Download Leaf inner structure and immunogold localization

Journal of Experimental Botany, Vol. 49, No. 329, pp. 1953–1961, December 1998
Leaf inner structure and immunogold localization
of some key enzymes involved in carbon
metabolism in CAM plants
Ayumu Kondo1,2, Akihiro Nose1 and Osamu Ueno2,3
1 Faculty of Agriculture, Saga University, Honjo, Saga 840–8502, Japan
2 Department of Plant Physiology, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305–8602,
Japan
Received 16 April 1998; Accepted 27 July 1998
Abstract
There are relatively few reports on the leaf structure
and in situ immunolocalization of carbon metabolism
enzymes in crassulacean acid metabolism (CAM)
plants, compared with reports on C plants. The leaf
4
inner structure and the subcellular location of some
key CAM enzymes for a phosphoenolpyruvate carboxykinase (PCK) CAM species, Ananas comosus, and three
malic enzyme (ME) CAM species, Mesembryanthemum
crystallinum, Kalanchoë daigremontiana, and K.
pinnata, was investigated by immunogold labelling and
electron microscopy in this study. The leaves of these
species had few intercellular air spaces in the mesophyll. A large vacuole occupied the mesophyll cells,
and many vesicles of various sizes occurred in the
cytosol. Immunocytochemical study revealed that
labelling was present for phosphoenolpyruvate carboxylase in the cytosol and for ribulose-1,5-bisphosphate carboxylase/oxygenase in the chloroplasts of the
mesophyll cells in all species. No specific labelling
for pyruvate orthophosphate dikinase (PPDK) was
observed in the PCK-CAM species. In the ME-CAM
species, the patterns of labelling for PPDK differed. In
M. crystallinum labelling for PPDK was present only in
the chloroplasts, whereas in the two Kalanchoë
species it occurred in the cytosol as well as in the
chloroplasts. These results suggest that the subcellular localization of PPDK varies with ME-CAM species,
in contrast to the conventional belief that it is localized
in the chloroplasts.
Key words: Crassulacean acid metabolism, immunolocalization, leaf inner structure, phosphoenolpyruvate
carboxylase, pyruvate orthophosphate dikinase.
Introduction
Crassulacean acid metabolism (CAM ) represents one of
three major photosynthetic modes, together with the C
3
and C pathways. In CAM plants, malic acid is accumu4
lated in the vacuoles of mesophyll cells at night as a result
of fixation of CO by phosphoenolpyruvate carboxylase
2
(PEPC ). During the day, malic acid is decarboxylated,
and the released CO is refixed in the C cycle (Osmond,
2
3
1978; Cushman and Bohnert, 1997). CAM plants can be
divided into two groups, phosphoenolpyruvate carboxykinase (PCK ) CAM and malic enzyme (ME ) CAM plants,
based on the decarboxylation process. In ME-CAM
plants, malic acid is decarboxylated by NAD(P)-ME and
generates pyruvate with CO . Pyruvate is phosphorylated
2
to PEP by catalysis of pyruvate orthophosphate dikinase
(PPDK ), and then it is conserved in gluconeogenesis. In
PCK-CAM plants, oxaloacetic acid produced from malic
acid is decarboxylated by PCK, and generates PEP with
CO without the catalysis of PPDK (Dittrich et al., 1973;
2,
Dittrich, 1976; Osmond, 1978; Holtum and Osmond,
1981; Winter and Smith, 1996). These biochemical processes operate within a single cell, but are separated
between day and night (Osmond, 1978). Compared with
leaves of C plants, leaves of CAM plants have a simple
4
inner structure ( Kluge and Ting, 1978). There are relatively few reports on leaf anatomy of CAM plants.
3 To whom correspondence should be addressed. Fax: +81 298 38 7408. E-mail: [email protected]
Abbreviations: CAM, crassulacean acid metabolism; ME, malic enzyme; PCK, phosphoenolpyruvate carboxykinase; PEPC, phosphoenolpyruvate
carboxylase; PPDK, pyruvate, orthophosphate dikinase; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase.
