Journal of Experimental Botany, Vol. 52, No. 358, pp. 1003±1013, May 2001
Ultrastructural localization of photosynthetic and
photorespiratory enzymes in epidermal, mesophyll,
bundle sheath, and vascular bundle cells of the C4 dicot
Amaranthus viridis
Osamu Ueno1
Department of Plant Physiology, National Institute of Agrobiological Sciences, Tsukuba,
Ibaraki 305-8602, Japan
Received 31 August 2000; Accepted 14 November 2000
Abstract
In the leaves of the NAD-malic enzyme (NAD-ME)type C4 dicot Amaranthus viridis L., there are chloroplasts in the vascular parenchyma cells (VPC),
companion cells (CC), ordinary epidermal cells (EC),
and guard cells (GC), as well as in the mesophyll cells
(MC) and the bundle sheath cells (BSC). However, the
chloroplasts of the VPC, CC, EC, and GC are smaller
than those of the MC and BSC. In this study, the
accumulation of photosynthetic and photorespiratory
enzymes in these leaf cell types was investigated
by immunogold labelling and electron microscopy.
Strong labelling for phosphoenolpyruvate carboxylase was found in the MC cytosol. Weak labelling
was observed in the CC and GC cytosol. Labelling for
pyruvate, Pi dikinase occurred to varying degrees in
the chloroplasts of all cell types except CC. Labelling
for the large subunit of ribulose-1,5-bisphosphate
carboxylaseuoxygenase was detected in the chloroplasts of all cell types except MC. For both NAD-ME
and the P-protein of glycine decarboxylase, intense
labelling was found in the BSC mitochondria; weaker
labelling was recognized in the VPC mitochondria.
These data indicate that when not only the MC and
BSC but also the other leaf cell types are included,
the cell-specific expression of the enzymes in C4
leaves becomes more complex than has been known
previously. These findings are discussed in relation
to the metabolic function of epidermal and vascular
bundle cells.
1
Key words: Amaranthus viridis, C4 plant, immunogold
localization, leaf component cells, photosynthetic and
photorespiratory enzymes.
Introduction
Leaves are composed of various kinds of tissues, including epidermis, mesophyll, bundle sheath, and vascular
bundles. In C3 plants, the major photosynthetic cells
are the mesophyll cells (MC). In C4 plants, however,
both the MC and the bundle sheath cells (BSC) are the
major photosynthetic cells. Differentiation of the two cell
types is essential for the operation of C4 photosynthesis
(Hatch, 1987; Dengler and Nelson, 1999). The primary
CO2 ®xation enzyme, phosphoenolpyruvate carboxylase
(PEPC), is localized in the MC. The decarboxylation
enzymes, such as NADP and NAD-malic enzyme (ME),
and ribulose-1,5-bisphosphate carboxylaseuoxygenase
(Rubisco), are distributed in the BSC (Hatch, 1987). The
photorespiratory enzyme glycine decarboxylase (GDC) is
also predominant in the BSC (Ohnishi and Kanai, 1983;
Morgan et al., 1993). The compartmentalization of these
enzymes has been extensively studied in C4 leaves, and
interest is growing in the molecular mechanism of the
structural and functional differentiation of C4 leaves
(Dengler and Nelson, 1999; Sheen, 1999).
In leaf cells, chloroplasts are not restricted to the
MC and BSC. The guard cells (GC) generally contain
Fax: q81 298 38 7408. E-mail: [email protected]
Abbreviations: BSC, bundle sheath cells; CC, companion cells; GC, guard cells; GDC, glycine decarboxylase; LS, large subunit; MC, mesophyll cells;
NAD-ME, NAD±malic enzyme; EC, ordinary epidermal cells; PEPC, phosphoenolpyruvate carboxylase; PPDK, pyruvate, Pi dikinase; Rubisco,
ribulose-1,5-bisphosphate carboxylaseuoxygenase; VPC, vascular parenchyma cells.
