3.4—
Chloroplast Development
Although the study of chloroplast development and the onset of photosynthesis in seedlings has attracted attention during essentially the whole of the twentieth century (Irving, 1910) it is in recent years that most interest has been shown. There have been two main methods of conducting this study, of which the first is essentially concerned with chloroplast development in plants grown under natural environmental conditions. Since natural conditions are normally neither constant nor consistent such studies have often been conducted in controlled environmental chambers set to a standard day length and standard conditions for day and night. When it became evident that illumination was the critical environmental factor controlling chloroplast development it became fashionable to study chloroplast development by allowing the seedlings to grow for an initial period of several days in complete darkness prior to their transfer to continuous illumination. Both approaches have their merits and it is clear to the present author that both are necessary to obtain an understanding of chloroplast development. Obviously seedling growth under natural environmental conditions shows the normal state of chloroplast development but it provides two main experimental difficulties. Firstly, under diurnal conditions there is a gradient of chloroplast development both within the plant and within the leaf, such that any experimental analysis is either very difficult or impossible. The second difficulty is that diurnal conditions make it difficult to distinguish the effects of illumination on chloroplast development. If seedlings are grown in continuous darkness for a sufficiently long period, their development reaches a stationary phase in which all of the developing plastids in a leaf or a section of a leaf show
the same stage of growth. On transfer to illumination the subsequent development then tends to occur in a synchronous manner, thus facilitating microscopic analysis and making biochemical analysis feasible. Such treatments may enable leaf chloroplast development to show synchrony like that obtainable in certain microbial cultures. It should also be pointed out that much progress in the study of photomorphogenesis has depended on illumination treatments of such darkgrown seedlings (Mohr, 1972). The behaviour of dark-grown seedlings in response to illumination does differ in a number of respects from seedlings grown under diurnal conditions of illumination, as discussed by Schiff (1975) for example.
3.4.1—
The Proplastid
Figure 3.1A shows an electron micrograph of a proplastid in a section of a primary leaf of a 6-day-old dark-grown bean seedling. The invaginations of the inner membrane of the envelope are interpreted as representing the formation of porous sheets of membrane which are shown in section. The proplastid also contains a starch grain and scattered ribosomes while the cytoplasmic ribosomes are more prominent and are apparently mostly present as polysomes.
3.4.2—
Etioplast Formation
Between 4 and 14 days of dark growth of Phaseolus vulgaris seedlings, the primary leaf primordia, which are already present in the dry seed, show a considerable amount of growth in increasing from a fresh weight of less than 1 mg to 30–40 mg while the cell number increases by more than 10 times and plastid number increases by 18 times (Bradbeer et al., 1974c). During this time the amount of membrane within the plastid increases considerably so that etioplasts like that in Fig. 3.2A are formed. The term etioplast was used first by Kirk and Tilney-Bassett (1967) and defines a structure which is typical of dark-grown seedlings, not normally found in plants grown under diurnal conditions of light and dark. In the bean etioplast about half of the membrane is organised in a regularly-arranged network of tubules called the prolamellar body with the remainder in concentrically arranged porous lamellar sheets (thylakoids). The prolamellar body shows a para-crystalline form and a knowledge of crystallography has contributed to the interpretation of its basic structure. In one plane the tubules form a mesh of hexagons, each individual hexagon being connected to the one immediately above by tubules arising from three alternate nodes, and to the one immediately below by tubules arising from the other three nodes (Weier & Brown, 1970). In planes cutting the hexagonal plane at 90º the arrangement of the tubules is approximately rectangular. Prolamellar bodies are frequently large and complex structures with evident discontinuities, but there are no published reports which account exactly for the structure of these large pro-
Figure 3.2
Stages of chloroplast development during the greening of the primary leaves
of 14-day-old dark-grown beans under continuous illumination of 3 mW. cm–2
A, no illumination; B, 105 minutes illumination; C, 4 hours illumination; D, 5
hours illumination; E, 15 hours illumination. Magnification × 25,000. Key
to lettering: PB, prolamellar body; other details as in Fig. 3.1.
lamellar bodies. Calculations based on measurements in the author's laboratory of electron micrographs of the prolamellar body structure described above show that 1 µm3 of the prolamellar body of 14-day-old dark-grown bean leaves should contain the equivalent of 44 µm2 of membrane.