© Oxford University Press 1998
1954
Kondo et al.
To elucidate a metabolic pathway, it is important to
determine the subcellular localization of the enzymes
involved. This is fairly clear for CAM enzymes. However,
this knowledge is based on the biochemical analysis of
cell fractions, and some conflicting results, such as in the
subcellular distribution of PEPC, have been reported
(Brandon, 1967; Spalding et al., 1979; Schnarrenberger
et al., 1980; Perrot-Rechenmann et al., 1982). Recently,
immunocytochemical techniques have been used.
Immunoelectron microscopy shows the subcellular localization of enzymes in plant cells at high resolution
( Herman, 1988). To our knowledge, no report describes
the cellular localization of CAM enzymes with immunogold labelling and electron microscopy, although some
immunofluorescent studies have been reported (PerrotRechenmann et al., 1982; Taborsky et al., 1983).
The intracellular localization of PEPC, ribulose-1,5bisphosphate carboxylase/oxygenase (Rubisco), and
PPDK in leaves of some CAM species with different
decarboxylation types was investigated here by immunogold electron microscopy. The inner structure of the
leaves in relation to the mechanism of CAM was also
examined.
Light and electron microscopy of leaf structure
Leaf samples were collected at 09.00 h. Small segments of leaves
were fixed 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-fixed in 2% OsO in buffer, as
4
described by Ueno (1992). They were then dehydrated through
an acetone series and embedded in Spurr’s resin. Ultrathin
sections were stained with uranyl acetate and lead citrate.
Semithin sections were stained with 1% toluidine blue O.
Antisera
Antisera raised against PEPC and PPDK from maize leaves
were generously provided by Dr M Matsuoka (Nagoya
University, Nagoya, Japan). Antiserum raised against the large
subunit of Rubisco from pea leaves was a kind gift of Dr S
Muto (Nagoya University).
Analysis by SDS-PAGE and Western blotting
Leaf samples (0.5 g fresh weight) were ground with 0.5 g of sea
sand and 25 mg of insoluble polyvinylpyrrolidone in 1 ml of
50 mM HEPES-KOH (pH 8.2) that contained 5 mM dithiothreitol, 0.2 mM disodium EDTA, 1 mM phenylmethylsulphonyl fluoride (PMSF ), 1% (v/v) Triton X-100, and 0.1%
(w/v) leupeptin. In the case of A. comosus, 1 mM p-( chloromercuric) benzoic acid (PCMB) was added to the buffer. Each
homogenate was centrifuged at 10 000 g for 10 min at 4 °C.
Each supernatant was subjected to SDS-PAGE and Western
blotting as described by Ueno (1992).
Materials and methods
Plant materials
Four species of CAM plants were examined; Ananas comosus
(L.) Merr. (cv. smooth cayenne N67–10), Mesembryanthemum
crystallinum L., Kalanchoë daigremontiana Hamet et Perr., and
K. pinnata (Lam.) Pers. A. comosus is a PCK-CAM plant, and
other species are ME-CAM plants (Dittrich et al., 1973;
Dittrich, 1976; Holtum and Osmond, 1981: Winter et al., 1982).
A. comosus was planted in 10 dm3 pots filled with sand. The
Kalanchoë species were planted in 1 dm3 pots filled with
vermiculite. Plants were grown in a naturally illuminated
greenhouse (20–30 °C ) for 3 months and watered every third
day. Compound fertilizer (Otsuka I, Otsuka Chemicals Co.,
Osaka, Japan) diluted to 0.1% was supplied every 2 weeks.
Plants were subsequently transferred to a growth chamber and
grown for 2 weeks. The temperature in the growth chamber
was maintained at 25 °C during the light period (14 h) and at
20 °C during the dark period (10 h). Photon irradiance was
provided by metal halide lamps at about 300 mmol m−2 s−1.
