Download Compartmentation of photosynthesis in cells and

Journal of Experimental Botany, Vol. 52, No. 356,
Compartmentation Special Issue, pp. 577±590, April 2001
Compartmentation of photosynthesis in cells and tissues
of C4 plants
Gerald E. Edwards1,5, Vincent R. Franceschi1, Maurice S. B. Ku1, Elena V. Voznesenskaya2,
Vladimir I. Pyankov3 and Carlos S. Andreo4
1
School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA
Department of Anatomy and Morphology, V. L. Komarov Botanical Institute of Russian Academy
of Sciences, Prof. Popov Street 2, 197376 St Petersburg, Russia
3
Department of Plant Physiology, Ural State University, Lenin Avenue 51, 620083 Ekaterinburg, Russia
4
Centro de Estudios FotosinteÂticos y BioquõÂmicos, (CEFOBI), Universidad Nacional de Rosario,
Suipacha 531, 2000 Rosario, Argentina
2
Received 31 March 2000; Accepted 24 November 2000
Abstract
Critical to defining photosynthesis in C4 plants is
understanding the intercellular and intracellular compartmentation of enzymes between mesophyll and
bundle sheath cells in the leaf. This includes enzymes
of the C4 cycle (including three subtypes), the C3
pathway and photorespiration. The current state of
knowledge of this compartmentation is a consequence of the development and application of
different techniques over the past three decades.
Initial studies led to some alternative hypotheses on
the mechanism of C4 photosynthesis, and some controversy over the compartmentation of enzymes. The
development of methods for separating mesophyll
and bundle sheath cells provided convincing evidence
on intercellular compartmentation of the key components of the C4 pathway. Studies on the intracellular
compartmentation of enzymes between organelles
and the cytosol were facilitated by the isolation of
mesophyll and bundle sheath protoplasts, which can
be fractionated gently while maintaining organelle
integrity. Now, the ability to determine localization of
photosynthetic enzymes conclusively, through in situ
immunolocalization by confocal light microscopy
and transmission electron microscopy, is providing
further insight into the mechanism of C4 photosynthesis and its evolution. Currently, immunological,
5
ultrastructural and cytochemical studies are revealing relationships between anatomical arrangements
and photosynthetic mechanisms which are probably
related to environmental factors associated with
evolution of these plants. This includes interesting
variations in the C4 syndrome in leaves and cotyledons of species in the tribe Salsoleae of the family
Chenopodiaceae, in relation to evolution and ecology. Thus, analysis of structure±function relationships using modern techniques is a very powerful
approach to understanding evolution and regulation
of the photosynthetic carbon reduction mechanisms.
Key words: Anatomy, C4 plants, chloroplasts, gene
expression, immunolocalization, photosynthetic enzymes,
ultrastructure.
C4 pathway of photosynthesis
In the 1960s, it was recognized that some plants have a
unique pathway of assimilating atmospheric CO2 (see the
historical account in Hatch, 1999, including the early
work of Karpilov, Kortschack et al., and Hatch and
Slack). As what is now termed the C4 pathway was being
identi®ed in various species, it was also recognized that
To whom correspondence should be addressed. Fax: q1 509 335 3184. E-mail: [email protected]
Abbreviations: NAD-ME, NAD-malic enzyme; NADP-MDH, NADP-malate dehydrogenase; NADP-ME, NADP-malic enzyme; PEP,
phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; PEP-CK, phosphoenolpyruvate carboxykinase; PPDK, pyruvate,Pi dikinase; PGA,
phosphoglyceric acid, Rubisco, ribulose 1,5-bisphosphate carboxylase-oxygenase; RuBP, ribulose 1,5-bisphosphate.
ß Society for Experimental Biology 2001
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Edwards et al.
it was associated with a special leaf anatomy. Large,
distinctive bundle sheath cells, with prominent chloroplasts, surround the vascular tissue, which, in turn, are
surrounded by a layer of chloroplast-containing mesophyll cells; more than a century ago Haberlandt called
this Kranz anatomy (Edwards and Walker, 1983).
Once the association between Kranz anatomy and
the ®xation of atmospheric CO2 into C4 acids was made,
there were immediate questions about the biochemical
mechanism of carbon assimilation and the role of the two
photosynthetic cell types. It became clear that C4 plants
have very high levels of phosphoenolpyruvate carboxylase (PEPC) compared to C3 plants, where ribulose
1,5-bisphosphate carboxylase-oxygenase (Rubisco) is the
primary carboxylase, which results in the ®xation of
atmospheric CO2 via the C3 pathway. Subsequently, three
C4 acid decarboxylases were identi®ed: NADP-malic
enzyme (NADP-ME), NAD-malic enzyme (NAD-ME),
and phosphoenolpyruvate carboxykinase (PEP-CK),
in that order, which were key to developing an understanding of the C4 mechanism (i.e. C4 plants ®x atmospheric CO2 into C4 acids via the C4 cycle, C4 acids
are decarboxylated and the CO2 donated to Rubisco
of the C3 pathway) (see the historical summary by Hatch,
1999). Since the 1960s, there have been parallel studies
on the biochemistry of C4 photosynthesis and the
compartmentation of metabolic processes of carbon
assimilation between mesophyll and bundle sheath cells.
These include enzymology of the C4 cycle, the C3 pathway of photosynthesis and photorespiration. More
recently, research on the C4 mechanism has been focused
on the molecular evolution of C4-speci®c genes and
their differential expression among various organs and
between the two photosynthetic cell types (Ku et al.,
1996; Sheen, 1999). This paper discusses the development
and application of techniques for studying compartmentation in C4 photosynthesis which have been
critical in elucidating this metabolic process, and which
continue to play a key role in advancing current understanding of the evolution and diversity of the C4 syndrome
in plants.
