J. Cell Sci. 6, 243-255 (i97°)
Printed in Great Britain
243
THE THREE-DIMENSIONAL ARRANGEMENT
OF INTERGRANAL LAMELLAE IN
CHLOROPLASTS
D. J. PAOLILLO, JR.
Department of Botany, University of Illinois, Urbana, Illinois, U.S.A. 61801
SUMMARY
The intergranal lamellae, or frets, are helically arranged around each granum. All helices
within a plastid are co-directional. The helices wind in the same direction from plastid to
plastid, from cell to cell, and from species to species, and are right-handed. The continuity of
the intergranal membranes in 3-dimensional space is facilitated by the fact that all the helices
within a plastid wind in the same direction. Because there are multiple frets around each
granum, the fretwork consists of a highly interlocked series of membranes. The incompleteness
of the integration of the fretwork suggests that continuity of the membranes from granum to
granum is obtained, to a large degree, by fusions of membranes rather than by continuous
growth and ramification of a simpler membrane system.
INTRODUCTION
The lamellar component of a chloroplast in a higher plant is differentiated into
regions of densely packed membranes, the grana, and a system of more loosely
arranged membranes, the fretwork, that interconnects the grana (Weier, 1961). HeslopHarrison (1963) postulated a high degree of membrane continuity within a chloroplast,
when he established that the fret membranes are tilted with respect to the cylindrical
grana. Wehrmeyer (1964) suggested a spiral-cyclical arrangement of frets around a
granum. Paolillo & Falk (1966) established the existence of multiple helical frets
around each granum of mesophyll plastids in corn. Paolillo & Reighard (1967)
extended the model of a granum with multiple helical frets to the grana of bean plastids
and verified the continuity of membranes and the loculi they confine. It was concluded that the multiple helical frets communicate with each other via the granum
compartments they are attached to, and the compartments communicate via the
attached frets. The model of a granum with multiple helical frets has now been
generalized for 7 species of flowering plants (Paolillo, MacKay & Reighard, 1969).
Sections of chloroplasts show that frets can be continuous from one granum to the
next in the profile of a plastid. The 3-dimensional form of these frets is hard to visualize
when one conceives of their helical arrangement around individual grana. Weier,
Stocking & Shumway (1967) have offered one visualization of the 3-dimensional
arrangement of frets among grana. The present report deals with a more precise
characterization of the form of the frets in 3-dimensional space.
16-2
D. J. Paolillo, Jr.
244
MATERIALS AND METHODS
The sources of sectioned materials used in this study are the same as those used in earlier
studies on the ultrastructure of chloroplasts in 7 species of flowering plants: hemp, elodea,
tobacco, bean, pea, spinach, and corn (Table 1; Paolillo et al. 1969). The 3-dimensional form of
the fretwork was reconstructed via the analyses employed elsewhere (Paolillo & Reighard, 1967;
Paolillo et al. 1969), by extending the observations simultaneously to adjacent grana. Directionality of the numerous helices within a plastid was determined by assessing the tilt of membranes
at near and far tangents of grana in serial sections (Fig. 5). In all cases, 50 % or more of the
grana within the sampled volume of a plastid could be evaluated for the directionality of the
helical frets (Table 1).
OBSERVATIONS
Earlier articles in this series have been concerned with the fretwork around individual grana. This paper deals with the fretwork between and among grana, but the
significance of the present work will be lost if overemphasis is given to the fretwork as
an entity apart from the grana. The argument that will be presented here is that the
form of the fretwork between and among grana is a consequence of the interaction of
the multiple helical frets around individual grana.
The directionality of the helices
Multiple helical frets are of general occurrence in the grana of flowering plants
(Paolillo et al. 1969). But helices can occur in two forms, the so-called left- and righthanded configurations. A left-handed helical fret is one that ascends to the left at the
Table 1. Sample sizes for 7 species for co-directionality of helical frets
(The frets were co-directional in each of the 57 plastids examined.)
