Annals of Botany 77: 629-637, 1996
Suppression of Granal Development and Accumulation of Rubisco in Different
Bundle Sheath Chloroplasts of the C4 Succulent Plant Portulaca grandiflora
DAISUKE NISHIOKA, HIROSHI MIYAKE* and TAKESHI TANIGUCHI
Laboratory of Plant Resources and Environment, School of Agricultural Sciences, Nagoya University, Chikusa,
Nagoya 464—01, Japan
Received: 25 September 1995 Accepted: 5 December 1995
Chlorenchyma of Portulaca grandiflora, an NADP-malic enzyme type succulent C4 plant, consists of several types of
green cells. Typical bundle sheath and mesophyll cells were located around the peripheral vascular bundles in the
cylindrical leaf. Water storage cells with small and few chloroplasts were located in the inner part of the leaf. Another
vascular bundle was located in the centre of the leaf, which was surrounded by water storage cells. Typical bundle
sheath cells were not observed around the central vascular bundle. Granal development was suppressed in the
chloroplasts of typical bundle sheath cells. Mesophyll chloroplasts and the chloroplasts in most of the water storage
cells possessed well-developed grana. However, granal development was suppressed in the chloroplasts of the
innermost water storage cells adjacent to the central vascular bundle. Immunogold localization revealed that ribulose
1,5-bisphosphate carboxylase/oxygenase (Rubisco) accumulated in bundle sheath chloroplasts and in the chloroplasts
of the water storage cells adjacent to the central vascular bundle. Labelling of Rubisco was markedly reduced in
mesophyll chloroplasts and in the chloroplasts of remaining water storage cells. Gradient in labelling of Rubisco and
granal development was not observed in the remaining water storage cells. Therefore, the accumulation of Rubisco
and the suppression of granal development were restricted to the chloroplasts in the cells adjacent to the vascular
bundles.
© 1996 Annals of Botany Company
Key words: Bundle sheath, C 4 plant, chloroplast, granum, immunogold localization, Portulaca grandiflora Hook,
Rubisco.
INTRODUCTION
Chlorenchyma of C4 plants consists of two distinct types of
photosynthetic cells (Laetsch, 1974). The bundle sheath cells
are just around the vascular bundle and the mesophyll cells
surround the bundle sheath cells. The C4 dicarboxylic acid
metabolism of photosynthesis is operated by the cooperative
function of the two cell types. Atmospheric CO2 is initially
fixed into C4 dicarboxylic acids in the mesophyll cells. The
C4 dicarboxylic acids are then transported to the bundle
sheath cells where they are decarboxylated to release CO2
and C3 compounds. The released CO2 is refixed via ribulose
1,5-bisphosphate carboxylase/oxygenase (Rubisco) into the
photosynthetic carbon reduction cycle and the remaining C3
compounds are transferred back to the mesophyll cells
where they are converted to phosphoenolpyruvate and used
as the acceptor of atmospheric CO2 (Hatch, 1987). Therefore, it is reasonable that Rubisco is specifically located in
bundle sheath chloroplasts while it is almost absent in
mesophyll chloroplasts (Slack, Hatch and Goodchild, 1969;
Hattersley, Watson and Osmond, 1977).
There are three biochemical subtypes in C4 species based
on the difference in decarboxylating enzymes in bundle
sheath cells (Gutierrez, Gracen and Edwards, 1974; Hatch,
Kagawa and Craig, 1975). Bundle sheath chloroplasts of
one of the subtypes, the NADP-malic enzyme (NADP-ME)
* For correspondence.
0305-7364/96/060629 + 09 $18.00/0
type, lack well-developed grana while mesophyll chloroplasts have typical grana. Although part of the thylakoid
system in bundle sheath chloroplasts develops granal stacks
during the early stages of leaf development, as reported by
Brangeon (1973), our previous quantitative analysis indicated that the average number of thylakoids per granum is
suppressed throughout the course of leaf development
(Nishioka et al., 1993). Therefore, granal development is
suppressed in bundle sheath chloroplasts from early in leaf
development of NADP-ME type C4 species. However, the
mechanism of suppression of granal development is not
known.