From measurements of electron micrographs and the dimensions of the plastids determined by light microscopy it has been possible to follow the changes in the area of the lamellar sheets and of the volume of the prolamellar bodies during etioplast development. Determination of the number of plastids in the leaf then enables quantities per plastid (Bradbeer et al., 1974c) to be expressed on a per leaf basis as shown in Fig. 3.3. In Fig. 3.3 the area of membrane in the prolamellar bodies has been calculated on the basis stated above that 1 µm3 of prolamellar body contains 44 µm2 of membrane. As this factor is likely to vary during development according to the degree of contraction of the prolamellar body some inaccuracy is inevitable from this source for samples other than the 14-day-old one. In particular the slight apparent fall in the total membrane, shown in Fig. 3.3, after 14 days of dark growth may represent a contraction of the prolamellar body without any change in its membrane content. In the experiment shown in Fig. 3.3 membrane formation occurred between 6 and 14 days of growth. The porous lamellar sheets were formed first and the data are consistent with the presumed formation of the prolamellar bodies by some sort of condensation of these sheets (Weier & Brown, 1970). The area of the lamellar sheets reached a peak at 12 days after which the continued formation of the prolamellar bodies seems to have resulted in some consumption of the lamellar sheets.
During dark development of the etioplast most of the chemical components of the chloroplast are formed; for example all of the photosynthetic carbon cycle enzymes are present in the etioplast (Bradbeer et al., 1974c). Substances not yet detected in etioplasts which have received no illumination are chlorophyll, the chloroplast pigment-protein complexes and cytochrome b –559HP . The etioplasts of beans appear to reach the peak of their development by 14 days of dark growth while retaining an ability to form chloroplasts on illumination; however 17-day-old leaves with 75% of the etioplast membrane in the prolamellar body green only feebly and 21-day-old dark-green bean leaves fail to survive when transferred to illumination. It should also be pointed out that more rapid greening occurs in leaves younger than 14 days.
Figure 3.3
The formation of internal membrane during etioplast development in the
mesophyll of primary leaves of Phaseolus vulgaris during growth in
continuous darkness at 23°C
porous lamellar sheets;
3.4.3—
The Conversion of Etioplasts into Chloroplasts
The formation of chloroplasts in dark-grown leaves requires the provision of an appropriate amount and quality of illumination. Continuous illumination of 3 mW. cm–2 by fluorescent tubes in a growth cabinet was used for the electron microscopic study of 14-day-old dark grown beans shown in Fig. 3.2. For this material fixed with glutaraldehyde-osmium tetroxide, the paracrystalline appearance of the prolamellar body is retained for about 30 minutes after the beginning of illumination, and then between 30 and 60 minutes there is a change from a regular to an irregular appearance like that shown in Fig. 3.2B (Bradbeer et al., 1974a). Transformation of the prolamellar body appears to occur much more rapidly, usually within less than 1 minute of illumination, if the leaves are fixed with permanganate but this rapid change is commonly regarded as an artefact in structural terms. On the other hand, it may result from an early photochemical reaction in the etioplast which renders the paracrystaline nature of the prolamellar body less stable (Berry & Smith, 1971). Subsequently the volume of the reacted prolamellar body falls and the area of the thylakoid sheets increases as shown in Fig. 3.2C & D for 4 and 5 hours of illumination
Figure 3.4
The effects of the transfer of 14-day-old dark-grown beans to continuous
illumination of 3 mW. cm–2 on the prolamellar body volume and the thylakoid area
of the plastids of the primary leaves.