Grana/plastid
Number of
r
plastids examined
Cannabis sativa (hemp)
Elodea canadensis (elodea)
Nicotiana rustica (tobacco)
Pliaseolus vulgaris (bean)
10
4
10
4
range
(X)
Sample size, as
% of all grana
5-10
13-22
9-23
13-33
(7)
(16)
(15)
(25)
(29)
69
63
58
83
5O
Pisum sativum (pea)
Spinacea oleracea (spinach)
Zea mays (corn)
10
7-47
6-18
(11)
62
10
9-22
(16)
59
910 grana from 7 species
57
5-47
(16)
05
9
near tangent of a granum, whereas a right-handed fret ascends to the right. It is not
necessary to designate which end of the granum stack is the top because turning the
stack over does not reverse the direction of the helices. Likewise, looking from the
opposite side of a granum (or from any 'side') does not alter the directionality of
the helices. The first property that must be determined is whether or not all helices
within a plastid are wound in the same direction, because a characteristic configuration
of the fretwork will result if they are.
Three-dimensional structure of plastids
245
The data presented in Table 1 allow the conclusion that it is very likely that all the
helices within a plastid are wound in the same direction. Even in the smallest samples,
elodea and hemp, the chances that there are an equal number of left- and right-handed
helices in one plastid are (£)64 and (£)70, respectively. The composite sample includes
910 grana from 57 plastids. Even a low frequency of assortment of left- and righthanded helices should be detected with this sample.
Table 2. Sample sizes for 5 species studied for absolute direction of helical frets
(All samples contained only right-handed frets.)
Number of
cells
Cannabis sativa (hemp)
Elodea canadensis (elodea)
Nicotiana rustica (tobacco)
Pisum sativum (pea)
Spinacea oleracea (spinach)
5 species
Number of plastids
in each cell
2
4, 2
2
2, 1
3
3
4
S. 3, 3
3, 3. 1
5, 3, 3, 1
14 cells
39 plastids
The number of grana for which the direction of the helical frets was determined
represents a high percentage of the total number of grana in the sections, averaging
65% for all data taken. This percentage is obtained using rapid-scoring techniques
that utilize the tangential sections to determine directionality. One simply determines
within the context of serial sections whether one is dealing with the near or far
tangent for each granum to determine the relative directionality of the helices (Fig. 5).
It is therefore possible to decide whether or not the helices are co-directional, even
when the absolute directionality is not known. The samples taken were well distributed
within the plastid profiles, including both adjacent grana and grana that were widely
separated from each other. Grana excluded from the determinations were simply those
that did not appear in tangential section in the series examined.
The observation that the helices are co-directional within a plastid is crucial to the
development of the arguments that follow. The question of whether left- or righthanded helices are involved is of minor consequence to the form of the fretwork,
because the models that would be developed in the two cases would be mirror images
of each other. However, the direction of the helices is worthy of at least preliminary
consideration in this report.
To evaluate the frequency of left- and right-handed helices, serial sections were cut
from 5 blocks that were trimmed asymmetrically so that the leading section and the
absolute left-right orientation of a ribbon could be recognized in the microscope. The
results of this evaluation are given in Table 2. In all 39 plastids examined, the helices
were right-handed. Up to 5 plastids were analysed from a given cell, and regardless of
proximity or separation of the plastids within the cell, the helices were co-directional.
Likewise, 2-4 cells were examined per series of sections, and the helical frets were
co-directional regardless of whether the cells were adjacent or separated from each
other by several intervening cells in the section. These results harmonize with some
246
D. J. Paolillo, Jr.
Three-dimensional structure of plastids
Fig. 2
247
Fig. 3
Fig. 1. Ideal or maximum integration of the fretwork around 4 grana in a line. At
right the grana stacks are omitted and the 8 frets required by this model are drawn in.
At left 7 of the frets are left out but the fret connexions to the grana are drawn in. The
grana are of equal diameter and are assumed to stand parallel, while the tilt of the fretwork is the same throughout. It is under these circumstances that maximum integration of the fretwork occurs.
Fig. 2. Co-directional, right-handed helices of the type found in the fretwork.
Fig. 3. Anti-directional helices (one left-handed, one right-handed). This situation is
not encountered in the fretwork.
additional observations on the series of sections that furnished the data for Table 1.