Fully expanded leaves of A. comosus and the fully expanded
fifth or sixth leaves (numbered from the apex) of Kalanchoë
were used for the experiments.
Seeds of M. crystallinum were germinated in 1 dm3 pots filled
with a mixture of soil and vermiculite (151, v/v) in the
greenhouse. During the 7 weeks after germination, a fifthstrength of Hoagland’s solution that contained 1 mM NaCl
was supplied every day. Subsequently, a fifth-strength of
Hoagland’s solution that contained 350 mM NaCl was supplied
every other day to induce CAM. After 6 weeks, the fully
expanded second or third leaves (numbered from the apex)
were used for the experiments. These leaves were confirmed
to have high activities of PEPC (above 1000 mmol mg−1
chlorophyll h−1), indicating full expression of CAM.
Immunoelectron microscopy
Preparations and immunostaining of samples for immunoelectron microscopy followed the method reported by Ueno (1992).
Leaf samples were collected at 09.00 h. Small segments of leaves
were fixed immediately in 3% glutaraldehyde in 50 mM sodium
phosphate (pH 6.8) for 4 h on ice, and were then washed in
phosphate buffer. They were dehydrated through an ethanol
series at −20 °C and embedded in Lowicryl K4M resin
(Chemische Werke Lowi, Wald Kraiburg, Germany) at −20 °C.
Ultrathin sections were collected on 100 mesh nickel grids
coated with Formvar. Sections on grids were incubated for
20 min in 0.5% (w/v) bovine serum albumin (BSA) in phosphatebuffered saline (PBS) that contained 10 mM sodium phosphate
(pH 7.2), 150 mM NaCl, and 0.1% (v/v) Tween 20, and then
for 3 h in antiserum that had been diluted with 0.5% BSA in
PBS. For controls, antiserum was replaced by non-immune
serum. The grids were washed four times with PBS and
incubated in a 40-fold-diluted suspension of 15 nm protein
A-colloidal gold particles (EY Laboratory Inc., San Mateo,
California, USA) for 30 min. After several washes with PBS
and distilled water, the sections were stained with uranyl acetate
and lead citrate.
Quantitative analysis of immunogold particles
The density of PPDK labelling was determined by counting the
gold particles on electron micrographs at 15 000× magnification
and calculating the number per unit area (mm2). For profiles of
chloroplasts, the areas occupied by starch grains were excluded
from estimation. Ten to twelve individual cells were examined
in several immunolabelled sections from each species.
Enzyme localization of CAM
Results
Structural features of leaves
Leaves of A. comosus possessed thick-walled epidermis,
and two or three layers of thick-walled hypodermal cells
were present just inside both the adaxial and abaxial
epidermis (Fig. 1A). Stomata were present only on the
abaxial epidermis. Water storage tissue, which is composed of colourless cells, existed inside the adaxial epidermis, and occupied a quarter to a third of the leaf
thickness. Spherical, swelling mesophyll cells were densely
packed and had few intercellular air spaces. Aerating
canals were present. Fibre strands were scattered in the
mesophyll ( Fig. 1A). In M. crystallinum, K. daigremontiana, and K. pinnata, stomata were present on both adaxial
and abaxial epidermis (not shown). The mesophyll exhibited almost no differentiation into palisade or spongy
tissue, and large swelling mesophyll cells were densely
packed ( Fig. 1B, C, D). In both species of Kalanchoë,
cells that contained dense material were arranged near
the abaxial epidermis ( Fig. 1C, D). The epidermis of M.
crystallinum was covered with many transparent bladder
cells (not shown).
A large vacuole occupied the mesophyll cells of the
four CAM species. The organelles thus appeared pressed
against the cell periphery ( Fig. 1). Most chloroplasts were
distributed at the sides facing the intercellular spaces. The
chloroplasts were granal and included starch grains and
plastoglobules ( Fig. 2). No peripheral reticulum was
observed in the chloroplasts. Many vesicles of various
sizes occurred in the cytosol of mesophyll cells. The
development of vesicles was prominent in M. crystallinum
and the Kalanchoë species, and the vesicles frequently
clustered around chloroplasts (Fig. 2B, C, D). In all four
species, the bundle sheath cells showed structural features
similar to those in adjacent mesophyll cells.