Initial studies on the compartmentation of
key enzymes, metabolites and understanding
of C4 photosynthesis
When Hatch and Slack found, by 14CO2±12CO2
pulse±chase experiments, the initial incorporation of label
into the C4-carboxyl carbon of oxaloacetate, malate
and aspartate, followed by labelling in the C-1 carboxyl
of phosphoglyceric acid (PGA) and then hexose-P in
sugarcane, they developed a working model for photosynthesis in C4 plants (Hatch and Slack, 1966). The model
included a `C4 cycle', with ®xation of atmospheric CO2
by pyruvate or phosphoenolpyruvate (PEP) to generate a
C4 acid, transfer of the C4-carboxyl to an acceptor to
form the C-1 carboxyl of 3-phosphoglyceric acid (PGA),
and then use of the 3-carbon product as the substrate
for ®xing another CO2. In this initial model of C4 photosynthesis, a transcarboxylase reaction was suggested
(direct transfer of carbon from a C4 acid to an acceptor
to form PGA). It was logical not to postulate C4 acid
decarboxylation, CO2 release and re®xation by Rubisco,
since in initial studies Rubisco activities from whole leaf
extracts were relatively low, and the ef®ciency by which a
C4 acid decarboxylase and re®xation of CO2 by Rubisco
could function was questionable (exposure of leaves to 5%
12
CO2 during the chase gave a similar labelling pattern).
At that time, the relationship between Kranz anatomy
and the biochemical pathway had not been established, and the mechanism of photorespiration via
ribulose 1,5-bisphosphate (RuBP) oxygenase in C3 plants
under limiting CO2 had not been discovered.
After the connection was made between plants ®xing
atmospheric CO2 into C4 acids and Kranz anatomy,
it was obviously important to determine the function of
the two chlorenchyma cell types, mesophyll and bundle
sheath cells. Already, a number of hypotheses had been
put forth about the mechanism of photosynthesis in
plants having Kranz type anatomy, prior to any knowledge of the biochemistry involved. Even Haberlandt
(Haberlandt, 1896), in his descriptions of plants with
Kranz anatomy in the late 1800s, suggested there might
be some co-operative function of the two cell types in
photosynthesis.
From the 1940s to the early 1960s, there was speculation that the mesophyll cells may assimilate atmospheric
CO2 and that the bundle sheath chloroplasts may only
function like amyloplasts to store starch, or possibly to
®x CO2 generated by respiration in vascular tissue
(see Rhoades and Carvalho, 1944, and other references
in Slack, 1969).
Subsequent to their 14CO2±12CO2 pulse-chase isotope
results, from 1967±1969 Hatch and Slack searched for
enzymes which might be involved in C4 photosynthesis
and found key enzymes, including PEPC, NADP-ME,
pyruvate,Pi dikinase (PPDK), and Rubisco (the latter
at low levels), along with evidence for other enzymes of
the C3 cycle. Recognition that sugarcane and other plants
with this pattern of photosynthesis have two types of
chlorenchyma cells (mesophyll and bundle sheath), led to
studies on compartmentation of the enzymes.
The earliest applications of techniques to study cellspeci®c compartmentation of enzymes in C4 plants were
the non-aqueous fractionation methods used by Slack
(Slack, 1969; Slack et al., 1969) and differential grinding
(BjoÈrkman and Gauhl, 1969). Application of non-aqueous
density fractionation (Slack et al., 1969; Slack, 1969)
provided important insight into the compartmentation
Compartmentation in C4 plant tissues
of some of the key enzymes of photosynthesis between
mesophyll and bundle sheath chloroplasts of maize and
amaranth. Using freeze-dried, macerated leaf tissue,
a step-wise fractionation was made in different densities
of hexane-carbon tetrachloride (1.30, 1.33, 1.36, and
1.40 g ml 1). The procedure employed gave pelleted
samples having densities of -1.30, 1.30±1.33, 1.33±1.36,
1.36±1.40, and )1.40 g ml 1. Earlier work with the nonaqueous technique (Stocking, 1959) had shown that
de-starched chloroplasts of tobacco (C3) are lighter
than most other cell constituents. Slack et al. showed
that the low density fraction,-1.30 g ml 1, was rich
in the starchless, grana-containing mesophyll chloroplasts, while the high density fractions, 1.36±1.40 and
)1.40 g ml 1, were rich in the agranal, starch-containing
bundle sheath chloroplasts (Slack et al., 1969). It was
concluded that PPDK, NADP-malate dehydrogenase
(NADP-MDH) and glycerate kinase occur in mesophyll
chloroplasts and that Rubisco and NADP-ME occur in
bundle sheath chloroplasts. NADP-triose-P dehydrogenase and PGA-kinase were found in both mesophyll and
bundle sheath chloroplast fractions. The non-aqueous
method gave variable results with PEPC, which, in some
preparations, was more associated with the lower density
mesophyll chloroplast fraction, while in other preparations it appeared at high densities. Although it was
suggested that the enzyme may be associated with the
bounding membrane of mesophyll chloroplasts, its compartmentation (cell type and intracellular localization)
was not clear. Subsequently, it became clear that this
enzyme occurs in the cytosol of mesophyll cells, based on
the fractionation of mesophyll protoplasts and in situ
immunolocalization studies (Gutierrez et al., 1974b;
Voznesenskaya et al., 1999; as discussed later). The
reasons for the variation with the non-aqueous technique,
and whether the enzyme may have some association with
the outer chloroplast envelope in situ, are unknown.
Slack et al. also fed 14CO2 to maize leaves for 25 s, and
then analysed the distribution of metabolites with the
non-aqueous density fractionation (Slack et al., 1969).
The major labelling of malate, aspartate, and PGA
occurred in the mesophyll chloroplast fraction while
the major labelling of fructose phosphates and ribulose
phosphates was in the bundle sheath chloroplast fraction.