In those materials, there were 9 instances in which 2-3 plastids from one cell could
be evaluated simultaneously, and in all cases the samples contained only co-directional
helices. In 2 instances, 2 plastids from different cells were available for simultaneous
evaluation, and in both cases the helices were co-directional.
Hence, one is inclined to postulate that the helices are all right-handed, winding in
the same direction from plastid to plastid, from cell to cell, and from species to species.
If the 39 plastids are regarded independently, the chance that left- and right-handed
plastids occur in equal numbers is (^)30. Adopting the most conservative stance
imaginable, one must at least admit that the probability that left- and right-handed
helices are equally frequent is small. Considering the present data, it seemed advisable
to construct the models presented in this paper from right-handed helices.
The form of the fretwork
In the discussion that follows, it will be useful to keep in mind that the number of
helical frets around a granum is directly proportional to the pitch of the helices and
the diameter of the granum, and inversely proportional to the ratio of granum compartments to frets at the margin of a granum. These are the parameters that are
measured in serial sections to determine the number of helices around a granum
(Paolillo et al. 1969). The parameters that determine the number of helices vary from
248
D. J. Paolillo, Jr.
granum to granum within a chloroplast (Paolillo & Falk, 1966; Paolillo & Reighard,
1967; Paolillo et al. 1969). The grana also vary in their orientation within a plastid.
The most regular form of the fretwork can be expected to occur between grana of
equivalent morphology that stand parallel to each other. The configuration imparted
to the fretwork under these circumstances will be discussed first. Subsequently,
sources of variability will be explored.
Figure 1 shows parallel grana of equal diameter. There are twice as many granum
compartments as frets at the margin of each granum (compartment-to-fret ratio,
2 :1). At any location, each fret is attached eight compartments higher or lower than
on the opposite side of the granum (tilt of 8, pitch of 16 compartments). These conditions fix the number of helical frets as 8 around each granum, but the arguments
that follow are independent of the number of frets. Furthermore, the planes of the
fretwork are parallel in this model, and it will suffice to characterize the shape of a
single fret and then to call attention to the way in which the frets are locked together.
Considering first one fret around the location of each of 2 adjacent grana (Fig. 2),
the helices are in register at each turn. Because they are co-directional, the gyres of
the helix appear to 'cross' each other (Fig. 2, at top), but it is readily seen that the
extended form of the helical fret membranes leads to a confluency of the frets at the
near and far tangents of the grana (Fig. 2, at bottom). With this confluency, tangential
sections would show a continuous slant from left to right across both grana. This
prediction is borne out by micrographs (Figs. 10, 11). For comparison, a model
constructed from anti-directional helices is presented (Fig. 3). Note that sections
tangential to both grana would reveal opposing slants in the continuous fretwork. The
properties of this model are not those of the grana seen in micrographs. All direct
reconstructions of serial sections lead to the visualization of the type of relationships
depicted in Fig. 2, and all implications of the model so constructed are borne out by
micrographs.
Figure 1, at bottom right, shows how 8 parallel frets are locked together to form the
complete fretwork around the locations of adjacent grana in the model. While the 8
frets are in a sense distinct, it should be remembered that they communicate via connexions to the granum compartments (Fig. 1 at left; see also Paolillo & Reighard, 1967).
Having settled on the form of the fretwork between adjacent grana, one must next
consider whether the model can be extended to include groups of 3 or more grana.
It can be argued that any group of grana can be subdivided into a finite number of
pairs of adjacent grana. The relationships within each pair will be given by the model
described above (Figs. 1, 2). Because one can choose overlapping pairs within a group,
the model should apply to clusters of grana as well as to individual pairs. Models have
been constructed to verify this theoretical argument. Figure 1 shows the model
extended to 4 grana in a line. The model can be extended in any direction, e.g. at
right angles to the plane of the printed page. When this is done, one notes that as the
model is rotated, the frets on the near side always tilt upwards to the right because the
model is constructed entirely of right-handed helices.
Three-dimensional structure of plastids
249
Variations from the ideal case
Three conditions have been fixed for developing the model in Fig. 1: (1) the grana
stand parallel to each other, (2) the pitch of the helices is the same on each granum,
and (3) the grana are of equal diameter. Our studies show that the helical frets may
have a different pitch on different grana, and that adjacent grana can vary in their
diameters and orientation within the chloroplast. It is necessary to consider how these
variations from the ideal case affect the model of the fretwork that has been presented.