Recently the mechanism of structural and functional
differentiation in chlorenchyma of C4 plants has become of
great interest. It is now recognized that cell type specific
expression of C4 photosynthetic genes is regulated by light
and cell position (Nelson and Langdale, 1989, 1992;
Langdale and Nelson, 1991). Most of the photosynthetic
enzymes, except Rubisco, are accumulated in appropriate
cell types under high light conditions but are almost absent
under low light or in the dark. On the contrary Rubisco is
accumulated in both mesophyll and bundle sheath chloroplasts in the dark or under low light conditions. Accumulation of Rubisco in mesophyll chloroplasts is repressed under high light conditions. However, repression of
Rubisco accumulation is not evident in the tissues where
more than two mesophyll cells are located between bundle
sheath cells such as in husk leaves of maize (Langdale et al.,
© 1996 Annals of Botany Company
630
Nishioka et al.—Granal Development and Rubisco in Portulaca
1988). Therefore, vascular bundles seem to play an
important role in positional regulation of photosynthetic
gene expression in C4 plants although the mechanism is not
understood. The regulation mechanisms such as suppression
of granal development in NADP-ME type C4 plants by
which cellular structures are regulated are unknown.
Portulaca grandiflora Hook is an NADP-ME type C4
plant (Gutierrez et al., 1974) with some traits of plants with
Crassulacean acid metabolism (CAM) (Ku et al, 1981). Its
succulent, cylindrical leaves have several types of green cells.
Bundle sheath cells around the peripheral vascular bundles
possess agranal chloroplasts typical of NADP-ME type C4
plants (Nishioka et al., 1993). Mesophyll cells with granal
chloroplasts incompletely surround the bundle sheath cells.
Water storage cells with few, small chloroplasts are located
inside the peripheral array of vascular bundles. Another
vascular bundle is located in the centre of the leaf and
surrounded by water storage cells. The characteristic
arrangement of green cells in P. grandiflora is of interest in
studying the positional regulation of C4 photosynthetic
development in relation to the location of vascular bundles.
Therefore, we examined the granal development and the
accumulation of Rubisco in chloroplasts of various cell
types of P. grandiflora to gain an insight into the positional
regulation of chloroplast differentiation.
equal volume of buffer solution containing 100 mM TrisHC1 (pH 6-8), 4 % SDS, 2 % mercaptoethanol, 20 % glycerol
and 0-1 % bromophenol blue, boiled for 3 min and
centrifuged at 10000 g for 1 min. The supernatant was used
for SDS-polyacrylamide gel electrophoresis on a 15%
polyacrylamide gel (Laemmli, 1970). The soluble proteins
separated were electrophoretically transferred to a nitrocellulose membrane (Towbin, Staehelin and Gordon, 1979).
Rubisco proteins on the membrane were immunochemically
detected with the rabbit antiserum and alkaline phosphataseconjugated goat anti rabbit IgG antibody.
Immunogold localization for electron microscopy
Small segments of Portulaca leaves were fixed in 3%
glutaraldehyde in 50 mM sodium phosphate buffer (pH 7-3)
for 3 h at 4 °C and washed in the buffer. They were
dehydrated in an ethanol series and embedded in LR White
resin. Ultrathin sections were collected on nickel grids
coated with Formvar. Sections on grids were incubated in
1 % bovine serum albumin in Tris-HCl buffered saline
(TBS) consisting of 50 mM Tris-HCl (pH 7-3) and 150 mM
NaCl for 2 h at room temperature. They were then incubated
in a 1:200 dilution of the antiserum in TBS for 2 h. For
control sections, antiserum was replaced with non-immune
rabbit serum. The sections were washed with TBS containing
0-1 % (v/v) Tween 20 and with TBS and incubated in a 1:50
M A T E R I A L S AND M E T H O D S
dilution of a suspension of goat anti rabbit IgG antibody15 nm colloidal gold complex for 2 h. They were washed
Plant materials
with TBS and distilled water, stained with uranyl acetate
Seeds of Portulaca grandiflora were sown in pots and grown and lead citrate and observed on a Hitachi H-600 electron
in a greenhouse. Middle portions of fully elongated microscope at 100 kV.