ß Society for Experimental Biology 2001
1004
Ueno
chloroplasts (Willmer and Fricker, 1996). The epidermal
cells of leaves of some submerged aquatic plants, which
lack stomata, are well known to include prominent
chloroplasts (Sculthorpe, 1967). In the leaves of some
dicot plants, the phloem parenchyma includes chloroplasts, irrespective of the difference in the photosynthetic
modes (Crookston and Ozbun, 1975). In leaves of the C4
dicot Amaranthus retro¯exus, all living cell types except
for sieve-tube members include chloroplasts (Fisher and
Evert, 1982). To elucidate the functional signi®cance of
chloroplasts in leaf cells other than the MC and BSC, it is
important to know how they accumulate photosynthetic
and photorespiratory enzymes. Besides the GC (Willmer
and Fricker, 1996), however, little is known about the
distribution of enzymes in these cells. It would be dif®cult
to isolate the vascular parenchyma cells (VPC) and the
ordinary epidermal cells (EC) without contamination
to study enzyme localization. On the other hand, such
information could contribute signi®cantly to the present
understanding of how the cell-speci®c expression of the
enzymes within C4 leaves is regulated.
The NAD-ME-type C4 dicot Amaranthus viridis has
chloroplasts in various leaf cells, as has A. retro¯exus.
This study used immunogold labelling and electron
microscopy to determine the cellular accumulation of
representative enzymes involved in C4 photosynthesis and
photorespiration in leaves of Amaranthus viridis, with
particular reference to leaf cells other than MC and BSC.
Materials and methods
Plant material
Seeds of Amaranthus viridis L. were sown in 4.5 dm3 pots ®lled
with ®eld soil. Plants were grown in a naturally illuminated
greenhouse maintained at 25±30u20 8C (dayunight temperature).
They were watered daily and supplied weekly with full-strength
Hoagland's solution. Fully expanded young leaves were
examined 2 months after planting.
Light and electron microscopy of leaf inner structure
Leaf samples were collected at 09.00 h. Small segments of leaves
were ®xed immediately in 3% glutaraldehyde in 50 mM sodium
phosphate (pH 6.8) for 1.5 h. They were then washed in
phosphate buffer and post-®xed in 2% OsO4 in buffer. They
were then dehydrated through an acetone series and embedded
in Spurr's resin. Semithin sections were stained with 1%
toluidine blue O. Ultrathin sections were stained with uranyl
acetate and lead citrate.
To determine the sizes of chloroplasts and mitochondria, the
long axes of chloroplasts and the diameters of mitochondria
were measured on electron micrographs that had been magni®ed
to 5600 3 for the chloroplasts of the MC and BSC, and
17 000 3 for those of the remaining cell types and for the
mitochondria of all cell types. The cited values are means of
16±27 measurements for chloroplasts and 22±30 measurements
for mitochondria.
Antisera
The following antisera were used for immunogold electron
microscopy: anti-pea Rubisco large subunit (LS) antiserum
(courtesy of S Muto, Nagoya University), anti-Amaranthus
tricolor NAD-ME antiserum (courtesy of T Murata, Iwate
University), anti-pea mitochondrial GDC (P-protein) antiserum
(courtesy of DJ Oliver, University of Idaho), and anti-maize
PEPC and PPDK antisera (courtesy of M Matsuoka, Nagoya
University). These antisera were the same as those used in
previous immunocytological studies (Ueno, 1992, 1998a, b;
Ueno and Agarie, 1997).
Protein A-immunogold electron microscopy
Leaf samples were collected at 09.00 h. Small segments of leaves
were ®xed with 3% glutaraldehyde in 50 mM sodium phosphate
(pH 6.8), dehydrated through an ethanol series, and embedded
in Lowicryl K4M resin (Chemische Werke Lowi GmbH,
Waldkraiburg, Germany), as previously described (Ueno, 1992).
Sections were immunolabelled with the antisera and 15 nm
protein A-colloidal gold particles (EY Lab. Inc., San Mateo, CA,
USA) by following the same procedure (Ueno, 1992). For
controls, antisera were replaced by non-immune serum. Crossreactivity of the antisera used was examined by Western blotting
of crude extracts of leaves after SDS-PAGE, as previously
described (Ueno, 1992). For both immunolabelling and Western
blotting, the antisera were used at a dilution of 1 : 1000 for
Rubisco LS and NAD-ME, and at dilution of 1 : 500 for PEPC,
PPDK and the P-protein of GDC.