At this time, two alternative hypotheses on C4 photosynthesis were proposed: (1) mesophyll chloroplasts
®x atmospheric CO2 through a C4 acid cycle and a
transcarboxylase reaction (e.g. with glycerate as a product
in the latter reaction, and its conversion to 3-PGA via
glycerate kinase, which would be consistent with labelling
of PGA in the mesophyll chloroplast), and bundle sheath
chloroplasts ®x mainly respired CO2 through RuBP
carboxylase, or (2) mesophyll chloroplasts ®x atmospheric CO2 via PEPC into C4 acids, and C4 acids donate
CO2 to Rubisco in bundle sheath cells via NADP-ME.
579
There are limitations with the non-aqueous method
which can complicate interpretations of the results: it
does not give a complete separation of any one cellular
compartment into one density fraction, adsorption of
cytosolic material to organelles may in¯uence their
density, and the yield of bundle sheath chloroplasts may
be low, in part by loss of starch from bundle sheath
chloroplasts, resulting in their partitioning to a lighter
fraction. For a review of the non-aqueous method see
Stitt et al. (Stitt et al., 1989), and for its use in studying the
distribution of metabolites between the chloroplasts
and extracellular compartments in maize mesophyll and
bundle sheath cells, see Weiner and Heldt (Weiner and
Heldt, 1992). In current applications of the non-aqueous
technique, tetrachlorethylene instead of carbon tetrachloride is used, due to the higher toxicity of the latter,
and an iterative mathematical approach can be used
more accurately to calculate the distribution between
compartments (Moore et al., 1997).
Other differential separation techniques were used
early on which added to the information provided by the
non-aqueous separation techniques. BjoÈrkman and Gauhl
showed that C4 plants have substantial Rubisco activity,
suggesting its function in photosynthesis in these species
(BjoÈrkman and Gauhl, 1969). They also employed a
sequential grinding procedure with Atriplex rosea (C4).
Using increasing force to break bundle sheath cells, they
found PEPC was high in the initial extract and low in the
terminal extract, while Rubisco was three times higher
in the terminal extract. On this basis, they suggested
PEPC was predominantly in mesophyll cells and that
Rubisco was highest in bundle sheath cells. Adapting this
differential grinding technique to maize and Gomphrena
globosa (C4), Berry et al. suggested NADP-ME as well
as Rubisco occurs in bundle sheath cells and that PEPC
occurs in mesophyll cells (Berry et al., 1970). The substantial activities of Rubisco and NADP-ME in the initial
extract (30±40% of Rubisco on a soluble protein basis)
left it uncertain whether the C3 pathway also functioned
in mesophyll cells. These approaches were limited by the
lack of strict isolation of individual mesophyll or bundle
sheath cells.
Mechanical isolation of mesophyll cells and
bundle sheath cells/strands
Separation of intact mesophyll and bundle sheath cells of
a C4 plant provided a direct means of studying intercellular compartmentation of enzymes between both cell
types and testing their functions in photosynthetic
metabolism (Edwards et al., 1970; Edwards and Black,
1971). A procedure combining gentle grinding in a mortar
and ®ltration through a series of nylon mesh ®lters with
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Edwards et al.
precise porosity, allowed for rapid isolation and puri®cation (c. 30 min) of individual mesophyll and bundle
sheath cells (the latter from bundle sheath strands)
(Edwards et al., 1970; Edwards and Black, 1971). This
was ®rst accomplished with the C4 monocot Digitaria
sanguinalis, an NADP-ME type species. Species of this
genera are particularly amenable to isolation of both
cell types, whereas in C4 species of other genera tested,
mesophyll cells are largely or completely broken during
mechanical treatment. From a series of studies with D.
sanguinalis, it became clear that enzymes of the carboxylation phase of the C4 pathway were in mesophyll cells,
and that light-dependent ®xation of CO2 into C4 acids
occurred when the cells were provided with pyruvate
and 14CO2. Bundle sheath cells showed the capacity for
light-dependent ®xation of CO2 via Rubisco into PGA.
Subsequently, bundle sheath strands isolated mechanically or enzymatically, were used in many studies on
C4 photosynthesis. These included studies on the capacity
for the Hill reaction with the addition of Hill oxidants,
on light-dependent CO2 ®xation, on metabolism of C4
acids by the three different C4 subtypes, and on the capacity for photorespiration via Rubisco under CO2-limited
conditions (Edwards and Huber, 1981; Edwards and
Walker, 1983; Hatch, 1999; Kanai and Edwards, 1999).
In C4 plants, there are numerous plasmodesmata
connections between mesophyll and bundle sheath cells.
When cells are isolated, these connections are severed
and apparently remain unsealed, and metabolites such as
malate and pyruvate can be taken into the cells through
the open plasmodesmata. This interesting feature extends
the types of experiments that can be done with the
isolated cells. It made possible studies to test the ability of
these cells to metabolize various suspected intermediate
compounds in the light (e.g. pyruvate and 3-PGA
by mesophyll cells; ribose-5-P and C4 acids by bundle
sheath cells), and to evaluate the effects of addition of
these substances on 14CO2 ®xation or photosynthetic
O2 evolution.
While mesophyll and bundle sheath cells isolated by
mechanical force were useful for studies on photosynthetic metabolism of these cell types, they also had
their limitations. First, preparations of the two cell types
were always cross-contaminated (particularly in bundle
sheath, e.g. 5±10% contamination by mesophyll cells),
making precise determination of compartmentation of
any enzymes between the two cell types dif®cult. Second,
due to potential physical damage of the isolated cells,
enzymes associated with the cytosol may be lost and
thus underestimated. Third, both cell types could only be
isolated effectively from Digitaria species by this mechanical means, such that comparative studies were limited
to this genera. Fourth, both mesophyll and bundle sheath
cells are very dif®cult to break, such that it was impossible to isolate intact organelles to study intracellular
compartmentation. As a consequence, methods to overcome these shortfalls for separation of intact cells or
organelles needed to be developed.
