Fine Structure of the Chromoplasts of Fruit of Solanum aviculare

Aust. J. Bot., 1978,26,783-92
Fine Structure of the Chromoplasts of Fruit
of Solanum aviculare Forth. var. brisbanense
D. J. SimpsonAyB,M. R. BaqarA3C
and T. H. LeeA
A
School of Food Technology, University of New South Wales,
Kensington, N.S.W. 2033.
Present address: Department of Physiology, Carlsberg Research Centre,
Gamle Carlsberg Vej 10, DK-2500 Valby, Copenhagen, Denmark.
Present address: Department of Chemical Technology,
Papua New Guinea University of Technology, Lae, P.N.G.
Abstract
Chromoplasts of ripe fruit of Solanum aviculare contain a large number of electron-translucent
structures, which distinguishes them from the chromoplasts of many other species. During the
chloroplast-chromoplast transformation, starch and grana disappear and plastoglobules accumulate.
As ripening progresses, the plastoglobules fill with increasing amounts of electron-translucent
structures which then protrude from the plastoglobules and eventually form a single small slab-shaped
structure, and the plastoglobule disappears. B-Carotene (86.4%) is the main carotenoid of the ripe
fruit, and small amounts of lutein, zeaxanthin, phytofluene, mutatochrome and neoxanthin are
present. On the basis of carotenoid composition and appearance under the electron microscope,
it is concluded that the translucent structures consist of a crystalline form of bcarotene.
Introduction
Chromoplasts have been classified into five categories on the basis of ultrastructure,
particularly those structures of the chromoplasts in which the carotenoid pigments
are deposited. The five classes of chromoplasts are fibrillar, crystalline, globular,
membraneous and reticulo-tubular (Sitte 1974).
The ultrastructural differences between crystals of lycopene and p-carotene in
chromoplasts are sufficiently large to be distinguished with the electron microscope
(Harris and Spurr 1969a, 1969b). In addition, p-carotene appears to crystallize in
the plastoglobules of the chromoplasts of some plant species (Harris and Spurr
19690; Devidt 1970; Harris 1970; Wrischer 1972), and these structures are quite
different from other carotene crystals in chromoplasts. Thus, sufficient criteria exist
to justify the classification of crystalline chromoplasts into at least three subcategories,
i.e. lycopene crystals, large p-carotene crystals, and small p-carotene crystals originating from plastoglobules.
Chromoplasts with structures resembling, but different from, the p-carotene crystals
which develop from plastoglobules of the chromoplasts of fruit of the high beta tomato
mutant have been reported in the fruit of Solanum pseudocapsicum (Salema 1968) and
in the receptacle of yellow-fruited peaches (Eymt 1971). A survey of tissues containing
chromoplasts revealed that the fruit of an orange-fruited variety of Solanum aviculare
contained spindle-shaped chromoplasts with an ultrastructure resembling that of S.
pseudocapsicum. The ontogeny and ultrastructure of these chromoplasts were investigated and correlated with their carotenoid composition.
D. J. Simpson et al.
Materials and Methods
Seeds of S. aviculare were germinated in moist sand and grown under glasshouse
conditions as described by Simpson et al. (1974). Fruits were sampled for electron
microscopy at the mature green, breaker and full-ripe stages of development. Tissue
was fixed for electron microscopy in PIPES-NaOH buffer according to the procedure
described by Simpson and Lee (1976). Fully ripe, orange-coloured fruits were frozen
and stored at - 10°C pending analysis of their carotenoid composition (Simpson et
al. 1974).
Results
The plastids of mature green fruit contained one or more large starch granules which
occupied most of the plastid volume. The thin layer of peripheral stroma contained
small grana connected by stroma lamellae. The onset of ripening was reflected in the
plastids, which began to accumulate uniformly electron-dense plastoglobules. As
ripening proceeded, the plastids lost starch and grana and the stroma increased in
volume and contained an increasing number of electron-dense plastoglobules (Fig. 1).
Subsequently, the plastoglobules developed electron-translucent regions of differing
shapes and sizes (Figs. 2, 5, 6). In the early stages of chromoplast development, the
electron-translucent regions were small and did not distort the shape of the plastoglobules. More than one of these regions was frequently present in each plastoglobule,
and the orientation or position of these regions in different plastoglobules was not
consistent (Fig. 3). Sometimes, however, the electron-translucent region was a single,
central cylindrical rod which protruded beyond the plastoglobule and elongated it
(Fig. 4). More usually, two or more of these regions developed within or at the periphery of the plastoglobule, often distorting its outline (Fig. 4). As the fruit continued
to ripen, a progressively greater proportion of the interior of the plastoglobules was
occupied by electron-translucent rods, usually with approximately parallel sides and
orientated in similar directions to one another within the same plastoglobule (Fig. 6).