Quantitative analysis of immunogold particles
The density of labelling was determined by counting the gold
particles on electron micrographs at 19 000 3 magni®cation and
calculating the number per unit area (mm2). For pro®les of
chloroplasts, the areas occupied by starch grains and crystalline
inclusions were excluded from estimation. Between 5 and 14
individual cells were examined in several immunolabelled
sections.
Results
Structural features of leaves
A brief summary of the structural features of the leaves
of A. viridis is followed by the results of immunocytochemical localization of enzymes.
The results of observation of A. retro¯exus leaves by
other authors (Fisher and Evert, 1982) was con®rmed in
this study. Leaves of A. viridis exhibit typical Kranz-type
anatomy (Fig. 1). The major chlorenchyma consisted
of MC and BSC. The MC showed a trend of radial
arrangement. Frequently, druse-containing cells were
present in the mesophyll. Large BSC surrounded the
vascular bundle. Stomata occurred more frequently on
the abaxial epidermis than on the adaxial. Under
light microscopy, the existence of chloroplasts was
easily recognized in the MC and BSC, but not in other
leaf cells.
In leaves of A. viridis, all living cells other than
sieve-tube members had chloroplasts, as observed in
Enzyme localization in Amaranthus leaf
1005
Table 1. Sizes of chloroplasts and mitochondria in various cells of
leaves of Amaranthus viridis
Cell type
Ordinary epidermal cells
Guard cells
Mesophyll cells
Bundle sheath cells
Vascular parenchyma cells
Companion cells
Chloroplasts
(mm)
Mitochondria
(mm)
3.70"1.04
2.70"0.71
7.99"1.05
13.03"3.25
4.91"1.16
2.87"0.54
0.29"0.05
0.34"0.11
0.36"0.10
0.80"0.30
0.39"0.08
0.36"0.05
(21)
(16)
(25)
(30)
(20)
(27)
(22)
(30)
(25)
(30)
(30)
(30)
Values are given as means"SD. Numbers in parentheses show the
numbers of organelles examined.
observed in the chloroplasts of the BSC, EC and MC
(Fig. 2B). Crystalline inclusions were rare in the MC
chloroplasts relative to the BSC and EC chloroplasts.
Fig. 1. Cross-section of a leaf of Amaranthus viridis. Asterisk indicates
a druse-containing cell. Bar ˆ 50 mm. MC, mesophyll cell; BSC, bundle
sheath cell; OEC, ordinary epidermal cell; St, stoma; V, vascular bundle.
A. retro¯exus leaves (Fisher and Evert, 1982). The MC
included large chloroplasts (Table 1). As pointed out
previously (Fisher and Evert, 1982), the degree of granal
development in the chloroplasts was lower in the adaxial
MC (Fig. 2A) than in the abaxial MC (data not shown).
The BSC included numerous chloroplasts in centripetal
positions (Fig. 1). The chloroplasts were larger than those
in the MC (Table 1). They had well-developed grana
and were elongated (Fig. 2B). In the BSC, the numerous
mitochondria, which were larger than those in the MC
(Fig. 2B; Table 1), were always located along the inner
tangential walls and innermost portions of the radial
walls (Fig. 2C).
In the vascular bundles, the VPC and CC included
considerable numbers of chloroplasts (Fig. 2C), smaller
than those of the MC and BSC (Table 1). The VPC
chloroplasts possessed well-developed thylakoids with
grana (Fig. 2C). Although the CC chloroplasts were also
granal, the degree of development was somewhat lower
than in the VPC chloroplasts (Fig. 2C, D). The VPC and
CC mitochondria were smaller than the BSC mitochondria (Table 1). The EC and GC also contained a few small
chloroplasts with less-well developed grana (Fig. 2E, F;
Table 1). In the EC, the chloroplasts were distributed
mainly along the inner tangential walls. The EC and GC
mitochondria were smaller than the BSC mitochondria
(Table 1).
