1967 117

The Reversion of Chromoplasts to Chloroplasts in Valencia Oranges

W. W. Thomson, L. N. Lewis and C. W. Coggins

Departments of Life Sciences and Horticultural Sciences, University of California, Riverside, Cal., U. S. A.

Received April, 28, 1966

Introduction

Schimper (1885) stated that chromoplasts, leucoplasts, and chloroplasts develop from each other, and gradually this concept gained general acceptance (Straus 1953). However, Frey-Wyssling, Ruch, and Berger (1955) questioned this concept. They proposed the following scheme for plastid development:

They maintained that development was in one direction, and that there was no reversion of chromoplasts to chloroplasts, or chloroplasts to leucoplasts, as was postulated by Schimper. They pointed out that once a leaf, which was fully developed, loses its green coloring and becomes mottled or chlorotic, it could not again become green. A young leaf that was never green because of a lack of certain elements could become green when supplied the deficient elements. In the first case, the chloroplasts were fully developed and then changed to chromo plasts. In the second case, the plastids had not developed but were arrested along the path of metamorphosis and therefore could continue their development once the proper conditions were present. In southern California, the rind of Valencia oranges reaches maximum orange color during the winter months but tends to regreen during the spring and summer. This regreening is partly influenced by temperature (Caprio, 1956) and nutrition (Jones and Embleton, 1959). Potassium gibberellate also enhances regreening (Coggins, Hield, and Garber, 1960, Coggins and Lewis 1962). There are two mechanisms which could account for reversion of orange colored fruit to the green state: one, the chromoplasts revert to chloroplasts -which would support Schimper's contention; or two, new chloroplasts are formed in some manner (MacKinney 1961), which would be support for the concept of Frey-Wyssling et al. This study of the ultrastructural changes in plastids during regreening of Valencia oranges was undertaken to determine which of the above theories was valid for this fruit.

Material and methods

Valencia oranges (Citrus sinensis L.), growing on large trees at Riverside, were used in this experiment. Pairs of fruit of approximate uniformity in color, size, and common light ex posure were selected. One fruit of each pair was dipped for about 30 seconds in 500 ppm acid equivalent potassium gibberellate solution containing a small amount of X-77 wetting agent (Coggins and Lewis 1962). The orange fruits were treated in mid-summer and rind samples were taken every two weeks for four months until the control fruits were puffy and generally overripe or senescent in appearance. Samples were taken from the middle (equator) of all fruits, and some samples were taken from the greener region near the button of the regreened fruit. 118 W. W. Thomson, L. N. Lewis and C. W. Coggins Cytologia 32

The rind was peeled from the fruit and most of the inner, colorless portion of the rind, the mesocarp, was trimmed away with a razor blade, The outer, colored part of the rind, the epicarp, was diced into small, approximately 2 mm square sections. These sections were fixed for 18-24 hours in 1% osmium tetroxide buffered at pH 7.0 with a phosphate buffer. The material was dehydrated in acetone and embedded in Maraglas (Bisalputra and Weier 1963). Thin sections were cut on a Porter-Blum MT-2 ultramicrotome, picked up on uncoated or Formvar coated grids, and studied with a Hitachi HU 11 electron microscope. The sections were stained on the grids with either 1% Ba(KMnO4)2 for 10-20 minutes or with a saturated aqueous solution at uranyl acetate for one hour followed by lead citrate (Venable and Cogge shall 1965) for 15-30 minutes.