uppermost leaves were used. Mature leaves of Nicotiana
The density of labelling was determined by counting the
tabacum L. (cv. Bright Yellow) and Zea mays L. (cv. Honey gold particles and measuring the sectional area of the
Bantam) were also used to check the reliability of antiserum. organelles. Areas occupied by starch grains were omitted
from the calculation of chloroplast area. The density of gold
particles on the mitochondria of bundle sheath cells was
used as the background value. About ten photographs were
Light and electron microscopy
estimated for each organelle.
Leaf tissues were fixed and embedded in Epon 812 or
Spurr's resin for conventional light and electron microscopy
as described previously (Nishioka et al., 1993).
RESULTS
Leaf anatomy
Antiserum
The leaves of P. grandiflora are cylindrical and composed of
various cell types. Vascular bundles are located peripherally
in a transverse section of a leaf (Fig. 1 A, VBp). Bundle
sheath cells surround the peripheral vascular bundle and
their chloroplasts are centripetally oriented (Fig. 1B, BS).
Mesophyll cells incompletely surround the bundle sheath
cells and occur in the areas between the bundle sheath and
the epidermis and between the bundle sheaths (Fig. 1 A, B,
Western blotting
MC). They are lacking at the inner side of the bundle
Leaf tissues (2 g f. wt) were frozen with liquid nitrogen sheath. Water storage cells (Ku et al, 1981) or watery cells
and ground in a mortar. Tissues were further ground in (Kim and Fisher, 1990) fill the inner portion of the leaf (Fig.
2-5 ml of buffer solution consisting of 5 ml of 50 mM Tris- 1A). They are highly vacuolated and contain few, small
HC1 (pH 7-5), 1 ml of 10 mM NaCl and 0-5 ml of 5 mM chloroplasts. Bundle sheath cells located towards the inner
sodium ascorbate. The homogenate was centrifuged at side of the leaf are somewhat different from other bundle
10000 g for 20 min. The supernatant was mixed with an sheath cells (Fig. 1B, BSi). They are not associated with
Rabbit antiserum raised against the whole molecule (large
and small subunits) of Rubisco of tobacco (cv. Bright
Yellow) was a generous gift from Professor A. Hirai,
Faculty of Agriculture, the University of Tokyo, Japan.
Nishioka et al.—Granal Development and Rubisco in Portulaca
631
VBp
j
FIG. 1. Transverse sections of Portulaca grandiflora leaves. A, Complete cross section with a peripheral vascular bundle (VBp) and a central
vascular bundle (VBc) labelled and mucilage cells (Mu) also visible, (magnification x 140). B, Region around a peripheral vascular bundle;
mesophyll cells (MC) incompletely surround a layer of bundle sheath cells (BS) with the bundle sheath cell located on the inner side of the leaf
(BSi) containing fewer chloroplasts than other bundle sheath cells, (magnification x 380). C, Region around the central vascular bundle, which
is embedded in water storage cells (W). A water storage cell adjacent to the central vascular bundle is labelled with We. (magnification x 380).