If two grana share a continuously integrated fretwork but do not stand parallel to
each other, the pitch of the fretwork will differ for the two grana. The effect will be to
increase the compartment-to-fret ratio where the pitch is greater. The number of
helices round a granum is inversely proportional to the ratio of compartments to
frets, whereas it is directly proportional to the pitch of the frets. Hence these 2 factors
can cancel each other to conserve the number of frets from granum to granum.
Variations in the number of frets on adjacent grana can occur without prohibiting
the integration of the fretwork. Figure 4 shows the arrangement of the fretwork
around two parallel grana with 8 helices (left) opposed to 6 (right), as might be the
case if the pitch of the helices differed on two adjacent and parallel grana. The model
shows that the form of the fretwork is not affected by the difference in number of
helices, when the spacing of frets (compartment-to-fret ratio) is appropriate. Assigning
designations ai-a8 to the frets on the left and bi-b6 to those at the right, one notes a
serial change in the match between any fret followed on the right, e.g. bi and the
a-frets (bi-ai; bi-aj; bi-a$; bi-aj; bi-ai.. .). The effect of this situation is to integrate
further the membranes of the fretwork.
If 2 adjacent grana are of different diameters, they will be surrounded by different
numbers of frets, if other things are equal. This does not interfere with the integration of the fretwork when the spacing of the frets along the margins of the two grana is
the same. Instead, the situation is resolved as in Fig. 4, although the cause of the
difference in number of helices is not the same.
It is evident that the individual variables that affect the number of helices around a
granum do not necessarily impose any limitation on the integration of the fretwork.
Acting in concert and over the range of their variability, however, they lead to a
degree of discontinuity among the fret membranes that cannot be overlooked. This
discontinuity, visible as interruptions in the sectional views of membranes, must not be
confused with simple perforation of the frets (Paolillo, Falk & Reighard, 1967). It is,
instead, to be construed as evidence of a failure in the integration of frets from adjacent
grana. In other words, careful analysis of adjacent grana shows unmatched frets, and
frets that come into contact with several of the frets of adjacent grana when followed
for relatively short distances in serial sections.
The impression that one gains from these analyses is that whatever continuity
exists in the fretwork between grana may be the result of a developing compatible
relationship among frets, as they grow outward from their respective grana to fuse.
Obviously it is to be anticipated that the orientation and construction of neighbouring
grana may be unfavourable for the development of perfectly matched fretwork, and
D. J. Paolillo, Jr.
250
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Three-dimensional structure of plastids
251
that all intermediate conditions of integration can exist. Therefore, although some parts
of the fretwork are well integrated, others are not. This situation is encountered
in all the species studied, but the fretwork generally presents a more orderly appearance
in corn, pea, bean and elodea than in hemp, spinach and tobacco.
The relationship of' columns' of grana
One of the characteristic features of median profile views of plastids in some species
is the relatively orderly arrangement of the grana, with the stacking of individual
grana into columns (Figs. 6, 7, grana a-d). Serial sections of these arrangements show
that the grana can be integrated by the fretwork in all directions. The serial sections in
Figs. 6-11 illustrate that certain of the frets wind from granum to granum in the
column, within the distance covered by the series. A similar relationship occurs where
the lamellar components converge at the periphery of the plastid profiles.
DISCUSSION
Heslop-Harrison's (1963) contention that the lamellar component of the chloroplast is a highly integrated unit is clearly correct. The 3-dimensional reconstructions
presented in this paper enhance the appreciation for the integrated character of the
grana-fretwork system. The reconstructions differ from others (Heslop-Harrison,
1966; Weier et al. 1967) in the emphasis that is given to 2 features of the fretwork: there
are multiple helices around each granum and the helices are co-directional. Weier
et al. (1967) have speculated that the biological significance of the helical arrangement
of frets is to integrate all levels in a granum with the stroma. The presumed benefit of
this integration is the exchange of products of the light and dark reactions. Firm evidence in support of this concept remains to be obtained, but the idea is most attractive.
