similar laminae

Fig. 10. Amounts of ATP and the energy charge ratios along the laminae of 7-day old light-grown barley seedlings

Fig. 11. Levels of inorganic phosphate and 3-PGA along similar leaf blades

blades of 7-day old light-grown barley

264 A.R. Wellburn et al. : Light-grown plastid development

was only slightly higher (4.8) nearer the meristem than in final mature tissue (3.5); quite unlike the precipitous decline of the chlorophyll a/b ratio from above 20 observed in greening tissues. Figure 7 also demonstrates the usefulness of chlorophyll/carotenoid ratios to follow the developmental process. Of the two shown, the newer expression of chlorophyll a t o fi-carotene (Lichenthaler et al. 1981) reaches a steady equilibrium from 2.75 cm upwards and better illustrates the transformations taking place along the leaf as the appropriate protein-pigment complexes are formed to cope with the low light environment. When the carotenoids are expressed on a membrane area basis (Fig. 8), lutein and neoxanthin appear to be concentrated with chlorophyll a (cf. Fig. 6) at 2.75 cm whilst the other two, fi-carotene and violaxanthin, are concentrated within the thylakoids earlier (i.e. lower down at 2.25 cm).

The remaining 3 Figs. 9-11 are of special interest to the starch distribution throughout the seedlings. Figure 9 shows the levels of activity of three of the main constitutive enzymes of starch metabolism ex- pressed on a protein basis. Both c~- and fl-amytase activities rise quickly to a peak around the late amoe- boid phase (1.25-1.75cm) and thereafter decline. Starch synthetase by contrast is highest near the mer- istem, falls dramatically at the amoeboid stage, with a late rise from reduced levels towards the tip.

Levels of nucleotides fluctuate along the laminae although a steady energy charge ratio is maintained along the blade (Fig. 10). The pool sizes of all adenine nucleotides (ADP and AMP not shown) are reduced over the middle range of segments (1.25-3.75 cm) where plastid division and pigment accumulations are greatest. Levels of ATP (Fig. 10) are highest in the early proplastid stages when the mitochondria/plastid ratios are high and later when photophosphorylation is possible.

The regulatory enzyme of starch biosynthesis, adenosine diphosphate glucose pyrophosphorylase (Ghosh and Preiss 1966) is activated not only by ATP but also by 3-phospho-glycerate (3-PGA) in an altos- teric manner and strongly inhibited by phosphate. Figure 11 shows that reduced levels of phosphate oc- cur in early proplastid stages when 3-PGA is high but that these concentrations are reversed over most of the regions where the amoeboid stages are con- verted into mature plastids. Only towards the tip are more favourable ratios re-established.

Discussion

In clear impression from these studies is that a well- ordered and inter-related progression of biochemical and ultrastructural changes occur along the monoeo-

tyledonous blade which may be recognized structur- ally and functionally as distinct phases (1 to 5), using the classification suggested by Whatley (1977).

The region (I) immediate above the meristem where vacuolation has just ceased contains eoplasts with little membrane or phosphate and high PGA levels - but no starch. The elevated levels of ATP may be associated with the high ratio of mitochondria to plastids. Furthermore, conditions appear to be fa- vourable for the synthesis of starch by activation of ADP glucose pyrophosphorylase leading to the accu- mulations characteristic of the amyloplast region above (2).

After this starch-rich region (2) the situation is rapidly reversed even with respect to the levels of activity of both starch synthetase and amylase en- zymes. Although phosphate rises, ATP and 3-PGA levels are most likely maintained by starch hydrolysis within the plastids rather than by mitochondrial activ- ity. The products and derived energy from the starch are probably utilized by the amoeboid proplastids (3) to divide and by the immature plastids (4) to develop grana initials and early accumulations of pig- ments. It is over these two phases that low levels of ATP (and 3-PGA) occur although overall energy charge ratios are maintained. This implies a signifi- cant reduction of sizes of all nucleotides when de- mands for transcription are likely to be greatest as components for photosynthesis and related activities are synthesized.

Individual stages within the mature chloroplasts (5) are more difficult to define. Studies of pigment distribution within membranes indicate a progressive change in composition. Early predominance of fl-car- otene and violaxanthin gives way to chlorophyll a and lutein (plus neoxanthin) with chlorophyll b added more gradually. Later additional membrane forma- tion spreads the pigments over a greater area.

Semi-crystalline inclusions have been observed at all 5 phases. Isolated PLBs from etiolated and green- ing tissue have been shown to be associated with pro-thylakoid vesicles (PTVs; Weltburn et al. 1977) and only the latter appear to be associated with pho- tochemical development (Wellburn and Hampp 1979). When similar preparations of internal plastid membranes were made from different segments of light-grown barley (1.25-2.25 cm), no trace of crystal- line-like bodies were found associated with the lamel- lae whilst examining over 1,000 lamellar systems in the electron microscope. This implies a high degree of instability of PTBs under conditions in which etio- plast or etiochloroplast membranes readily retain PLBs (Wellburn et al. 1977). It is possible that PTBs are converted completely into PTVs in vitro. We often found vesicles adhering to where PTBs ought to have

A.R. Wellburn et al. : Light-grown plastid development 265

been by comparison to the in situ appearance of the lamellar systems, If this is so then PTBs are probably whoMy engaged in photochemical assembly and are not dual systems like PLBs which contain additional material such as steroidal saponins (Kesselmeier and Budzikiewicz 1979).