Starch grains were observed more or less in the
chloroplasts of all cell types (Fig. 2). The amounts
of starch grains ranged in the following order: GC)
BSC)VPC)CC)EC)MC. Crystalline inclusions were
Western blot analysis
The cross-reactivity between soluble proteins from leaves
of A. viridis and the antisera used was examined by
Western blotting (Fig. 3). The antisera against PEPC,
PPDK, Rubisco LS, and the P-protein of GDC recognized the respective enzymes from A. viridis, generating
strong single bands on the blots. The antiserum against
NAD-ME gave two adjacent bands on the blot which
differed in molecular size and thickness. These large
and small bands corresponded to the a and b subunits
of NAD-ME protein, respectively (Long et al., 1994).
These results thus provide a reliable basis for immunocytochemical localization of the photosynthetic and
photorespiratory enzyme proteins in leaf sections. When
sections were incubated with non-immune serum, they
showed only non-speci®c and negligible labelling with
gold particles (Fig. 4A).
Cellular localization of photosynthetic and
photorespiratory enzymes
Strong labelling for PEPC was found in the MC cytosol
(Fig. 4B, C; Table 2). Table 3 shows the densities of labelling. The strength of labelling did not differ between the
adaxial and abaxial MC. No signi®cant labelling for
PEPC was observed in the BSC (Fig. 4C), VPC (Fig. 4D),
or EC (Fig. 4B). However, weak labelling for PEPC
was present in the CC cytosol (Fig. 4E). In addition, weak
labelling was occasionally seen in the GC cytosol
(Fig. 4F).
Labelling for PPDK was observed in the chloroplasts
of all leaf cell types except CC (Figs 4G±I, 5A, B; Table 2).
Table 4 shows the densities of labelling for PPDK in
these cells. The strongest labelling for PPDK was found
in the MC chloroplasts (Fig. 4G). Weaker labelling
occurred in the BSC chloroplasts (Fig. 4H). The VPC
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Fig. 2. Chloroplasts of various leaf cells in A. viridis. (A) Chloroplast of an adaxial mesophyll cell. (B) Chloroplasts and mitochondria of a bundle
sheath cell. Unlabelled arrow indicates a crystalline inclusion. (C) Chloroplasts of vascular parenchyma cells and companion cells. Inner parts
of bundle sheath cells are also seen at the left margin, in which large mitochondria are present along the cell walls. (D) Chloroplasts of a companion
cell. (E) Chloroplast of an ordinary epidermal cell. (F) Chloroplast of a guard cell. Bar for C ˆ 2 mm. Bars for others ˆ 1 mm. c, chloroplast;
mt, mitochondrion; s, starch grain; BSC, bundle sheath cell; CC, companion cell; ST, sieve-tube member; VPC, vascular parenchyma cell.
chloroplasts were also weakly labelled for PPDK
(Fig. 4I). Interestingly, the EC and GC chloroplasts
were also labelled for PPDK (Figs 4G, 5B)Ðthe EC
strongly so (Table 4). The crystalline inclusions of
chloroplasts were labelled for PPDK in the EC
(Fig. 5D), but not in the MC (Fig. 5C) or BSC (data
not shown).
Labelling for Rubisco LS was found in the stroma
of the chloroplasts of all leaf cell types except MC
(Fig. 5E±J; Table 2). Table 4 shows the densities of labelling. Strong labelling occurred in the BSC chloroplasts
(Fig. 5F), but weaker labelling was also present in the
VPC and CC chloroplasts (Fig. 5G, H). Signi®cant
labelling for Rubisco LS was also found in both the
EC and GC chloroplasts (Fig. 5I, J) Ð strongly so in the
EC chloroplasts (Table 4). The crystalline inclusions in
the chloroplasts of the MC, BSC and EC were not
labelled for Rubisco LS (Fig. 5E, F, I).
Labelling for NAD-ME was observed in the BSC
and VPC mitochondria; it was stronger in the former
(Fig. 6A, C; Table 2, Table 5). No signi®cant labelling for
NAD-ME was observed in the mitochondria of the MC
(Fig. 6B), CC (Fig. 6C), EC (Fig. 6D), or GC (Fig. 6E).