respectively and in Fig. 3.4. Figure 3.4 shows that the fall in the prolamellar body volume and the increase in the area of the thylakoid sheets approximately correspond with each other with the actual increase of 21.5 µm2 thylakoid/plastid (43 µm2 membrane) accounting for the loss of 0.99 µm3 of prolamellar body per plastid (43.6 µm3 membrane). After 10 hours of illumination, appression of the thylakoids becomes more obvious, the formation of grana occurs, and the further increase in thylakoid membrane may be presumed to have resulted from de novo membrane synthesis. Figure 3.2E shows the stage of development after 20 hours of illumination and Fig. 3.1B that after 48 hours of illumination. During the course of bean chloroplast development under these conditions, de novo membrane synthesis results in an 8-fold increase in thylakoid area.
The illumination of 14-day-old dark-grown beans results in a synchronous development of the etioplasts in the primary leaf mesophyll cells. There is no division of these cells, less than 10% of the plastids divide, and the rather amoeboid etioplasts double in diameter to give typical discoid chloroplasts. The small amount of chloroplast division does not seem to be typical of developing leaves in which replication of chloroplasts is usual. Greening leaves show a lag before the onset of photosynthesis. The length of this lag depends upon the nature of the plant material, the conditions and duration of dark growth, the
conditions for greening, the intensity of the illumination used to assay photosynthesis and the sensitivity and nature of the method of assay. Consequently, published reports on the duration of this lag show a range of values; however, all agree that at least one hour of illumination, and usually more, is required before photosynthetic CO2 -fixation can be detected. For the bean leaves studied in the author's laboratory, the lag has been at least 5 hours, after which the photosynthesis of the developing chloroplasts becomes increasingly important as the source of the energy and the carbon requirements, of their own development (Bradbeer, 1976).
The effects of different light treatments on the form of the resultant chloroplast have been considered in an earlier section (3.2.4.3). There have been a number of studies in which light of different wavelengths has been used for the illumination of dark-grown seedlings in an attempt to determine an action spectrum of the light dependent reactions involved in chloroplast development. Henningsen (1967) obtained a sharp peak at 450 nm in the action spectrum for a stage termed vesicle dispersal in the development of bean plastids. Unfortunately this developmental stage may be another artefact of permanganate fixation as thylakoid extrusion does not involve such a stage when seen in glutaraldehydeosmic acid fixed material and studies with this latter fixative have not so far observed any corresponding action spectrum. However Henningsen's experiments have not been exactly replicated with the latter fixative and his data may well indicate a so far undefined photoresponse. Most studies of the effects of light quality on chloroplast development have used rather wide bands of wave-lengths and have concentrated on the role of phytochrome in chloroplast development. It has been shown in a number of laboratories that phytochrome has an important role in chloroplast development, and a number of aspects of chloroplast development are promoted by short treatments with red light and reversed by short exposure to far-red. In bean, the presumably-active form of phytochrome, Pfr (see chapter 12) promotes plastid expansion, plastid division, the formation of plastid membrane and the synthesis of chloroplast proteins (Bradbeer, 1971; Bradbeer et al., 1974b). These reactions may be regarded as 'slow' reactions in that they require several hours to become evident. In addition to phytochrome, 'slow' reactions may possibly also be effected by a red-light-absorbing photoreceptor other than phytochrome, and by a blue-light-absorbing photoreceptor. Most of the 'rapid' changes in etioplasts, which occur within three hours of illumination, do not seem to involve phytochrome; such changes as prolamellar body transformation and loss are sensitive to a wide range of wavelengths whilst thylakoid extrusion seems to depend on a red-absorbing photoreceptor other than phytochrome. Only two 'rapid' effects of phytochrome on etioplast fine structure have so far been defined; they are the Pfr-promoted crystallization of the prolamellar body in mustard cotyledons (Kasemir et al., 1975) and the Pfr-inhibited recondensation of the prolamellar body in bean primary leaves (Bradbeer & Montes, 1976). Although Wellburn and Wellburn (1973) have implicated Pfr in 'rapid' changes in isolated and
in situ etioplasts their method of analysis does not permit them to define these changes with any precision and their conclusions seem to require reconsideration.