All the helices within a plastid are co-directional and probably right-handed. Our
first reconstruction (Paolillo & Falk, 1966) was based on sections that did not lend
themselves to the discovery of these properties of the fretwork, and a right-handed
model was illustrated fortuitously. The detailed reconstruction prepared later
(Paolillo & Reighard, 1967) is left-handed for artistic considerations only. The
co-directionality of helices within a plastid was not anticipated until the analyses for
the present paper were well under way. However, this characteristic of the fretwork
is fundamental to an accurate 3-dimensional reconstruction.
Fig. 4. Integrated fretwork with 8 helices (left) opposed to 6 (right), around the
locations of adjacent parallel grana of equal diameter. The helices are numbered
arbitrarily. The partner to which any fret is matched changes in a regular pattern,
e.g. bi—ai, bi-aj, bi-a5, bi-a3, bi-ai, etc., because of simple geometric requirements.
But this does not prevent the integration of the fretwork.
Fig. 5. Sets of data consistent with co-directional helices. Set I is consistent with
right-handed helices, set II with left-handed helices, when the absolute direction of
the series of sections is known. If the absolute direction is not known, sets I and II
are interchangeable. Within the sets, i represents the sequence of appearances starting
at the ' near' tangent and ii represents the sequence working toward the ' far' tangent,
for grana followed simultaneously in the same series of sections. All data from sections
of known absolute sequence complied with the requirements of set I.
252
D. J. Paolillo, Jr.
The finding that 5 samples (from 5 species) contained only right-handed helical
frets was also not anticipated, and a source of chagrin, because until that time all our
detailed drawings and 3-dimensional models had been constructed with the lefthanded helices. The co-directionality of helices offers evidence for a guiding principle
in the construction of the fretwork, perhaps controlled by some physical-chemical
property of membrane subunits that comprise the lamellae. Such a control mechanism
could help explain the presumed dismantling and reassembly of grana-fretwork
systems reported by Izawa & Good (1966).
Variability in the characteristics that determine the relationship of a granum to the
fretwork at its periphery is the basis for only part of the variability that exists in the
integration of the fretwork between and among grana. The major discontinuities seem
to be brought about by differences in orientation among the grana. All of the observations available are compatible with the conclusion that the fretwork develops its
complexity initially in the vicinity of the individual grana and only later between grana.
Some ontogenetic studies support this conclusion (Muhlethaler & Frey-Wyssling,
1959) but a thorough analysis of development using the semi-quantitative methods we
have applied in our studies is yet to be accomplished.
Wehrmeyer's (1964) theory of spiral cyclical growth of fret membranes can be
adapted to explain how the fretwork could be built up to its multiple condition around
the grana. The rough outline of events must start with the initiation of grana on
primary growth layers (von Wettstein, 1959), followed by the building up of average
granum height and the formation of multiple helices by complex spiral-cyclical
growth, and concluded with the outgrowth of frets to populate the intergranal
regions, with their fusions linking the grana together. The difference between these
ideas and those of other authors is largely in the emphasis placed on the role of
membrane fusion in establishing continuities during spiral-cyclical growth and
afterwards. Figure 2 illustrates the feasibility of this idea at the final stage of the
developmental process. Viewing the figure from top to bottom as a series in time, one
may visualize how the expansion and fusion of membranes results in a continuous
fretwork between grana.
The form and integration of the fretwork described here are in no way in conflict
with the known flexibility of the membranous component of chloroplasts. There
remains the question of whether or not the details of grana-fretwork relationships are
static or dynamic in a normal plastid after the chloroplast has matured. This question
remains unanswerable at the present time.
In closing it is necessary to emphasize that Fig. 1 represents only the ideal or
maximum degree of integration in the fretwork. More often than not the integration is
only partly realized, as is illustrated by grana c, d and e in Figs. 6-11. Although these
grana have linked fretworks, their frets are not completely matched. A much better
integration is represented by the fretworks around grana a, b,/and g in Figs. 6-11.