The fully mature chromoplasts (Figs. 7, 8) contained electron-transparent structures which had developed from the electron-translucent regions of the plastoglobules.
These structures were either long thin rods with parallel sides or broad slabs with
non-parallel sides the same length as the rods (Fig. 7). Both types had an electrondense border and were probably the same structure viewed from different angles of
section. By this stage, most of the electron-dense material of the plastoglobules had
disappeared, although some was still visible in a few chromoplasts (Fig. 8). Another
feature of mature chromoplasts was the presence of numerous small vesicles in an
otherwise dense stroma (Fig. 8), as well as an electron-dense membraneous body
resembling a thylakoid plexus. The vesicles could first be seen when the electrontranslucent regions began to form within the plastoglobules (Fig. 6). Developing and
mature chromoplasts frequently contained one or more electron-transparent regions
that had a finely fibrillar ultrastructure and resembled the areas of presumptive DNA
often seen in chloroplasts. Under the light microscope, the mature chromoplasts
Fig. 1. Chromoplast from pale orange S. aviculare fruit. Grana have disappeared and electrondense plastoglobules have accumulated. x 28,200. Scale: 1.0 pm.
Fig. 2. Chromoplast from a cell layer deeper below the surface of pale orange fruit in which the
plastoglobules have developed electron-translucent regions. x 35,000. Scale: 1 . 0 pm.
Structure of Chromoplasts of Solanum auiculave
786
D. J. Simpson et al.
Structure of Chromoplasts of Solanum at.iculave
appeared to be of a bright orange colour. They were generally round in contour with
a grana-like appearance and occasionally showed elongated extremities, which gave
them a spindle shape.
Electron micrographs of the unusual plastoglobules of the chromoplasts of S.
aviculare fruits, taken at higher magnification, are shown in Figs. 9-12. The electrontranslucent material, which was usually circular in transverse section (Fig. 3), was
often found in several parallel rods within each plastoglobule (Figs. 5, 6, 9), although
at more advanced stages it protruded beyond the boundary of the plastoglobule (Fig.
10) with further development. The electron-dense material of the plastoglobule was
spread out along the electron-translucent rods (Fig. 11) and then diasppeared altogether, leaving the long rods or slabs with an electron-dense border free in the stroma
(Fig. 12).
The major carotenoid of the mature fruit was P-carotene, with small amounts of
its oxygenated derivatives zeaxanthin, neoxanthin and mutatochrome, as well as
lutein and phytofluene (Table 1). The total concentration of carotenoid was high
(1293 pg/g fresh wt.), so that the level of p-carotene within each chromoplast would
also be high.
Table 1. Carotenoid composition of ripe fruit of
Solanum aviculare
Carotenoid
Phytofluene
8-Carotene
[-Carotene
Mutatochrome
Lutein
Zeaxanthin
Neoxanthin
Percentage of total carotenoid
3
86.4
trace
1.2
5
4
0.4
Total carotenoids 1293 pg/g fresh wt.
Discussion
The electron-translucent structures which characterize the chromoplasts of the
fruit of S. aviculare have been shown to originate in, and develop from, the plastoglobules. In the mature chromoplast these structures occupy a large proportion of
the plastid volume, and no other features are found in these chromoplasts in which
carotenoids are known to be localized. The plastoglobules of chromoplasts are known
to contain carotenoids (Lichtenthaler 1970a, 1970b), and it has been suggested that
p-carotene crystallizes out of the plastoglobules of the chromoplasts of ripe high beta
tomato fruit (Harris and Spurr 1969~).In view of the high concentration of p-carotene
Figs. 3-6. Progressive stages of development of crystalloid structures from plastoglobules.
Fig. 3. Plastoglobules in chlorochromoplast of turning fruit showing an early stage of the formation
of electron-translucent regions, x 44,800. Scale: 0.5 pm.
Fig. 4. Plastoglobules from which electron-translucent, fibril-like structures are protruding.
x 44,800. Scale: 0.5 pm.