Labelling for the P-protein of GDC was found in the
Enzyme localization in Amaranthus leaf
Fig. 3. Western blots of proteins extracted from leaves of A. viridis.
Total soluble protein (7.0 mg for all enzymes except Rubisco LS
at 1.5 mg) was subjected to SDS-PAGE, blotted on nitrocellulose
membranes, and identi®ed with antisera against PEPC (1), PPDK (2),
NAD-ME (3), Rubisco LS (4), and the P-protein of GDC (5).
BSC and VPC mitochondria; it was stronger in the
former (Fig. 6F, H; Table 2, Table 5). No signi®cant
labelling for the P-protein of GDC was recognized in
the mitochondria of the MC (Fig. 6G), CC (Fig. 6I) or
EC (Fig. 6J). In the GC mitochondria, however, weak
labelling for the P-protein of GDC was occasionally
found (Fig. 6K).
Sieve-tube members are also living cells in leaves,
and contain plastids but not chloroplasts. They showed
no labelling for any of the antisera used here (data not
shown).
Discussion
The inner structure of A. viridis leaves exhibited features
typical of NAD-ME-type C4 dicot plants (Gutierrez et al.,
1974; Hatch et al., 1975). The cellular localization of
photosynthetic enzymes in the MC and BSC also showed
characteristics of typical NAD-ME plants: PEPC is
localized in the MC, whereas NAD-ME and Rubisco
are distributed in the BSC. However, PPDK, which is
involved in the regeneration of PEP from pyruvate, was
found in both the MC and BSC chloroplasts, although
the chloroplasts of the MC accumulated more PPDK
than those of the BSC. A similar pattern of PPDK
localization was reported for some NADP-ME-type and
NAD-ME-type C4 species of grasses and dicots (Aoyagi
and Nakamoto, 1985; Ueno, 1998b). The predominant
accumulation of the P-protein of GDC in the BSC
1007
mitochondria of A. viridis coincided well with the
results for C4 plants from previous studies (Morgan
et al., 1993).
The VPC included many chloroplasts, in which
Rubisco and PPDK were accumulated. In addition,
NAD-ME and the P-protein of GDC accumulated in
the VPC mitochondria. These patterns of enzyme
accumulation were similar to those of the BSC, although
the levels of accumulation were less in the VPC than in
the BSC. Therefore, it seems likely that the VPC have a
function similar to that of the BSC. The CC have dense
cytosol with many mitochondria and are thought to be
metabolically active; they are intimately involved in
phloem loading (Grusak et al., 1996). In this study
labelling for PEPC was found in the CC, although the
functional role of PEPC is unknown. Recent immunohistochemical studies have reported the accumulation of
PEPC in the vascular tissues of germinating wheat grains
(Gonzalez et al., 1998) and of developing grape berries
(Famiani et al., 2000). In the latter case, PEPC may play
an important role in the metabolism of assimilates during
delivery to sink tissues (Famiani et al., 2000). The CC
contained a considerable number of granal chloroplasts
that accumulated Rubisco. It is unlikely that CC have a
function similar to that of BSC, because the mitochondria
are substantially lacking NAD-ME, and the CC are
located within the innermost of the vascular bundles. The
presence or absence of chloroplasts in CC and VPC is
not associated with the difference in photosynthetic
modes (Crookston and Ozbun, 1975). Therefore, the CC
chloroplasts, together with the VPC chloroplasts, may be
responsible for the recycling of metabolically released
CO2 (Crookston and Ozbun, 1975).
The metabolic pathways in the GC that may operate
during stomatal opening and closing have been proposed
mainly from labelling studies of metabolites and detection
of enzyme activities (Willmer and Fricker, 1996). There is
still considerable debate over whether the Calvin±Benson
cycle is present in GC chloroplasts (Willmer and Fricker,
1996). However, the accumulation of Rubisco in GC
chloroplasts (Zemel and Gepstein, 1985; Vaughn, 1987)
and the activity of Rubisco and other Calvin±Bensoncycle enzymes in GC (Shimazaki et al., 1989) have been
demonstrated. The accumulation of Rubisco in the GC
chloroplasts of A. viridis was also found unambiguously.