The function of the model in Fig. 1, and especially that in Fig. 2, is to affirm the concept that the integration that does exist in the fretwork is an outcome of the codirectionality of the helices. Whatever frets are matched between adjacent grana follow
the form demonstrated by these models.
Three-dimensional structure of plastids
253
This investigation was supported by National Science Foundation Grant GB 6520. The
author also wishes to express his thanks to the staff artist, Mr Stanley Jones, for his assistance in
preparing the illustrations, and to Mrs Norma Chao MacKay for excellent technical assistance.
REFERENCES
HESLOP-HARRISON, J. (1963). Structure and morphogenesis of lamellar systems in granacontaining chloroplasts. I. Membrane structure and lamellar architecture. Planta 60, 243-260.
HESLOP-HARRISON, J. (1966). Structural features of the chloroplast. Sci. Prog. 54, 519—541.
IZAWA, S. & GOOD, N. E. (1966). Effect of salts and electron transport on the conformation of
isolated chloroplasts. II. Electron microscopy. PL Physiol., Lancaster 41, 544-552.
MOHLETHALER, K. & FREY-WYSSLING, A. (1959). Entwicklung und Struktur der Proplastiden.
J. biophys. biochem. Cytol. 6, 507-512.
PAOLILLO, D. J., JR. & FALK, R. H. (1966). The ultrastructure of grana in mesophyll plastids of
Zea ways. Am.J. Bot. 53, 173-180.
D. J., JR., FALK, R. H. & REIGHARD, J. A. (1967). The effect of chemicalfixationon
the fretwork of chloroplasts. Trans. Am. microsc. Soc. 86, 225-232.
PAOLILLO, D. J., JR., MACKAY, N. C. & REIGHARD, J. A. (1969). The structure of grana in
flowering plants. Am.J. Bot. 56, 344-347.
PAOLILLO, D. J., JR. & REIGHARD, J. A. (1967). On the relationship between mature structure
and ontogeny in the grana of chloroplasts. Can. J. Bot. 45, 773-782.
WEHRMEYER, W. (1964). Zur Klarung der strukturellen Variabilitat der Chloroplastengrana des
Spinats in Profil und Aufsicht. Planta 62, 272-293.
WEIER, T. E. (1961). The ultramicrostructure of starch-free chloroplasts of fully expanded
PAOLILLO,
leaves of Nicotiana rustica. Am. J. Bot. 48, 615—630.
T. E., STOCKING, C. R. & SHUMWAY, L. K. (1967). The photosynthetic apparatus in
chloroplasts of higher plants. Brookliaven Symp. Biol. 19, 353-374.
WETTSTEIN, D. VON (1959). The formation of plastid structures. Brookliaven Symp. Biol. 11,
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[Received 5 May 1969)
WEIER,
254
I
D. J. Paolillo, Jr.
.1
Q
Three-dimensional structure of plastids
Figs. 6—11. Serial sections from a chloroplast of Alaska pea. The wall-facing side of
the disk-shaped plastid was to the left. In Figs. 8 and 10 the outlines of the grana not in
the sections have been drawn in with dashed (approaching near tangent) and dotted
(beyond the far tangent) lines to show the slope of the fretwork across the tangents of the
grana. Compare with Fig. 5. The outlines in Fig. 8 are forgranaa and6. In Fig. 10 they
are for grana d,/and g. Some of the frets are numbered for identification. The series
illustrates the following points: (1) grana a, b, c and d are integrated by their fretworks.
Between Figs. 6 and 7 some of the frets change their association from one granum to
another. (2) The slope of the fretwork is continuous when the section is tangential to
adjacent grana. This occurs regardless of direction, because Figs. 7, 8 show the alternative spatial relationship of two grana (a, b) to that shown in Figs, io, 11 (/, g). (3)
The integration of the fretwork is imperfect. The large expanse of fretwork in Figs.
8-10 is well integrated for grana a, b, f and g. However, the fretworks of c, d and e
integrate imperfectly with each other and still less perfectly with the fretworks of
a, b, f and g. (4) The apparent tilt of the fretwork at the near tangent is opposite the
apparent tilt at the far tangent. Compare locations for g r a n a / a n d g against din Fig. 10.
See also Fig. 5. x 75 000.
255