Light-grown systems such as that described here resemble more closely the development of greening algal mutants (Wellburn et al. 1980) rather than etio- lated tissue and offer the prospect of study of many interesting features of plastid morphogenesis as they relate to plants in the field. The involvement of mito- chondria and the early importance of respiratory ac- tivity await further study. The commonality of the r61e of starch in providing metabolites and energy and the nature of the source of the starch clearly deserve more detailed attention than could be encom- passed within this initial study. Other questions which suggest themselves include the sequence of formation and the composition of the chlorophyll-carotenoid- protein complexes, the function of the PTBs and the controlling inter-relationships between the different plant hormones.

References

Abbott, I.R., Matheson, N.K. (1972) Starch depletion in germinat- ing wheat, wrinkled-seeded peas and senescing tobacco leaves. Phytochemistry 11, 1261-1272

Baker, N.R., Leech, R.M. (1977) Development of photosystem I and photosystem II activities in light-grown maize (Zea mays). Plant Physiol. 60, 640-644

Boffey, S.A., Selld6n, G., Leech, R.M~ (1980) Influence of cell age on chlorophyll formation in light-grown and etiolated wheat seedlings. Plant Physiol. 65, 680-684

Chapman, D.J., Leech, R.M, (1979) Changes in pool sizes of free amino acids and amides in leaves and plastids of Zea mays during leaf development. Plant Physiol. 63, 567 572

Esau, K. (1953) Plant Anatomy. Wiley & Sons. New York GaudilI+re, J.P. (1974) Amelioration du dosage spectrophotome-

trique des chlorophyll a e t b e t des carot~noides totaux darts des extracts foliares. Physiol. Veg. 12, 585-599

Ghosh, H.P., Preiss, J. (1966) Adenosine dipbosphate glucose pyro- phosphorylase. A regulatory enzyme in the biosynthesis of spin- ach leaf chloroplasts. J. Biol. Chem. 241, 4491-4504

Hampp, R., Wellburn, A.R. (1980) Translocation and phosphory- lation of adenine nncleotides by mitochondria and plastids dur* ing greening. Z. Pflanzenphysiol, 98, 289-303

Hampp, R., Ziegler, H. (1980) On the use of Arena protoplasts to study chloroplast development. Planta 147, 485494

Hawke, J.C., Rumsby, M.G., Leech, R.M. (1974) Lipid biosynthe- sis in green leaves of developing maize. Plant Physiol. 53, 555-561

Kasemir, H. (1979) Control of chloroplast formation by light. Cell Biol. Int. Rep. 3, 197-2[4

Kesselmeier, J., Budzikiewicz, H. (1979) Identification of saponins as structural building units in isolated prolamellar bodies from etioplasts of Ave~a sativa L. Z. Pflanzenphysio[. 91,333-344

Leese, B.M., Leech, R.M., Thompson, W.W. (1971) Isolation of plastids from different regions of developing maize leaves. In : Proc. of IInd Int. Congress on Photosynthesis, vol. 3, pp. 1485-1493, Forti, G,, Avron, M., Melandin, A., eds. Junk, The Hague

Lichtenthaler, H.K., Burkard, G., Kuhn, G., Prenzel, U. (1981) Light-induced accumulation and stability of chlorophylls and chlorophyll-proteins during chloroplast development in radish seedlings. Z. Naturforsch. 36, 421420

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with Folin Phenol reagent. J. Biol. Chem. 193, 265-275

Newcomb, E.H., Mersey, B.G., Cline, K.C. (1981) Plastid tubules associated with the inner membrane of the chloroplast envelope. In: Proc. of Vth Int. Congress on Photosynthesis, Akoyunog- lou, G., ed. (in press)

Ozbun, I.L., Hawker, J.S., Preiss, J. (1972) Soluble adenosine di- phosphate glucose-e-l,4-glucan-~-4-glycosyl-transferase from spinach leaves. Biochem. J. 126, 953-963

Platt-Aloia, K.A., Thomson, W.W. (1977) Chloroplast develop- ment in young sesame plants. New Phytol. 78, 599 605

Rascio, N., Orsenigo, M., Arboit, D. (1976) Prolamellar body transformation with increasing cell age in the maize leaf, Pro- toplasma 90, 253-263

Robertson, D., Laetsch, W.M. (t974) Structure and function of developing barley plastids. Plant Physiol. 54, 148-159

Taussky, H.H., Shorr, E. (1953) A microcolourimetric method for the determination of inorganic phosphorous. J. Biol. Chem. 202, 675-685

Weliburn, A.R., Hampp, R. (1979) Appearance of photochemical function in prothylakoids during plastid development. Biochim. Biophys. Acta, 547, 380-397

Wellburn, A.R., Higginson, C., Robinson, D., Walmsley, C. (1981) Biochemical explanations of more than additive inhibitory ef- fects of low atmospheric levels of sulphur dioxide plus nitrogen dioxide upon plants. New Phytol. 88, 223-237

Wellburn, A.R., Quail, P.H., Gunning, B,E.S. (1977) Examination of ribosome-like particles in isolated prolamellar bodies. Planta 134, 45 52

Wellburn, F.A.M., Wellburn, A.R. (1979) Conjoined mitochondria and plastids in the barley mutant 'Albostrians'. Planta 147, 178-179

Wellburn, F.A.M., Wellburn, A.R., Senger, H. (1980) Changes in ultrastructure and photosynthetic capacity within Scenedes- mus obliquus mutants C-2A', C-6D and C-6E on transfer from dark grown to illuminated conditions. Protoplasma 103, 35-54

Whatley, J.M. (1974) Chloroplast development in primary leaves of Phaseolus vulgaris. New Phytol. 78, 1097-i 110

Whatley, J.M. (1977) Variations in the basic pathway of chloroplast development. New Phytol. 78, 407420

Wrischer, M. (1966) Nenbildung yon Prolamellark6rpern in Chloroplasten. Z. Pflanzphysiol. 55, 296-299

Received 8 October; accepted l December 1981