Figs. 5, 6. Plastoglobules containing several electron-translucent regions which occupy an increasingly larger proportion of the plastoglobules as ripening proceeds. x 44,800. Scale: 0.5 pm.
D. J. Simpson et al.
in S. aviculare fruit, in which it accounts for 86.4 % of the total carotenoids (Table I),
it seems plausible that the electron-translucent structures in the plastoglobules are
partially or wholly p-carotene. Carotenoids are not osmiophilic compounds, since
plastoglobules isolated from chromoplasts and rich in carotenoids are electron-translucent (Lichtenthaler 1970~).The crystalloid structures observed in high beta tomato
fruit chromoplasts have been assumed to consist of crystalline p-carotene (Harris and
Spurr 1969a), and are also electron-translucent.
The initial stages of fibril formation from plastoglobules in fibrillar chromoplasts
are similar to early stages in the development of some of the structures in S. aviculare
chromoplasts (Fig. 4). These structures are not developing fibrils, however, since
fibrils are not found in mature chromoplasts. In addition, only one fibril develops
from each plastoglobule in most fibrillar chromoplasts (except asparagus fruit,
Simpson et al. 1977b), whereas usually two or more electron-translucent regions are
seen in each plastoglobule of chromoplasts of S. aviculare fruit.
The electron-dense border, which persists when these structures are found free in
the stroma, is possibly a coating of plastoglobule material, since it cannot be distinguished inside plastoglobules. It is also conceivable that the electron-dense material
is due to surface staining by osmium tetroxide or a densely staining stroma component,
but it is clear that they are not membranes. The ease of fixation of these structures is
surprising in view of the extreme difficulty in fixing large crystals of /?-carotene
(Ben-Shaul et al. 1968) or lycopene (Harris and Spurr 1969b). The uniformity of shape,
however, suggests that they are crystalline, and their small and uniform size is probably
a consequence of having originated from large numbers of small plastoglobules. The
appearance of electron-translucent regions occurs almost synchronously in all plastoglobules and they develop at similar rates, presumably until carotene synthesis is
limited by precursor availability. This results in the formation of a large and uniformsized population.
The chromoplasts of the fruit of S. aviculare and S , pseudocapsicuvlz (Salema 1968)
and the orange receptacle of Prunus persica (EymC 1971) are strikingly similar in
ultrastructure and sufficiently different from all other types to justify placing them in
a separate subcategory. The chromoplasts of high beta tomato fruit (Harris and Spurr
1969~)are similar but different from those exemplified by S. aviculare. The chromoplasts of the anther of Raphanus (Dickinson 1973; Dickinson and Lewis 1973) may
also contain crystalline /?-carotene, but the electron-translucent regions in the plastoglobules disappear as the chromoplasts mature and they do not develop into the
structures seen in S. aviculare chromoplasts.
It has been shown that for fibrillar chromoplasts of Capsicum at least (Simpson
et al. 1977a), the formation of plastoglobules does not depend on the disintegration
of photosynthetic lamellae, so their components are being actively synthesized during
chromoplast development. The site of synthesis of carotenoids within the chromoplasts has not yet been determined, nor is it known how carotenoids are deposited
Fig. 7. Mature chromoplast from ripe fruit. The chromoplast stroma is almost completely occupied
by the numerous small, electron-translucent structures that have developed from plastoglobules.
These structures are thin and surrounded by an electron-dense border when sectioned transversely,
and wider with less parallel sides and more diffuse edges when sectioned longitudinally, x 23,200.
Scale: 1 . 0 fim.
Fig. 8. As in Fig. 7, showing the presence of electron-transparent vesicles in the chromoplast
stroma. x 20,000. Scale: 1. 0 pm.
Structure of Chromoplasts of Solanum aviculave
D. J. Simpson et al.
Figs. 9-12. High-magnification electron micrographs showing the later stages in the development
of the electron-translucent structures from plastoglobules. x 127,000. Scale: 0.2 pm.
Structure of Chromoplasts of Solanum aviculare
in the different forms in which they are found in chromoplasts. Eilati et al. (1972)
have postulated that the accumulation of xanthophylls in plastoglobules is facilitated
by their esterification in chromoplasts, thereby increasing their lipophilic nature.
Perhaps when esterification is not possible, as with carotenes, increased synthesis
leads to crystallization.