In GC, PEPC is thought to be involved in the synthesis
of malate, a metabolite that accumulates during
stomatal opening (Willmer and Fricker, 1996). In the
GC cytosol of A. viridis, labelling for PEPC was also
detected, but the density was weaker than would be
expected from the pivotal role of PEPC. At present, the
reason is not clear. It may be due to the difference in
the immunological traits of PEPC isoforms, although
stomatal PEPC has not yet been suf®ciently characterized
(Denecke et al., 1993). The GC chloroplasts of A. viridis
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Ueno
Fig. 4. Immunogold labelling of various leaf cells in A. viridis with non-immune serum (A) and with antisera to PEPC (B±F) and to PPDK (G±I).
(A) Bundle sheath cell. (B) Ordinary epidermal cell and mesophyll cell. (C) Mesophyll cell and bundle sheath cell. (D) Vascular parenchyma cell.
(E) Companion cells. (F) Guard cell. (G) Ordinary epidermal cell and mesophyll cell. (H) Bundle sheath cell. (I) Vascular parenchyma cell. Bars for B
and G ˆ 1 mm. Bars for others ˆ 0.5 mm. CW, cell wall; N, nucleus. For other abbreviations, see legends to Figs 1 and 2.
showed signi®cant labelling for PPDK. Schnabl found
PPDK activity in pellets of GC protoplasts of Vicia faba,
and suggested that PPDK may be responsible for the
formation of PEP from pyruvate generated by malate
decarboxylation (Schnabl, 1981). However, labelling for
NAD-ME was not found in the GC mitochondria of A.
viridis. It remains to examine whether NADP-ME is
present.
Whether or not photorespiration is present in the GC
also remains unknown (Willmer and Fricker, 1996). The
GC possess three kinds of organelles required for the
operation of the glycolate pathway: chloroplasts, peroxisomes and mitochondria (Willmer and Fricker, 1996).
There is an indication that oxygenase activity of Rubisco
occurs in the GC (Cardon and Berry, 1992). Only weak
labelling was found for the P-protein of GDC in the
Enzyme localization in Amaranthus leaf
1009
Table 2. Summary of the cellular labelling of photosynthetic and photorespiratory enzymes in various cells of leaves of A. viridis
Cell type
PEPC
(Cytosol)
Ordinary epidermal cells
Guard cells
Mesophyll cells
Bundle sheath cells
Vascular parenchymal cells
Companion cells
qu
qqq
q
PPDK
(Chlt)
Rubisco LS
(Chlt)
qq
q
qqq
q
q
qqq
quqq
qqq
qq
q
Parentheses show the cell fraction in which the labelling is present. Chlt, chloroplasts; Mit, mitochondria. q and
labelling: qqq indicates strong labelling, indicates weak or no labelling.
Table 3. Immunogold labelling of PEPC in various cells of leaves
of A. viridis
Cell type
Ordinary epidermal cells
Guard cells
Mesophyll cells
Bundle sheath cells
Vascular parenchyma cells
Companion cells
No. of gold particles (mm 2)
Cytosol
Organelles
0.8"0.9 (11)
2.1"1.2 (7)
104.8"20.0 (9)
0.1"0.1 (8)
0.5"0.6 (6)
3.3"1.1 (14)
0.9"0.7
0.9"0.9
0.5"0.5
0.3"0.2
0.3"0.2
0.5"0.6
(8)
(7)
(10)
(7)
(6)
(12)
Numbers of gold particles per unit area (mm 2) are given as
means"SD. Numbers in parentheses show the numbers of cell pro®les
examined.