In summary, by 1970, application of non-aqueous and
mechanical isolation techniques produced a view of the
C4 photosynthetic mechanism in NADP-ME species like
sugarcane, sorghum, and D. sanguinalis (Slack et al., with
non-aqueous fractionation, BjoÈrkman and Gauhl, and
Berry et al., with bundle sheath strands, and Edwards
et al., with mesophyll and bundle sheath cells). It formed
the basis of the pathway for NADP-ME species as it
is known today (Hatch et al., 1971). However, there was
controversy over compartmentation and function of
mesophyll and bundle sheath cells in C4 photosynthesis
for several years. For example, there was evidence that
chloroplasts isolated from young maize leaves were
capable of photosynthesis by the Calvin cycle, and
when leaves of maize were incubated in the absence of
CO2, PEP was depleted, which was unexpected if atmospheric CO2 was ®xed by PEPC (see results of Gibbs
and colleagues in Hatch et al., 1971, and later evidence
and explanation for this effect, Usuda, 1987; Leegood and
von Caemmerer, 1988). Application of differential grinding techniques resulted in different proposals about
compartmentation of PEPC and Rubisco. This included
a radically different scheme of C4 photosynthesis based
on results from differential grinding techniques with
NADP-ME species like sugarcane and Pennisetum
purpureum (Coombs and Baldry, 1972). It was suggested
that CO2 was ®xed by PEPC in epidermal cells, that
malate donated CO2 to Rubisco in mesophyll cells for
®xation through the C3 pathway, and that the bundle
sheath chloroplasts only functioned to store starch
(consistent with earlier speculations on the role of
bundle sheath chloroplasts by Rhoades and Carvalho,
1944). Soon, a new technical approach, the enzymatic
isolation and separation of mesophyll protoplasts and
bundle sheath cells, ®rmly established the co-operative
function of these cell types in C4 photosynthesis; not
only in the NADP-ME type species, but also in other C4
subgroups as well.
Enzymatic isolation of mesophyll and
bundle sheath preparations
Enzymatic isolation of mesophyll protoplasts and bundle
sheath strands from C4 plants (free of epidermal tissue),
using a mixture of fungal cellulase-pectinase (Kanai and
Edwards, 1973a, b), was a technical advancement which
allowed the properties of these cells to be explored in
various C4 species. The method was especially successful
with C4 monocots, including species of all three subgroups. There was a clear separation of the two cell types
with very little cross contamination, and use of the dye
Compartmentation in C4 plant tissues
Evans Blue (which is excluded from mesophyll protoplasts retaining their selective permeability) showed a
high degree of intactness (Kanai and Edwards, 1973b).
Signi®cantly, this technique allowed studies on the intercellular distribution of enzymes between the cell types
(e.g. photosynthetic, photorespiratory, glycolytic, and
enzymes of nitrogen and sulphate assimilation) (Edwards
and Walker, 1983). With respect to C4 photosynthesis,
these results showed that enzymes of the carboxylation
phase of the C4 cycle, including PEPC, are in mesophyll
cells, while C4 acid decarboxylases, along with phosphoribulokinase and Rubisco, are in bundle sheath cells
(Kanai and Edwards, 1973c; Ku et al., 1974). This
included species used in the studies of Baldry and Coombs
that speci®cally addressed their proposal. Research on
these preparations included studies on the photochemical
properties (relative levels of PSI and PSII, and capacity
for photochemistry), and on the effects of addition
of various metabolites on photosynthetic metabolism
(Edwards and Walker, 1983).
Isolated bundle sheath strands have been used for
many years to study their photosynthetic metabolism
(e.g. ®xation of CO2 and utilization of C4 acids as donors
of CO2 to the C3 pathway). Methods include enzymatic
isolation, as described above, mechanical isolation or
combining enzymatic treatment with subsequent mechanical isolation. With some species, the latter method
has the advantage of providing a purer bundle sheath
preparation and a shorter preparation time. Methods of
isolation and photosynthetic studies with bundle sheath
strands have been described earlier (Edwards and
Huber, 1981; Hatch, 1987; Furbank et al., 1990; Meister
et al., 1996).
Mesophyll protoplasts can be broken gently while
maintaining the integrity of chloroplasts (intactness 90%
or greater) and other organelles (e.g. mitochondria
and peroxisomes) by passing them several times through
a 20 micron nylon mesh. Mesophyll protoplast extracts
(containing functional chloroplasts and cytosolic PEPC)
were used to study the energetics of pyruvate conversion
to PEP and reduction of oxaloacetate to malate (Edwards
and Huber, 1979). These results showed that mesophyll
chloroplasts are capable of generating ATP not only by
linear electron ¯ow to NADP, but also by PSI-dependent
cyclic electron ¯ow and by the O2-dependent Mehler
peroxidase reaction. Mesophyll protoplasts were valuable
for studying the intercellular compartmentation of
photosynthetic enzymes in species representing the three
C4 subgroups, and for characterizing certain chloroplast
transporters associated with the C4 pathway (Edwards
and Huber, 1979, 1981; Kanai and Edwards, 1999).
A number of photosynthetic enzymes in C4 plants
are regulated by light±dark transitions, including several
enzymes of the C4 cycle. Mesophyll and bundle sheath
preparations have been of value in studying the mechanism
581
of light activation of several enzymes (Usuda et al., 1984;
Nakamoto and Edwards, 1986). In this regard, mesophyll
protoplasts of sorghum and D. sanguinalis have been
used to study the signal transduction pathway controlling phosphorylationudephosphorylation of PEPC (Pierre
et al., 1992; Giglioli-Guivarc'h et al., 1996).
In 1984, Jenkins and Russ developed a mechanical
procedure for isolating functional mesophyll chloroplasts
from maize and several other C4 species (Jenkins and
Russ, 1984). The procedure (preparation time of about
20 min by maceration of maize leaves in a Sorvall
blender, and puri®cation by centrifugation of the intact
chloroplasts through a 32% Percoll gradient) provided
good yields of chloroplasts (80±90% intactness), with
negligible contamination by bundle sheath chloroplasts.