Immunogold localization of photosynthetic enzymes
Western blotting was used to examine the cross-reactivities
of proteins extracted with antisera against PEPC, PPDK,
and the large subunit of Rubisco ( Fig. 3). The antisera
against PEPC and the large subunit of Rubisco gave
strong single bands on the blots ( Fig. 3), which indicates
that they were a reliable tool for immunocytochemical
localization of these enzymes in sections of leaves. The
PCK-CAM species A. comosus showed no band for
PPDK. The ME-CAM species M. crystallinum showed a
single band for PPDK and the two Kalanchoë species
showed strong bands, which consisted of two or more
sub-bands (Fig. 3). The largest sub-band suggested a
larger molecular size than PEPC.
When leaf sections were incubated in non-immune
serum, only non-specific and negligible labelling with
collodial gold was observed (Fig. 4A). Immunogold label-
1955
ling for the large subunit of Rubisco was present only in
the chloroplasts ( Fig. 4B, C ). Labelling for PEPC was
present only in the cytosol, including the fraction that
included vesicles ( Figs 4D, E, F, 5A). No significant
labelling for PEPC was observed in the epidermal cells,
the vascular bundles, or the water storage tissues (not
shown).
Leaves of A. comosus showed no significant labelling
for PPDK (Fig. 5B), which was consistent with the result
of Western blotting. In M. crystallinum, labelling for
PPDK was present only in the chloroplasts ( Fig. 5C ); in
Kalanchoë it occurred in the cytosol as well (Fig. 5D, E ).
In the Kalanchoë species, labelling for PPDK was also
found in the fraction of cytosol that included vesicles,
showing similar density of labelling to the other fraction
of cytosol (Fig. 5E). In the epidermal cells and vascular
bundles, no labelling for PPDK was observed (not
shown).
The density of labelling of PPDK was calculated for
the mesophyll cells of the three ME-CAM species
( Table 1). In M. crystallinum, the density of labelling was
high in the chloroplasts, but negligible in the cytosol and
other organelles. In Kalanchoë, the density of labelling in
the cytosol was approximately twice that in the chloroplasts, indicating that the amount of accumulation of
PPDK differs between two subcellular sites.
Discussion
Reports on the subcellular localization of PEPC in CAM
plants conflict. Brandon (1967) found PEPC in the mitochondrial fraction in leaves of Bryophyllum calycinum (=
Kalanchoë pinnata). By contrast, Schnarrenberger et al.
(1980) reported that PEPC is present in the chloroplasts
in K. pinnata and Crassula lycopodioides. An immunofluorescent study detected PEPC mainly in the cytosol
but also slightly in the chloroplasts in K. blossfeldiana
(Perrot-Rechenmann et al., 1982). In general, however,
PEPC is considered to be present in the cytosol in CAM
plants (Spalding et al., 1979; Winter et al., 1982; Taborsky
et al., 1983). This study confirms that PEPC is localized
unambiguously in the cytosol of the mesophyll cells in all
four species examined.
No labelling for PPDK was found in leaves of A.
comosus, as PCK-CAM plants lack PPDK activities
(Holtum and Osmond, 1981; Black et al., 1996). Previous
studies indicated that PPDK is distributed in the chloroplasts in ME-CAM plants (Spalding et al., 1979; Winter
et al., 1982). This study confirmed such localization of
PPDK in M. crystallinum. However, it also revealed that
PPDK is found not only in the chloroplasts but also in
the cytosol of the Kalanchoë species. Compared with
studies of C plants, there are relatively few reports on
4
PPDK in CAM plants ( Kluge and Osmond, 1971;
Sugiyama and Laetsch, 1975). Recently, a chloroplastic
1956
Kondo et al.