The ripe fruits of S. aviculare are characterized by a high concentration of /-carotene,
which is typical of tissue in which the chromoplasts contain crystalline /-carotene. An
intriguing question is what determines whether the crystals form as large structures
in the stroma, as in carrot root, or in plastoglobules, as in fruit of high beta tomato,
or S. aviculare.
Acknowledgments
We wish to thank Dr M. R. Dickson, Mr A. B. Martin and Mrs K. Greenland
of the Biomedical Electron Microscope Unit, University of New South Wales, for
their assistance and advice. Seeds of S. aviculare were kindly supplied by Mr D. E.
Symon, Department of Agronomy, Waite Agricultural Research Institute, Adelaide.
We are grateful for financial assistance during the course of this work in the form of
a CSIRO Post-graduate Studentship to D. J. S. and a Colombo Plan Fellowship
awarded by the Australian Government to M.R.B.
References
Ben-Shaul, Y., Treffry, T., and Klein, S. (1968). Fine structure of carotene body development.
J. Micvosc. (Oxford) 7, 265-74.
DevidC, Z. (1970). Ultrastructural changes of plastids in ripe fruit of Cuvcurbita pep0 var. ovifeva.
Acta Bot. Croat. 29, 57-62.
Dickinson, H. G. (1973). The role of plastids in the formation of pollen grain coatings. Cytobios 8,
25-40.
Dickinson, H. G., and Lewis, D. (1973). The formation of the tryphine coating the pollen grains
of Raphanus, and its properties relating to the self-incompatibility system. Pvoc. R. Soc. London,
Ser. B 184, 149-65.
Eilati, S. K., Budowski, P., and Monselise, S. P. (1972). XanthophyIl esterification in the flavedo
of citrus fruit. Plant Cell Physiol. 13, 741-6.
EymC, J. (1971). La structure des plastes dans les parenchymes des coupes rkeptaculaires florales
du Picher Pvunus pevsica chez les variCtCs a fruits A chair blanche et A chair jaune. C.R. Acad.
Sci., Ser. D 272, 1232-5.
Harris, W. M . (1970). Chromoplasts of tomato fruits. 111. The high-delta tomato. Bot. Gaz. 131,
163-6.
Harris, W. M., and Spurr, A. R. (1969~).Chromoplasts of tomato fruits. I. Ultrastructure of lowpigment and high-beta mutants. Carotene analyses. Am. J. Bot. 56, 369-79.
Harris, W. M., and Spurr, A. R. (19696). Chromoplasts of tomato fruits. 11. The red tomato.
Am. J. Bot. 56, 380-9.
Lichtenthaler, H . K. (1970~).Formation and function of plastoglobuli in plastids. 7th Congr. Int.
Microsc. Electr., GrenobIe, Vol. 3, pp. 205-6.
Lichtenthaler, H. K. (1970b). Die Lokalisation der Plastidenchinone und Carotinoide in den Chromoplasten der Petalen von Sarothamnus scoparius (L.) Wimm ex Koch. Planta 90, 142-52.
Salema, R. (1968). Amido. estudo ultrastruttural da sua biogknese em plantas superiores. Broteria,
Sev. Cienc. Nut. 38. 1-126.
Simpson, D. J., Baqar, M. R., and Lee, T. H. (1977~).Chemical regulation of plastid development.
111. Effect of light and CPTA on chromoplast ultrastructure and carotenoids of Capsium annuurn.
Z. Pflanzenphysiol. 82, 189-209.
Simpson, D. J., Baqar, M. R., and Lee, T. H. (1977b). Fine structure and carotenoid composition
of the fibrillar chromoplasts of Asparagus oficinalis L. Ann. Bot. 41, 1101-8.
D. J. Simpson et al.
Simpson, D. J., and Lee, T. H. (1976). Plastoglobules of leaf chloroplasts of two cultivars of Capsicum
annuum. Cytobios 15, 139-47.
Simpson, D. J., Rahman, F. M. M., Buckle, K. A., and Lee, T. H. (1974). Chemical regulation of
plastid development. 11. Effect of CPTA on the ultrastructure and carotenoid composition of
chromoplasts of Capsicum annuum cultivars. Aust. J. Plant. Physiol. 1, 135-47.
Sitte, P. (1974). Plastiden-Metamorphose und Chromoplasten bei Chrysosplenium. Z. Pflanzenphysiol.
73, 243-65.
Wrischer, M. (1972). Transformation of plastids in young carrot callus. Acta Bot. Croat. 31, 41-6.
Manuscript received 6 February 1978