In 1979, bundle sheath and mesophyll protoplasts
were isolated from Panicum miliaceum, an NAD-ME
type monocot, and functional chloroplasts were isolated
from both protoplast types (Edwards et al., 1979). Subsequently, both mesophyll and bundle sheath protoplasts
were isolated from Portulaca grandi¯ora, a succulent
NADP-ME C4 dicot (Ku et al., 1981), Flaveria trinervia,
an NADP-ME type dicot and Atriplex spongiosa, an
NAD-ME type dicot (Moore et al., 1984), and several
PEP-CK monocots (Ku et al., 1980; Chapman and
Hatch, 1983; Watanabe et al., 1984). The bundle sheath
protoplasts are larger and more dense than the mesophyll
protoplasts and can be separated by density gradient
centrifugation. In these studies, bundle sheath protoplasts
have been used to investigate the intracellular compartmentation of various enzymes associated with pathways
of C4 acid decarboxylation, CO2 ®xation, and photorespiration. As noted earlier, it is dif®cult to isolate intact
and functional chloroplasts and other organelles from
bundle sheath strands; but there has been some success
with Flaveria bidentis (Meister et al., 1996) and maize
(Kanai and Edwards, 1999). As protoplasts were being
used for studies on C4 photosynthesis, they were also
employed to resolve questions on compartmentation
of enzymes and metabolites, and the mechanism of
photosynthesis in CAM and C3 species (Robinson and
Walker, 1979; Edwards and Walker, 1983; GardestroÈm
and Wigge, 1988; Ku et al., 1980; Stitt et al., 1989; Winter
et al., 1982).
Mesophyll and bundle sheath preparations enzymatically isolated from greening maize seedlings have been
used extensively by Sheen as a means of investigating gene
regulation and signal transduction (Sheen, 1995). Her
studies with maize mesophyll protoplasts, as a single-cell
transient expression system, have provided novel
information about sugar sensing and feedback inhibition
of transcription of photosynthetic genes. This simple
technique is also useful for rapid identi®cation of
promoter enhancer or suppresser elements for gene
transcription (Sheen, 1991; Imaizumi et al., 1997) and for
582
Edwards et al.
isolation of cell-speci®c transcriptional factors regulating
the expression of C4 photosynthesis genes. Mesophyll
protoplasts isolated from greening, etiolated maize
seedlings are very active and have high transcriptional
activity. After introduction of the gene into isolated
protoplasts by electroporation, for high levels of
expression of the inserted gene it is essential to maintain
viability of the protoplasts during the following 24 h
incubation under low light. Increasing the pH of the
incubation medium from 5.8, a pH traditionally used
for tissue culture, to 7.0 or 8.0, greatly enhanced the level
of gene expression by 10- or 20-fold, respectively
(M Ku, M Taniguchi, M Matsuoka, T Sugiyama,
unpublished data). In addition, inclusion of 10 mM KCl
and 10 mM NaHCO3 at pH 7.0 in the incubation
medium further stimulated gene expression by more than
60-fold. Thus, maintenance of ion homeostasis and
photosynthetic viability by the isolated mesophyll protoplasts is important for the cells to express the introduced
gene. The major limitation of this technique for promoter
analysis is that development- and tissue-speci®c, and
to some extent cell-speci®c, regulation of gene transcription cannot be examined. Stable transformants will
be required for these analyses.
In situ methods for studying compartmentation
of mRNA and protein in C4 photosynthesis
While study of cellular compartmentation of photosynthetic enzymes in C4 leaves can be facilitated by
separation and isolation of relatively pure cell types or
organelles from mature tissues, this approach is timeconsuming and, in some cases, it is not applicable to
very young leaves or other tissues. In addition, the speci®c
intracellular localization of a given enzyme among
the many compartments within a cell type cannot be
determined with certainty. There are two cytological
approaches to solve this problem in studying the expression and compartmentation of photosynthetic enzymes
in C4 plants. In situ hybridization and immunolabelling
have been developed to detect the site of expression of
a speci®c mRNA or protein, respectively, directly in the
tissues of interest. These complementary techniques are
particularly useful for detecting distribution of speci®c
transcripts and proteins directly in developing and mature
leaves and cotyledons of C4 plants.
Distribution of C4-specific mRNA
Initially, Sheen and colleagues (Sheen, 1999) ®rst extracted
total RNA from isolated mesophyll protoplasts and
bundle sheath strands and examined the cell-speci®c
differential expression of various C4 photosynthesis
genes. Subsequently, in situ hybridization was employed
directly on leaf sections to detect speci®c mRNA.
For in situ hybridization of mRNA, plasmids
(DNA template) have been used to generate sense and
antisense RNA probes for Rubisco LSU and SSU, PEPC
and PPDK. The sense strand probe is a critical control
for this technique. Both radioactive and stable-labelled
probes can be generated. For obvious reasons, stablelabelled probes, such as those tagged with digoxigenin,
are easier to work with and have found considerable use
for studies on C3 and C4 species. During in vitro synthesis
of probes, digoxigenin-11-UTP is added to generate
`dig-labelled probes' which can later be detected by a
secondary reaction. Paraf®n sections are prepared and
hybridized with labelled transcripts under carefully
controlled conditions. Hybridized transcripts are detected
using anti-digoxigenin antisera conjugated to alkaline
phosphatase (other probes can also be used) in combination with a colour detection system (Wang et al.,
1992, 1993). In more recent investigations, rhodamineconjugated secondary antibodies were used, and
sections were analysed using a confocal imaging system
(Ramsperger et al., 1996). Localization of mRNAs for
enzymes of the C4 pathway and Rubisco of the C3 pathway has been studied in several C4 species, with the most
detailed studies conducted on maize and Amaranthus
hypochondriacus (Wang et al., 1992, 1993; Long and
Berry, 1996; Ramsperger et al., 1996; Langdale et al.,
1988; Sheen, 1999; Dengler and Nelson, 1999).