Fig. 1. Cross-sections of leaves of CAM species. (A) Ananas comosus. (B) Mesembryanthemum crystallinum. (C ) Kalanchoë pinnata. (D) K.
daigremontiana. Bars=200 mm. AC, aerating canal; D, cell containing dense material; F, fibre strand; M, mesophyll cell; St, stoma; VB, vascular
bundle; W, water storage tissue.
gene for PPDK, which includes a domain representing a
transit peptide, has been isolated from M. crystallinum
( Fisslthaler et al., 1995). In this plant, PPDK is encoded
probably by a single gene ( Fisslthaler et al., 1995). This
agrees with the results of this study. The Kalanchoë species
accumulated more PPDK in the cytosol than in the
chloroplasts. Further research is necessary to determine
whether the difference in the blotting band patterns of
PPDK in the ME-CAM species reflects a difference in
the subcellular patterns of PPDK. In the amphibious
Enzyme localization of CAM
1957
Fig. 2. Ultrastructure of mesophyll cells of CAM species. (A) A. comosus. (B) M. crystallinum. (C ) K. daigremontiana. (D) K. pinnata. The cytosol
contains vesicles of various sizes. Bars=0.5 mm. C, chloroplast; Cw, cell wall; mt, mitochondrion; P, plastoglobule; S, starch grain; Vc, vesicle.
1958
Kondo et al.
Fig. 3. Western blots of proteins extracted from leaves of K. daigremontiana (A), K. pinnata (B), M. crystallinum (C ), and A. comosus (D).
Total soluble protein (15 mg for PEPC, 30 mg for PPDK, and 1.5 mg for
the large subunit of Rubisco) was subjected to SDS-PAGE, blotting on
nitrocellulose membranes, and identification with antisera against PEPC
(1), PPDK (2), and the large subunit of Rubisco (3).
Table 1. Immunogold labelling of PPDK in mesophyll cells of
ME-CAM species
Numbers of gold particles per unit area ( mm−2) are given as means±sd.
Numbers in parentheses show the numbers of cell profiles examined.
Species
M. crystallinum
K. daigremontiana
K. pinnata
No. of gold particles ( mm−2)
Cytosol
Chloroplasts
Other
organelles
0.1±0.1
(10)
26.6±2.6
(10)
18.3±1.5
(10)
13.8±1.4
(11)
13.2±1.2
(10)
8.0±1.3
(10)
0.3±0.5
(12)
0.2±0.6
(10)
0.4±0.6
(10)
sedge Eleocharis vivipara, which expresses the C -like
4
mode under terrestrial conditions and C -like mode under
3
submerged conditions, PPDK is also accumulated in both
the chloroplasts and the cytosol of the photosynthetic
cells ( Ueno, 1996). The genes encoding the chloroplastic
and the cytosolic PPDK have been isolated, and the
expression patterns of the genes differ between the growth
forms (Agarie et al., 1997). In CAM plants also, the
degree of CAM expression is controlled by growth conditions. Therefore, some environmental factors may
be involved in the expression pattern of PPDK in
ME-CAM plants.
It is important to know whether the cytosolic PPDK
protein is an active form so as to elucidate the metabolic
pathway in these CAM plants. In C plants, PPDK is
4
involved in the regeneration of PEP and is localized in
the chloroplasts (Hatch, 1987). In CAM plants, by contrast, PEP generated by PPDK is not used directly as
substrate for PEPC, as it enters the gluconeogenic pathway to synthesize sugars and glucan. Instead, PEP for
PEPC is derived from the glycolytic breakdown of storage
carbohydrate (Holtum and Osmond, 1981; Smith and
Bryce, 1992). It is unclear what role the cytosolic PPDK
plays in the carbon metabolism of Kalanchoë species,
even if it is an active form. In wheat seed, cytoplasmic
PPDK was detected and may be involved in the synthesis
of seed storage protein (Aoyagi and Bassham, 1984;
Aoyagi and Chua, 1988). The chloroplasts isolated from
the CAM leaves of M. crystallinum showed high activity
of pyruvate uptake under light, and the light-enhanced
uptake of pyruvate into chloroplasts may be associated
with the light activation of PPDK ( Kore-eda et al., 1996).