632
Nishioka et al.—Granal Development and Rubisco in Portulaca
kDa
1
2
3
25-
FIG. 2. Diagram of various cell types between a peripheral vascular
bundle (VBp) and the central vascular bundle (VBc) in a cross section
of a Portulaca grandiflora leaf. BS, bundle sheath cell; BSi, bundle
sheath cell on the inner side of the leaf; MC, mesophyll cell; W1 to W.,,
water storage cells numbered away from VBp; We, water storage cell
adjacent to the central vascular bundle. Intervening water storage cells
may occur between W3 and We.
mesophyll cells and appear intermediate between a bundle
sheath cell and a water storage cell. They contain few
chloroplasts, which are centripetally oriented. Another
vascular bundle is in the centre of the leaf (Fig. 1 A, VBc).
It is surrounded by water storage cells and typical bundle
sheath cells are not observed (Fig. 1 C). However, chloroplasts in the water storage cells just adjacent to the central
vascular bundle show a tendency to be located near to the
vascular bundle (Fig. 1C, We).
Based on the above observations, we distinguished the
following cell types for further investigation on chloroplast
differentiation (Fig. 2): typical bundle sheath (BS) and
mesophyll cells (MC) around the peripheral vascular bundle
(VBp), bundle sheath cells locating on the inner side (BSi)
and water storage cells (W). Water storage cells were further
classified into Wx to W3 according to their position between
the peripheral and the central vascular bundles (VBc) with
the outermost one being W,. The innermost one, just
adjacent to the central vascular bundle, was designated We.
W3 and We do not always abut on each other but may be
separated by water storage cells.
Reactivity of antiserum
The cross-reactivities of anti Rubisco serum with leaf
proteins were examined by western blotting (Fig. 3).
Although the antiserum had been raised against Rubisco of
tobacco, it also reacted well with Rubisco proteins from P.
grandiflora and Zea mays. Strong single bands which
corresponded to the large subunits of Rubisco appeared on
the blots (lanes 4 to 6). The small subunits of Rubisco were
not stained in the present conditions although the antiserum
FIG. 3. SDS-PAGE (lanes 1 to 3) and western blots (lanes 4 to 6) of
protein extracts from leaves of tobacco (lanes 1 and 4), Portulaca
grandiflora (lanes 2 and 5) and maize (lanes 3 and 6). Antiserum was
raised against the complete Rubisco molecule of tobacco.
had been raised against whole molecule of Rubisco. We
concluded that this antiserum was reactive and specific
enough to examine the accumulation of Rubisco in various
chloroplasts of P. grandiflora leaves.
Chloroplast structure and Rubisco accumulation
Mesophyll chloroplasts of P. grandiflora possessed welldeveloped grana (Fig. 4 A) but their stroma was scarcely
labelled with the colloidal gold particles indicating sparse
accumulation of Rubisco (Fig. 4B). On the contrary,
chloroplasts of typical bundle sheath cells possessed only
few, rudimentary grana (Fig. 4C) and were densely labelled
with the gold particles (Fig. 4D). Chloroplasts of the bundle
sheath cells located on the inner side of the leaf (BSi)
showed somewhat developed grana (Fig. 5 A) but they were
not so extensive as those in mesophyll chloroplasts. These
chloroplasts were densely labelled with the gold particles
(Fig. 5B). Chloroplasts in the water storage cells (WJ next
to BSi were slender and possessed well-developed grana
(Fig. 5C). Gold particles were scarcely observed on these
chloroplasts (Fig. 5D). The structure and the distribution of
gold particles of chloroplasts in the inner water storage cells
(W2 and W3) were essentially the same as those in Wx (Fig.
6 A and B). However, chloroplasts in the innermost water
storage cells (We) adjacent to the central vascular bundle
possessed reduced grana (Fig. 6C) and showed a high
density of labelling with the gold particles (Fig. 6D).
Quantitative analyses of granal development and density
of gold particles supported the above observations (Table
Nishioka et al.—Granal Development and Rubisco in Portulaca
633
wmWmm
FIG. 4. Mesophyll chloroplasts (A, B) and bundle sheath chloroplasts (C, D) in Portulaca grandifiora leaves observed by conventional electron
microscopy (A, C) and immunogold labelling for Rubisco (B, D). Grana (G) are developed well in mesophyll chloroplast (A) but are rudimentary
in bundle sheath chloroplasts (C). Note high density of gold particles in bundle sheath chloroplasts (D) compared with mesophyll chloroplast (B).