One of the most interesting problems in understanding the developmental aspect of C4 photosynthesis is
elucidating the initial C4 gene expression patterns and
post-transcriptional regulation in developing organs. In
mature leaves, it has been demonstrated that mRNAs for
the small and large subunit of Rubisco, and the NADPME (maize) and NAD-ME (amaranth) are expressed
exclusively in bundle sheath cells, while PPDK, PEPC
and NADP-MDH are expressed in mesophyll cells.
However, there is only partial information on the
control of expression of mRNA and synthesis of these
proteins during development. There are differences in the
environmentally- and developmentally-dependent signals
controlling the expression of these genes in the few species
studied, and in situ hybridization may help us to
understand the regulation of C3uC4 gene development
under different conditions further. In situ hybridization is a powerful technique for tissue and cell-speci®c
localization of gene expression. However, it must be
remembered that the intensity of the signal seen does
not necessarily translate into differences in protein accumulation due to translational regulation, and it does not
give information on subcellular distribution of the protein
encoded by the transcript. This is particularly illustrated
by observations of macromolecular traf®cking of both
Compartmentation in C4 plant tissues
protein and mRNA between highly differentiated cells
such as companion cells and sieve elements (Kuhn et al.,
1997; Xonocostle-Cazares et al., 1999). A different in situ
technique, immunolocalization, can be used to clarify
such relationships.
Immunolocalization of C4 photosynthesis
proteins
As discussed earlier, following the discovery of a C4
pathway and Kranz anatomy, several in vitro isolation
techniques were employed to determine the intercellular
and intracellular compartmentation of enzymes of C4
photosynthesis. However, a leaf is a complex organ
with many tissue types and considerable variation in size
of veins and relative distribution of cell types. Thus,
isolation techniques tend to give a limited picture of
compartmentation.
Immunocytochemistry, a technique that was developed
to take advantage of antibodies as very speci®c `stains'
for probing tissue and cellular structure and function, was
soon employed to con®rm and expand the cell isolation
results. This technique combines the high chemical speci®city of antibodies and the high spatial resolution of
microscopy. In 1977, Hattersley et al. used immuno¯uorescent labelling to study localization of Rubisco in
plants. Antibodies to Rubisco were used to locate the
enzyme in leaves of 42 species (Hattersley et al., 1977).
They used an indirect labelling approach, whereby leaf
segments were ®xed in ethanol; then, hand-cut leaf
blade sections were rinsed brie¯y in buffered saline and
incubated for 1 h with antiserum. After rinsing, the leaf
blade sections were incubated in the dark in ¯uorescein
isothiocyanate (FITC)-labelled sheep anti-rabbit immunoglobulin. Rinsed sections were mounted in 50% glycerol
(aqueous) containing 1% thymol and then sections were
observed with a Zeiss Photomicroscope III set up for
epi¯uorescence, and photographed within 24 h of preparation. All 29 species having a distinct Kranz anatomy
showed high levels of ¯uorescence for bundle sheath cells
when sections were treated with Rubisco antibodies and
limited ¯uorescence from mesophyll cells. Thus, evidence
for high Rubisco protein was demonstrated in BSC across
various C4 species by this in situ method indicating this
is a consistent feature in evolution of C4 plants.
Direct in situ methods have obvious advantages over
in vitro methods, where uncertainties may exist about
stability of proteins during isolation, and degree of purity
of the fractions obtained. In addition, there are many
variations in Kranz anatomy, and not all species are
amenable to cell isolation. For example, among the
Poaceae (the family with the largest number of C4 species)
there are three C4 subtypes which have `classical' anatomical and structural properties, including NADP-ME
583
species having a single bundle sheath layer, and NAD-ME
and PEP-CK types having a double bundle sheath
layer with the C3 cycle in the outer layer (Gutierrez
et al., 1974a; Dengler and Nelson, 1999). There are other
C4 species of Poaceae which have `non-classical' variants
in the type of Kranz anatomy, including the aristidoid
type which has a double chlorenchyma sheath. In the
Chenopodiaceae (which has the largest number of C4
species among dicot families) there are four variants of
Kranz anatomy including atriplicoid, kochioid, salsoloid,
and suaedoid types (Carolin et al., 1975). Thus, in situ
immunolocalization is a valuable tool for studying
compartmentation of enzymes across taxonomic groups
where C4 photosynthesis has evolved multiple times
resulting in variations in anatomy and biochemistry.
With subsequent studies on immunolocalization of
enzymes, a consistent feature across the different types
of C4 plants analysed is the selective, high level of PEPC
in mesophyll cells, and localization of Rubisco in bundle
sheath cells, and both malic enzymes, NAD-ME and
NADP-ME, in bundle sheath cells (Dengler and Nelson,
1999; Drincovich et al., 1998; Maurino et al., 1997; Sinha
and Kellogg, 1996; Madhavan et al., 1996; Voznesenskaya
et al., 1999). Initial studies of several C4 monocots on the
localization of PPDK, the enzyme which regenerates
PEP, showed high activity in mesophyll cells, with little
or no activity in bundle sheath cells. However, studies of
species among four different lineages of C4 evolution in
the grasses show variation in PPDK localization from
mesophyll, to bundle sheath, to both cell types (Sinha
and Kellogg, 1996). Since two ATP are required per
pyruvate converted to PEP via this enzyme, it would be
of interest to examine the energetics and function of the
C4 cycle between mesophyll and bundle sheath cells of
such species.
Immunolocalization techniques for studies of compartmentation in C4 plants have improved signi®cantly
since the initial work (Hattersley et al., 1977).
While immuno¯uorescence on unembedded or paraf®nembedded tissue samples is still a very powerful technique, the resolution is somewhat limited due to section
thickness and problems with structural preservation at the
subcellular level. In addition, auto¯uorescence of tissues
in the absence of antibody can limit interpretations of the
absolute compartmentation of proteins of interest.