Table 4. Immunogold labelling of PPDK and Rubisco LS in
various cells of leaves of A. viridis
Enzyme and cell type
PPDK
Ordinary epidermal cells
Guard cells
Mesophyll cells
Bundle sheath cells
Vascular parenchyma cells
Companion cells
Rubisco LS
Ordinary epidermal cells
Guard cells
Mesophyll cells
Bundle sheath cells
Vascular parenchyma cells
Companion cells
No. of gold particles (mm 2)
Chloroplasts
Cytosolqother
organelles
26.5"7.8
5.3"1.9
91.7"8.3
5.4"1.5
2.6"0.8
0.6"0.6
(11)
(6)
(11)
(10)
(10)
(10)
0.4"0.7
0.7"0.6
0.6"0.7
0.2"0.2
0.3"0.3
1.0"0.2
41.5"13.6 (9)
14.3"3.1 (5)
0.7"0.3 (13)
37.9"5.6 (10)
18.0"4.8 (7)
8.4"1.8 (9)
ND (9)
0.2"0.2
0.1"0.2
ND (9)
0.1"0.2
0.1"0.1
(12)
(7)
(7)
(5)
(8)
(8)
(6)
(8)
(9)
(6)
Numbers of gold particles per unit area (mm 2) are given as
means"SD. ND, not detectable. Numbers in parentheses show the
numbers of chloroplasts or cell pro®les examined.
GC mitochondria. Further investigation is necessary to
estimate the extent of photorespiration in the GC.
The EC have a cuticle on their outer tangential walls,
which are thought to be almost impermeable to water
and CO2. However, the inner tangential walls of the EC
may be permeable. In this study, the EC included
NAD-ME
(Mit)
GDC
(Mit)
uq
qqq
qq
qqq
qq
refer to the relative intensities of
granal chloroplasts that showed a high density of
labelling for Rubisco and accumulated starch grains.
The concentration of CO2 in mesophyll in C4 leaves is
about 100 ml l 1, lower than in C3 leaves (Osmond et al.,
1982). It is uncertain whether the EC chloroplasts can
directly incorporate CO2 from the intercellular spaces of
the mesophyll. More interestingly, the EC chloroplasts
accumulated considerable PPDK. To solve the functional
role of PPDK, it may be necessary to understand it
in relation to adjacent tissues, such as the MC and GC
(Outlaw and Fisher, 1975).
In leaves of A. viridis, crystalline inclusions were
observed in the chloroplasts of BSC, MC, and EC.
They were not labelled for Rubisco LS. Although early
reports on crystalline inclusions in spinach chloroplasts
suggested that they consisted of Rubisco (Sprey, 1977;
Sprey and Lambert, 1977), a recent immunocytochemical
study could not con®rm this (Shojima et al., 1987). In
A. viridis, the crystalline inclusions were labelled for
PPDK in the EC chloroplasts, but not in the MC or
BSC chloroplasts. These data suggest that the crystalline inclusions may consist of different components,
depending on the cell type. More detailed biochemical
characterization of these crystalline inclusions will be
required.
Recent studies have shown that cell-speci®c expression
of the enzymes involved in C4 photosynthesis is regulated
largely at the transcriptional level. Environmental and
developmental factors are also involved in the C4 pattern
of gene expression (Dengler and Nelson, 1999; Sheen,
1999). Proximity of cells to a vascular bundle may
be required for the C4 pattern of gene expression, and
the developing vascular centre may play a major role in
the differentiation of the C4 pattern (Langdale and
Nelson, 1991; Dengler and Nelson, 1999). It is unknown
whether such a regulatory mechanism is also applicable to
leaf component cells other than the photosynthetically
active MC and BSC. Rather, the enzyme expression
in these cells may be modulated under the cells' own
mechanisms, because the GC and CC have distinctive
physiological functions. It should also be noted that
isogenes (isoforms) of enzymes differing from those in
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Fig. 5. Immunogold labelling of various leaf cells in A. viridis with antisera to PPDK (A±D) and to Rubisco LS (E±J). Unlabelled arrows indicate
crystalline inclusions. (A) Companion cell. (B) Guard cell. (C) Chloroplast containing a crystalline inclusion of a mesophyll cell. (D) Chloroplast
containing crystalline inclusions of an ordinary epidermal cell. (E) Chloroplast containing a crystalline inclusion of a mesophyll cell. (F) Chloroplast
containing crystalline inclusions of a bundle sheath cell. (G) Vascular parenchyma cell. (H) Companion cell. (I) Ordinary epidermal cell. (J) Guard cell.