(magnifications A, x 14100. B, x 19 500. C, x 14000. D, x 26200).
634
Nishioka et al.—Granal Development and Rubisco in Portulaca
FIG. 5. Chloroplasts in the bundle sheath cells located on the inner side (A, B) and the water storage cells abutting on the inner bundle sheath
cells (C, D) in Portulaca grandiflom leaves observed by conventional electron microscopy (A, C) and immunogold labelling for Rubisco (B D)
Note rudimentary grana (A) and high density of gold particles (B) in bundle sheath chloroplasts compared with well-developed grana (C)'and
low density of gold particles (D) in chloroplasts of the water storage cells, (magnifications A, x 19600. B, x 24000. C, x 14500. D, x 36400).
635
Nishioka et al.—Granal Development and Rubisco in Portulaca
v;.;T
B
D
FIG. 6. Chloroplasts in the third inner water storage cells (A, B) and the innermost water storage cell (C, D) in Portulaca grandiflora leaves observed
by conventional electron microscopy (A, C) and immunogold labelling for Rubisco (B, D). Note well-developed grana (A) and low density of gold
particles (B) in chloroplasts of water storage cells compared with relatively reduced grana (C) and relatively high density of gold particles (D) in
chloroplasts of the innermost water storage cells, (magnifications A, x 20500. B, x 31 700. C, x 17600. D, x 29 100).
Nishioka et al.—Granal Development and Rubisco in Portulaca
636
1. Development of grana and immunogold labelling of Rubisco in chloroplasts in cell types of P. grandiflora leaves.
Values are mean + s.e. Means with different superscript letters are significantly different at P = 0-05 by Duncan's multiple
range test
TABLE
Cell type*
No. thylakoids per
granum
No. gold particles per
stromal area (/mi2)t
BS
BSi
MC
2-2±01 c
2-6±01 c
5-O±O-3a
a
b
11
96-9±10
83-3+l-3
We
3-7 + 0-4
4-3 + 0-2*
31 ±0-2
d
4-0±0-4 b
3-8±O-5
d
4-0 + 0-411
fl
3-2 + 0-3
2-6 + 0-2°
50-5± l-3e
* For abbrevations see Fig. 2.
t Background value (mitochondria of BS) is 0-7 + 0-2.
1). The number of thylakoids per granum was significantly
reduced in the chloroplasts of BS and BSi compared with
that of MC and water storage cells Wj to W3. The
development of grana in the chloroplasts of We was also
suppressed. The greater density of gold particles for Rubisco
was observed in the chloroplasts of BS. The density in the
chloroplasts of BSi was somewhat smaller than that of BS.
The density of gold particles was extremely low in the
chloroplasts of MC and water storage cells Wx to W3
although it was still higher than the background value
(mitochondria of bundle sheath cells). However, considerable accumulation of gold particles was apparent in the
chloroplasts of We. Positional gradients in granal development and labelling of Rubisco were not observed in
the water storage cells W1 to W3.
DISCUSSION
Immunocytochemical techniques using immunogold labelling for electron microscopy have been widely used to
localize macromolecules in plant tissues (Herman, 1988).
These techniques offer high resolution compared with
immunolocalization using fluorescent antibodies and in situ
localization using radioactive probes. It is possible, with
immunogold localization, to compare quantitatively the
intensity of labelling in organelles in different tissues. The
reactivity of antiserum raised against Rubisco of tobacco
was strong enough to localize Rubisco of other plant
species. Therefore, we used immunogold localization to
examine the accumulation of Rubisco in the chloroplasts of
various tissues in P. grandiflora.