3.4.4—
The Formation of Chloroplast Components in Greening Leaves
The transfer of dark grown plants to continuous illumination results in substantial increases in the amounts of most of the etioplast constituents which are also found in the chloroplasts (see for example Bradbeer et al., 1969; Gregory & Bradbeer, 1973). A few components, namely chlorophyll, the chloroplast pigment-protein complexes and cytochrome b– 559HP , which cannot be detected in dark-grown etioplasts, appear as a result of illumination.
The photosynthetic enzymes have almost exclusively been studied by determination of enzyme activity and thus any change in activity may result from either a change in the amount of enzyme protein or a change in the activity of the enzyme protein. Since, during the greening process, there is considerable protein synthesis it was considered that the simultaneous increases in enzyme activity probably resulted from protein synthesis. Subsequent investigations have established that enzyme activation is also responsible for a substantial part of the increase in the activities of certain enzymes in greening leaves.
Ribulosebisphosphate carboxylase is the most abundant protein in the chloroplast and probably consists of 8 large subunits (molecular weight about 5.2 × 104 ) and 8 small subunits (molecular weight about 1.3 × 104 ) which give a molecule of about 5.2 × 105 in molecular weight. By the use of two-dimensional polyacrylamide gel electrophoresis, Arron and Bradbeer (1975) found that the commencement of the synthesis of both subunits in bean leaves coincided with the beginning of illumination but that there was a lag before enzyme activity increased. Smith et al., (1974) reached a similar conclusion from experiments with barley in which they measured newly-synthesized ribulosebisphosphate carboxylase by labelling it during its biosynthesis and trapping the labelled substance with a specific antibody. In both bean and barley, synthesis of the enzyme occurs early in greening with a corresponding increase in enzyme activity, while late in greening after the enzyme synthesis has ceased considerable activation occurs. The mechanism of the activation is not known although the effect of illumination may be mediated by a small (molecular weight 5 × 103 ) constituent (Wildner et al., 1972).
In contrast, rather more is known about the activation of phosphoribulokinase and triosephosphate dehydrogenase, where a pretreatment of the extracted enzyme with 6 mM ATP prior to assay brings about activation, (Wara Aswapati et al., 1977). Activation may also be brought about by NADPH and sulphydryl reagents. On illumination of etiolated leaves, the activity of each of these enzymes shows a lag of several hours before it increases; however, preincubation of these extracts with ATP shows that the increases in activity seem to commence from the beginning of illumination (Fig. 3.5). It is concluded that synthesis of
Figure 3.5
The development of phosphoribulokinase activity in primary
leaves of 14-day-old dark-grown beans on transfer to
continuous illumination of 3 mW.cm–2 .
after pretreatment of the extract with 6 mM ATP;
no pretreatment;
leaves left in dark. After Wara-Aswapati (1973).
these two enzymes commences with the beginning of illumination but that activity does not develop until photophosphorylation commences, thus accounting for the lag in Fig. 3.5 for the extracts which were not activated prior to assay. Thus, for the phosphoribulokinase in greening bean leaves Fig. 3.5 shows that there was a 10-fold increase in enzyme protein and a 9-fold increase in the enzyme activity of the protein, thus accounting for a 90-fold increase in total enzyme activity.
There is the further example of ATPase, which is a polypeptide complex in which illumination brings about an increase in enzyme activity with comparatively little new synthesis of the protein being apparent (Gregory & Bradbeer, 1975). In this case the effect of illumination seems to be more direct and is probably necessary for the onset of photophosphorylation rather than a result of it.