Observations

Chromoplasts in the orange fruit contained many large, electron dense globules (Fig. 1, l). These gloubles varied in size, but often were as much as 800 mu in diameter. The chromoplasts contained few membranes or lamellae and the membranes were frequently quite long and extended inward from the chromoplast membrane (Fig. 1, arrow). In the yellow-green fruits that had started to regreen, the plastids showed several changes from the mature chromoplasts. They still possessed many large electron-dense globules (Figs. 2, 3 and 4, 1) but several small grana were present (Figs. 2, 3, and 4, g). Long invaginations of the inner part of the plastid membrane were often observed in these plastids (Fig. 2, arrow) and some of these invaginations were associated with the developing grana. Small vesicles were observed which appeared to have formed from the inner part of the plastid membrane (Fig. 3, arrows). Small vesicles were also found free in the peripheral stroma (Fig. 3) and some vesicles had a close association with the granal membranes (Figs. 2, 3 and 4, v). Frequently, the fret membranes were attached or closely associated with the electron dense globules (Fig. 3, c). Plastids in the fruits that had regreened to a light green color had well developed grana (Figs. 5 and 6, g) and fret membranes (Figs . 5 and 6, f). The amount of grana-fretwork system showed a wide range of variation in the different plastids. Some contained only a relatively small amount of internal membranes (Fig. 5) while in other plastids the grana-fretwork system occupied a great portion of the plastid volume (Fig. 6). These plastids contained many electron dense globules (Figs. 5 and 6, 1) and frets also extend to and were associated with osmophilic globules (Fig. 6, arrows) . The regreened fruits taken at the time the experiment was terminated were green to light green in color, but none were as deep green as immature fruits. The chloroplasts in these fruits showed a wide variation in structure . Many were virtually identical to those in the lightly regreened fruit (Figs . 5 and 6). These had a well developed grana-fretwork system and several large electron -dense globules. In other chloroplasts, the globules were considerably reduced in size and number (Fig. 7, 1), and there was a considerable development of the grana fretwork system. The general ultrastructure of these chloroplasts was similar to that of chloroplasts in a deep green immature fruit (Fig. 8). 1967 The Reversion of Chromoplasts to Chloroplasts in Valencia Oranges 119

Discussion

As men tioned in the introduction, Frey-Wyssl ing, Ruch, and Berger (1955) have suggested that the reversion of one plastid form to an earlier deve lopmental stage does not occur, but evidence in support of their theory is not deci sive. Toyama and Ueda (1965) fol lowed the development of chromo plasts from chloroplasts in aging Ginkgo leaves and they evidently did not find any indication of Figs. 1 and 2. 1, chromoplast from a mature orange fruit. Note the a reversal of large osmiophilic globules, 1, and the sparse amount of membrane chromoplasts within the plastid. The arrow indicates the attachment of a long membrane invagination with the plastid membrane. Starch, st. •~30,000 to chloro 2, a chromoplast from a slightly regreened orange. Note the many vesi plasts. They cles, v, in close association with the internal membranes. The arrow concluded indicates the invagination of the inner portion of the plastid membrane. Osmiophilic globules, l, grana, g•~36,700. that Ginkgo 120 W. W. Thomson, L. N. Lewis and C. W. Coggins Cytologia 32 chromoplasts can not revert to chloroplasts. Orsenigo (1963)studied the bleaching of the green tepals of Hyacinthus and found that during the change from green to white the membrane system within the chloroplast, (i.e. grana and intergranal membranes) gradually dis appears and that in the bleached tepals the limiting membrane of the plastids disintegrates. He concluded that the chlo roplasts did not revert to leucoplasts during this transforma tion. How ever, many of the micro graphs of plastids in the partially bleached tepals are virtually identical in structure to Figs. 3 and 4. 3, chromoplast from a yellow-green furit. Small vesicles proplastids apparently pinching off from the plastid membrane, arrows. Many and indeed vesicles are free in the peripheral stroma and others, v, are closely

associated with the developing grana, g. Note the close association of leucoplasts frets at c with the osmiophilic globules, l. Starch, st •~23,000. 4, (Granick another plastid from a slightly regreened fruit. Small grana are present, 1961) and, g, and some vesicles, v, are closely associated with the developing grana. Starch, st., osmiophilic globules, 1. •~18,000. although the chloroplasts eventually break down during the bleaching of the tepals, they appear to revert to leucoplasts during this transformation. All evidence from our studies indicates that during regreening of Valencia oranges the chromoplasts revert to chloroplasts. 1967 The Reversion of Chromoplasts to Chloroplasts in Valencia Oranges 121

Osumi (1961) described the chromoplasts from the juice sacs of Citrus unshiu as spindle-shaped with the carotenoid apparently bound to long fibrils within the chromoplast. However, the most characteristic feature of the mature chromoplasts from the rind of Valencia oranges is numerous large osmiophilic globules. The plastids in the yellow green and light-green fruits that had regre ened also contained several large osmiophilic globles as well as moderate to well deve loped grana fretwork systems. Thus, the

plastids in the fruits that had regreened to a light green color repre sent a form between chromo

plasts and Figs. 5 and 6. 5, chloroplast from a light green fruit that had regrecned. chloroplasts. Note the many, small but well developed grana, g and the intergranal con