Ku et al. (1981) isolated protoplasts from three distinct
cell types of P. grandiflora leaves, i.e. bundle sheath cells,
mesophyll cells and water storage cells and detected high
RuBP carboxylase activity only in bundle sheath protoplasts. In our study, using immunogold labelling, we
distinguished several other cell types. The greatest accumulation of Rubisco was observed in the chloroplasts of
typical bundle sheath cells. The accumulation was somewhat
reduced in the bundle sheath cells facing the inner side of the
leaf (BSi), which are not in contact with mesophyll cells.
Rubisco was extremely reduced in the chloroplasts of
mesophyll cells and water storage cells (Wx to W3). However,
considerable accumulation of Rubisco was apparent in the
chloroplasts of innermost water storage cells (We) adjacent
to the central vascular bundle. Therefore, the existence of
vascular bundles seems to affect the genetic expression of
Rubisco in cells and chloroplasts. Occurrence of adjacent
mesophyll cells may also promote the accumulation of
Rubisco.
A C4 grass Aristida latifolia Domin has double bundle
sheaths. Ueno (1992) observed the highest accumulation of
Rubisco in the chloroplasts of the inner bundle sheath cells,
a slightly lower accumulation in the outer bundle sheath cells
and practically no accumulation in mesophyll cells using
immunogold labelling. Cheng et al. (1988) discriminated
outer large mesophyll cells and inner small mesophyll cells
in addition to bundle sheath cells in a C4-like dicotyledon
Flaveria brownii A. M. Powell. They isolated protoplasts
from these three cell types and detected a gradient of RuBP
carboxylase activity increasing towards the nearest vein.
These observations also suggest that the location of vascular
bundles influences the accumulation of Rubisco. However,
the gradient of Rubisco accumulation was not detected and
the accumulation was restricted to the cells adjacent to
vascular bundles in P. grandiflora.
A trend similar to Rubisco accumulation was observed in
the suppression of granal development in chloroplasts of P.
grandiflora. Granal development was suppressed in the
chloroplasts of bundle sheath cells and innermost water
storage cells adjacent to the central vascular bundle. The
chloroplasts of remaining water storage cells not abutting
on vascular bundles possessed developed grana although
they were not so extensive as those of mesophyll chloroplasts. In addition, the chloroplasts of innermost water
storage cells tended to be located near the vascular bundle,
which is a characteristic feature of bundle sheath chloroplasts of C4 dicotyledons (Carolin, Jacobs and Vesk, 1978).
The photosynthetic role of the innermost water storage
cells is questionable because of their distance from mesophyll
cells and sparse occurrence of chloroplasts in these cells.
However, positional regulation for the differentiation of
bundle sheath cells seems to be exerted on these cells from
the central vascular bundle. Therefore, the present observations support the idea that C4 plants utilize vascular
bundles as positional landmarks for the differentiation of
photosynthetic cell types (Nelson and Langdale, 1992).
Langdale and Nelson (1991) and Nelson and Langdale
(1992) postulated that positional regulation from a vascular
bundle does not operate beyond two cell distances and that
Nishioka et al.—Granal Development and Rubisco in Portulaca
C3 development is the default scheme in the absence of
regulation. In fact Langdale et al. (1988) examined
photosynthetic gene expression in various leaf-like organs
such as husk leaves of maize and detected Rubisco in
mesophyll cells in the organs where more than two mesophyll
cells were located between adjacent bundle sheaths. In P.
grandiflora, however, the positional regulation seems more
effective than in maize. The accumulation of Rubisco was
not detected in water storage cells between a bundle sheath
cell and an innermost water storage cell although there were
usually more than three water storage cells between them.
ACKNOWLEDGEMENTS
We thank Prof. A. Hirai, Faculty of Agriculture, the
University of Tokyo for the gift of antiserum. We also
thank Prof. A. Watanabe, Graduate School of Science, the
University of Tokyo for helpful advice in western blotting.
This work was supported by Grant-in-Aid for Scientific
Research from the Ministry of Education, Science and
Culture, Japan.
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