Initial improvements in immunocytochemistry dealt
with changes in ®xation protocols and the chemical
®xatives used (formaldehyde, paraformaldehyde, glutaraldehyde, and combinations of these), and the testing of
various embedding media which allowed better structural
preservation compared to free hand sectioning (paraf®n,
epoxy and acrylic resins). In particular, the use of resinembedded leaf material for light and electron microscopy
has greatly improved the ability to resolve the distribution patterns of various enzymes of the C4 pathway
584
Edwards et al.
at the cellular and subcellular levels. The development
of improved ®xation techniques (including freezesubstitution and microwave processing), new resins
designed for retention of antigen recognition, availability
of improved ¯uorescent probes, new gold probes and
silver enhancing techniques, and laser scanning confocal
microscopy, have allowed cellular and subcellular localization of a range of relevant enzymes of the C4 pathway
in a large number of species. For the application of
immunolocalization techniques in studies with C4 plants
see the following studies (Perrot-Rechenmann et al., 1982,
1983; Bauwe, 1984; Reed and Chollet, 1985; Rawsthorne,
1992; Wang et al., 1993; Dengler et al., 1995; Sinha and
Kellogg, 1996; Ueno, 1992, 1996, 1998; Maurino et al.,
1997; Drincovich et al., 1998; Voznesenskaya et al., 1999).
Currently, one of the most precise methods is the
immunocytochemical technique which uses protein
(Protein A, G, or IgG)-conjugated gold particles as
a secondary probe. This method has been applied
successfully to the study of compartmentation of enzymes
of carbon assimilation in C3±C4 and C4 plants, and
its undoubted advantage is the possibility for its use
not only for light microscopy level investigations
(using normal light, epipolarization or confocal imaging
systems), but also at the electron microscopy level for
establishing the intracellular and organellar localization
of different enzymes (Rawsthorne, 1992; Ueno, 1992,
1996, 1998; Maurino et al., 1997; Drincovich et al., 1998;
Voznesenskaya et al., 1999). Fixation of material with the
paraformaldehydeqglutaraldehyde mixture and embedding it in Lowicryl or L.R. White acrylic resin gives good
preservation of tissues and organelles, and does not
require removing the resin. In particular, the development
of gold probes for the immunolocalization allowed for
TEM-level studies of the subcellular distribution of
the enzymes of the C4 pathway. In combination with
silver enhancing procedures, gold probes provide a high
resolution localization technique at the light microscope
level (Maurino et al., 1997; Drincovich et al., 1998;
Voznesenskaya et al., 1999).
An example of the spatial resolution and preciseness
of immunolabelling, even at the light microscope level,
Fig. 1. Re¯ected-transmitted overlay imaging of silver enhanced-immunogold localization of Rubisco and PEPC in 1 mm resin sections of leaves of
the C4 plant maize and C3 plant rice. Yellow colour is the signal from the silver-enhanced gold particles. (A) Rubisco in the maize leaf is restricted to
the chloroplasts of the bundle sheath cells (BS). (B) Rubisco in rice is primarily in the chloroplasts of the mesophyll cells (M). (C) PEPC in maize is
restricted to the mesophyll cells. Note the label is in the cytoplasm and that the mesophyll chloroplasts are unlabelled. (D) Preimmune serum control
for maize leaf gives essentially no signal. BS, bundle sheath cell; E, epidermal cell; M, mesophyll cell. Divisions on bar are 10 mm increments.
Compartmentation in C4 plant tissues
using gold probes are demonstrated in Fig. 1. Figure 1A
and B illustrate immunogold labelling of Rubisco in the
C4 plant maize versus the C3 plant rice. It shows Rubisco
is exclusively con®ned to chloroplasts of the bundle
sheath cells of mature maize leaves. While rice has a
distinct bundle sheath around the vascular tissue, these
sheath cells are less developed and have few chloroplasts;
the primary site of labelling of Rubisco is seen in the
chloroplasts of the upper layer of mesophyll cells. For
all immunolocalization studies it is important to run
preimmune (or non-immune) controls to be sure there is
little, or no, non-speci®c labelling of the tissue in the
absence of the primary antibody, which is illustrated in
Fig. 1D for maize. Figure 1C shows the immunolocalization of PEPC in maize. The label is restricted to the
mesophyll cells and close examination of these sections
shows the label is in the cytoplasm while the chloroplasts
are unlabelled. PEPC immunolabelling was also done on
rice, but as expected there was very little, or no, labelling,
consistent with low PEPC activity in the C3 rice leaf
(not shown).
In studying compartmentation of enzymes of photosynthetic carbon metabolism in different chloroplastcontaining tissues of various species, the partitioning
of carbon into carbohydrates, including starch, is also of
interest. It is thought sucrose is predominantly synthesized in mesophyll cells; evidence with various C4 species
shows starch is normally synthesized in bundle sheath
cells. However, enzymes for their biosynthesis are found
in both cell types (Leegood and Walker, 1999). Using the
PAS procedure for staining polysaccharides, Fig. 2
illustrates the heavy deposition of starch in bundle sheath
chloroplasts of maize, the site of Rubisco localization,
as well as the appearance of starch in the guard cells,
585
while mesophyll cells are essentially devoid of starch.
Such cytochemical techniques combining chemical selectivity and spatial resolution can be of signi®cant value
in combination with immuno and in situ techniques. For
determining the distribution of metabolites between
mesophyll and bundle sheath cells during C4 photosynthesis, different rapid fractionation techniques were
developed earlier (Leegood, 1985; Stitt and Heldt, 1985),
but these rely on the ability to obtain fractions enriched in
the respective cell types, which, as pointed out above, is
not possible with many species.
The current state of these microscope-based methods
and their ability to solve some of the intriguing problems
of C4 photosynthesis is illustrated in a recent study
(Voznesenskaya et al., 1999) involving several species
in the tribe Salsoleae of the family Chenopodiaceae. In a
given species, different photosynthetic vegetative organs
often have the same type of CO2 ®xation (Edwards and
Walker, 1983). However, unlike other species previously
examined, some chenopods having C4 type photosynthesis in leaves have C3 metabolism in cotyledons.