An analysis of metabolite transport is necessary to understand the role of the distinct PPDKs in the Kalanchoë
species.
ME-CAM plants occur in several families, including Agavaceae, Aizoaceae, Cactaceae, Crassulaceae,
Orchidaceae, and Liliaceae (Dittrich et al., 1973; Dittrich,
1976). This study examined only one species from the
Aizoaceae and two species from the Crassulaceae. More
extensive studies would be required to understand the
taxonomic variation in the subcellular localization of
PPDK in ME-CAM plants. CAM plants are divided into
four groups based on carbohydrate partitioning strategies
(Christopher and Holtum, 1996). According to this classification, all three ME-CAM species examined here are
ME starch formers. Thus no relationship could be found
between the localization patterns of PPDK and the types
of carbohydrate partitioning strategies.
The CAM species examined had succulent leaves, in
which highly vacuolate mesophyll cells were densely
packed and had few intercellular air spaces. These structual features have also been observed in leaves of other
CAM plants ( Kluge and Ting, 1978). Recently, a very
low internal conductance to CO diffusion has been
2
reported for leaves of K. daigremontiana (Maxwell et al.,
1997). The mesophyll exhibited almost no differentiation
into palisade and spongy tissues. In leaves of CAM
species in Peperomia, three types of parenchyma tissue
differentiate. These tissues differ not only in the levels of
starch content and granal development (Gibeaut and
Thomson, 1989) but also in the pattern of accumulation
of CAM enzymes (Nishio and Ting, 1987; Ting et al.,
1994). In leaves of the CAM species examined here, such
distinct cellular differentiation was not observed. Balsamo
and Uribe (1988a, b) reported that cytosolic vesicles
associated with chloroplasts are often observed in the
mesophyll cells of K. daigremontiana, and they also exhibit
ATPase activity, as do the tonoplasts of the central
vacuoles. In this study, many vesicles of various sizes
were also observed in the cytosol of mesophyll cells and
tended to cluster around the chloroplasts. The fraction
of cytosol including these vesicles also accumulated PEPC
and PPDK, as did the other fraction of the cytosol. The
structure may extend the surface area of the tonoplast,
Enzyme localization of CAM
1959
Fig. 4. Immunogold labelling of mesophyll cells of CAM species. (A) Labelling of K. daigremontiana with non-immune serum. Only non-specific
labelling of gold particles (arrows) is present. (B) and (C ) Labelling of A. comosus (B) and K. pinnata (C ) with antiserum to the large subunit of
Rubisco. Note dense labelling in the chloroplasts. (D–F ) Labelling of A. comosus (D), M. crystallinum (E ), and K. daigremontiana (F ) with
antiserum to PEPC. Note dense labelling in the cytosol. Bars=0.5 mm. Abbreviations as in Fig. 2.
1960
Kondo et al.
Fig. 5. Immunogold labelling of mesophyll cells of CAM species. (A) Labelling of K. pinnata with antiserum to PEPC. (B–E) Labelling of A.
comosus (B), M. crystallinum (C ), K. daigremontiana (D), and K. pinnata ( E ) with antiserum to PPDK. A. comosus shows no significant labelling.
Note labelling in the chloroplasts in M. crystallinum and labelling in both the chloroplasts and cytosol of both Kalanchoë species. Bars=0.5 mm.
Abbreviations as in Fig. 2.
Enzyme localization of CAM
which may result in efficient transport of metabolites
between the cytosol and the vacuole.
Acknowledgement
We are grateful to Drs T Yanagita and K Wasano (Saga
University) and Dr M Hayashi ( Kagoshima University) for
their encouragement. Part of this study was supported by a
grant-in-aid from the Science and Technology Agency of Japan
( Enhancement of Center-of-Excellence) to OU.
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