In only a nections, the frets, f. Starch, st., osmiophilic globules, _??_. •~25,800. 6, a few of the chloroplast from a light green fruit that had regreened which contain., large grana, g, and a well developed fret system, f. Note the large osmiophilic chloroplasts globules, l, and the close association of frets, arrows, with some of these in the regre globules. •~25,000. ened fruits is there a reduction in the number of globules. The latter chloroplasts have a fully developed grana-fretwork system and closely resemeble the structure of chloroplasts in immature green fruits. This series of transition forms between chromoplasts and chloroplasts in which the plastid color correlates with the color 122 W. W. Thomson, L. N. Lewis and C. W. Coggins Cytologia 32

of the regreened fruit is highly suggestive that the chromoplasts revert to chloro plasts during regreening. However, the regreening of Valencia oranges is a slow process occurring over a period of three to four months, while the greening of young leaves and etiolat ed seedlings and the cor related con version of proplastids to chloro plasts gene rally takes place in less than a week. It is possible that a con version of proplastids to choloro plasts was missed be cuase we sampled at biweekly intervals. However, no orangelles which could be classified

Figs. 7 and 8. 7, a chloroplast from a regreened fuirt , Note the well as proplas developed grana, g, and fret system, f, and the small size and number of tids were the osmiophilic globules, 1. •~20,800. 8 , a chloroplast from a deep, green immature fruit. Note the striking similarity in structure between this observed in

chloroplast and that in Figure 7. Starch, st. •~28 ,000. the nume rous samples taken and studied nor was there any indication of a proplastid to chloroplast conversion. Thus, we feel that our conclusion supports Schimper's (1885) statement 1967 The Reversion of Chromoplasts to Chloroplasts in Valencia Oranges 123 that chromoplasts can develop into chloroplasts. However, there are three types of chromoplasts: 1) chromoplasts which contained microscopic crystals of carotenoids; 2) chromoplasts which contained microscopic and submicroscopic yellow globules, and 3) chromoplasts which contained bundles of orange-red pigmented submicroscopic filaments (Frey-Wyssling and Kreutzer 1958a, 1958b, Steffen and Walter 1958). The chromoplasts in the rind of Valencia oranges are of the second or globular type. Whether the other types of chromoplasts, or if all globular types, can revert to chloroplasts is not known. During regreening some vesicles apparently form from the inner portion of the plastid membrane; some are found in the stroma and other vesicles are associated with the developing grana. This distributive pattern suggests that vesicles pinch off from the plastid membrane, drift inward and fuse together to form the grana. Several investigators have reported that during the development of chloroplasts from proplastids, the grana are constructed from vesicles which pinch off from the plastid membrane (von Wettstein 1959, Muhlethaler and Frey-Wyssling 1959). Therefore, it seems that the formation of grana in the Valencia orange during reversion of chromoplasts to chloroplasts follows the same developmental pattern as in the conversion of proplastids to chloroplasts. Although the grana-fretwork system appears to be constructed from portions of the plastid membrane, there is also a gradual reduction in the size and number of the large osmiophilic globules during the reversion of chromoplasts to chloroplasts. Since the frets are closely associated with these globules as well as with the grana, it appears that much of the material of these globules is utilized in the formation of the grana. Exactly when the chromoplasts have reached the point where they cannot be induced to revert to chloroplasts was not determined in this study. Since the reversion appears to be dependent on the formation of vesicles and invaginations of the inner part of the plastid membrane, then reversibility appears to be a function of the plastid membrane. We suggest that when vesicles can no longer form, because of a change in structural or chemical properties of the plastid membrane, then the plastid is irreversibly senescent.

Summary

The ultrastructural reversion of chromoplasts to chloroplasts in regreening Valencia oranges is described. During reversion of chromoplasts to chloroplasts the grana-fretwork system is evidently built up from small vesicles which pinch off from the inner plastid membrane. As the chromoplast regreens, the large osmio

philic globules become reduced in size and number, and there may be a utilization of the lipid material of these globules during the formation of the internal membrane system. It is suggested that irreversible senescences of the chromo

plasts probably is dependent on changes in the plastid membrane. 124 W. W. Thomson, L. X. Lewis and C.W. Coggins Cytologia 32

Acknowledgement

This study was supported in part by a Research Grant. GB 4513, from the National Science Foundation.

Literature cited

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