Bars for F and H ˆ 1 mm. Bars for others ˆ 0.5 mm. For abbreviations, see legend to Fig. 2.
the MC and BSC may be expressed in these other cell
types. Considering not only the MC and BSC but also the
other leaf cell types, it is apparent that the mechanism
regulating the cellular expression of the photosynthetic
enzymes in the C4 plant is more complex than was known
previously.
There is a close positive correlation between the
accumulation levels of GDC in mitochondria and the
photosynthetic activities of the host cells (Tobin et al.,
1989; Oliver, 1994). Recent immunocytochemical studies
of C3 leaves have shown strong labelling for GDC in
the mitochondria of the MC, while in those of the
non-photosynthetic cells the labelling is very low (Tobin
et al., 1989) or not found (Guinel and Ireland, 1996;
Ueno and Agarie, 1997). There is evidence that the
expression of GDC is co-ordinated with that of Rubisco
Enzyme localization in Amaranthus leaf
1011
Fig. 6. Immunogold labelling of various leaf cells in A. viridis with antisera to NAD-ME (A±E) and to the P-protein of GDC (F±K). (A) Bundle sheath
cells. (B) Mesophyll cells. (C) Vascular parenchyma cell and companion cell. (D) Ordinary epidermal cell. (E) Guard cell. (F) Bundle sheath cells.
(G) Mesophyll cells. (H) Vascular parenchyma cell. (I) Companion cell. (J) Ordinary epidermal cell. (K) Guard cell. Bars for G and J ˆ 0.25 mm.
Bars for others ˆ 0.5 mm. For abbreviations, see legend to Fig. 2.
(Srinivasan and Oliver, 1995) and chloroplast development (Archer et al., 2000). In the MC of C3±C4 intermediate and C4 plants, however, the accumulation of the
P-protein of GDC is reduced (Morgan et al., 1993). The
trend in the cellular accumulation of the P-protein
of GDC in the BSC, VPC, CC, and GC of A. viridis
also seems to re¯ect the degree of photosynthetic activity
in respective cell types. An approximate positive correlation is found between the densities of labelling for
Rubisco LS and those for the P-protein of GDC in
these cell types. In the EC mitochondria, however,
no signi®cant labelling for the P-protein of GDC was
detected, despite the fact that the EC chloroplasts
were densely labelled for Rubisco LS. The reason is
unclear, but it may be because the EC contain few
organelles and are not photosynthetically active, even
though they accumulate abundant Rubisco in individual
chloroplasts.
1012
Ueno
Table 5. Immunogold labelling of NAD-ME and the P-protein of GDC in various cells of leaves of A. viridis
Enzyme and cell type
NAD-ME
Ordinary epidermal cells
Guard cells
Mesophyll cells
Bundle sheath cells
Vascular parenchyma cells
Companion cells
The P-protein of GDC
Ordinary epidermal cells
Guard cells
Mesophyll cells
Bundle sheath cells
Vascular parenchyma cells
Companion cells
No. of gold particles (mm 2)
Mitochondria
Cytosolqother organelles
0.7"2.6 (10)
1.1"3.8 (8)
0.9"3.2 (11)
41.4"8.9 (15)
35.8"15.7 (14)
1.4"2.3 (14)
0.5"0.4
0.7"0.4
0.9"0.8
0.4"0.2
1.1"0.6
0.6"0.4
(5)
(5)
(9)
(5)
(6)
(7)
1.5"2.4 (10)
3.5"5.2 (38)
1.7"2.9 (16)
44.6"12.7 (30)
26.3"13.2 (11)
1.8"3.1 (14)
0.3"0.4
1.0"1.0
1.2"0.9
0.4"0.2
1.2"1.3
1.3"0.9
(5)
(5)
(7)
(13)
(8)
(5)
Numbers of gold particles per unit area (mm 2) are given as means"SD. Numbers in parentheses show the numbers of mitochondria or cell pro®les
examined.
Acknowledgements
The author is grateful to Drs DJ Oliver, T Murata,
M Matsuoka, and S Muto for providing the antisera. Part of
this study was supported by a grant-in-aid from the Ministry
of Agriculture, Forestry and Fisheries of Japan (BDP-01-I-1-3).
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