Initially, Butnik discovered non-Kranz cotyledons in
some C4 chenopods, for example, in the Salsola genus
(Butnik, 1984). Later studies using the `pulse±chase',
14
CO2±12CO2, technique and analysis of isolated enzymes
provided evidence for C3 and C4 type photosynthesis in
cotyledons among species of the tribe Salsoleae having
C4 photosynthesis in leaves (Pyankov et al., 1999, 2000).
Anatomically, four types of cotyledons were identi®ed,
two C3 types, having dorsoventral and isopalisade
mesophyll structure, and two Kranz types (atriplicoid and
salsoloid). Immunolocalization studies of these tissues
are important for evaluating the mechanism of photosynthesis for several reasons. As noted, there is large
Fig. 2. Periodic acid-Schiff's staining for polysaccharides in a 1 mm thick resin section of maize leaf. Cell walls and starch are stained pink-red. Starch
is localized in the bundle sheath cells, but not mesophyll cells. Some starch can also be seen in the guard cells of stomates. BS, bundle sheath cell;
E, epidermal cell; M, mesophyll cell. Divisions on bar are 10 mm increments.
586
Edwards et al.
diversity in anatomy. Among species of the tribe Salsoleae,
leaves and cotyledons can have up to four chloroplastcontaining tissues: hypoderm, mesophyll, bundle sheath,
and water storage. Thus, the role of each cell type in
photosynthesis needs to be identi®ed. Cotyledons in some
species are very small and are dif®cult to characterize
photosynthetically by most techniques. Also, in some
species, like Haloxylon, where the carbon isotope
fractionation values in cotyledons are intermediate
between C3 and C4 plants (Pyankov et al., 1999), immunolocalization studies can help resolve the mechanism of
carbon ®xation (Voznesenskaya et al., 1999).
In the study of Voznesenskaya et al. (Voznesenskaya
et al., 1999), immunocytochemical localization of four
main photosynthetic enzymes (Rubisco, PEPC, NAD-ME,
and NADP-ME) was determined in four species from
the tribe Salsoleae exhibiting C4 type CO2 ®xation of
the NAD- or NADP-ME subtype. The species studied
have in common salsoloid structure in leaves (or stem in
the case of Haloxylon), but with different leaf anatomy
in cotyledons: C3 dorsoventral, C3 isopalisade, C4 atriplicoid, and C4 salsoloid. It was shown that, irrespective
of the nature of assimilating organs having Kranz
anatomy (leaf, cotyledon or stem), Rubisco was strongly
localized to bundle sheath cells, and PEPC was localized
in mesophyll cells, while malic enzymes were restricted
to bundle sheath cells. Both types of cotyledons with C3
anatomy showed ordinary C3 Rubisco localization in all
mesophyll cells and the absence of C4 enzyme labelling.
Electron microscopy revealed the localization of
NAD-ME in mitochondria, while NADP-ME was in
chloroplasts of bundle sheath cells in the respective C4
types. Staining for polysaccharides showed sites of starch
accumulation, which generally paralleled the localization
of Rubisco. It was apparent that in some C4 organs, the
hypoderm and water storage tissue also have chloroplasts
which contain Rubisco, which store starch, and which,
thus, perform C3 photosynthesis.
The immunolocalization approach is also important
for addressing the question of whether CAM occurs in
succulent species of Chenopodiaceae. A degree of CAM
has been suggested, but not proven, in some Chenopodiaceae species because they have water storage tissue
with a signi®cant number of chloroplasts (Zalenskii and
Glagoleva, 1981; Bil' et al., 1983). However, little or no
PEPC or malic enzyme protein was detected by immunolocalization in water storage cells, suggesting there is no
CAM or donation of CO2 from C4 acids to Rubisco in
these cells. Rather, the occurrence of Rubisco in water
storage tissue of some species suggests a role for re®xation
of respired CO2 from vascular tissue which is centrally
located in the leaf (Voznesenskaya et al., 1999).
At this point, the limiting factor for use of immunocytochemistry in photosynthesis research is not so much
the methodology but the availability of antibodies to the
enzymes of importance to the pathways being studied.
While a number of companies are actively involved in
producing antibodies to thousands of proteins for animal
and human research purposes, most antibodies for plant
biology research are produced by individual researchers
and are of limited availability.
The in situ immunolocalization method for analysing
compartmentation of enzymes of C4 photosynthesis
is also ideal for developmental studies (Maurino et al.,
1997) and studies with C3±C4 intermediates (Hattersley
et al., 1977; Bauwe, 1984; Reed and Chollet, 1985; Hylton
et al., 1988, Drincovich et al., 1998). It is particularly
dif®cult to separate the cell types in the intermediate
species because the bundle sheath cells and Kranz
anatomy are less developed than in C4 plants. A common
feature of intermediates, demonstrated with immunocytochemistry, is localization of Rubisco in both mesophyll and bundle sheath cells. In this case, the Rubisco in
bundle sheath is the site of re®xation of photorespired
CO2 (where glycine decarboxylase of the photorespiratory
pathway is speci®cally localized), and ®xation of CO2
delivered to the bundle sheath by a limited C4 pathway
in certain C3±C4 intermediates.
Cell- and organ-specific expression of
C4-specific genesÐgene promoter analysis
in transgenic C4 plants
Recent molecular studies on the C4 mechanism have
focused on evolution of C4-speci®c genes from existing
genes in ancestral C3 plants, and regulation of their
expression in C4 plants (Ku et al., 1996; Rosche et al.,
1998; Sheen, 1999). Relative to the corresponding genes
in C3 plants, the key features of C4-speci®c genes are highlevel expression, and organ- and cell-speci®c expression.
During the course of evolution, C4-speci®c genes acquired
modi®cations in their promoter regions which regulate
the speci®c patterns of expression. Homologous transgenic C4 plants have been used to identify the promoter
elements of C4 genes that are essential for directing
organ-speci®c and cell-speci®c expression. In this
approach, a fragment of the gene promoter region is
fused to a reporter gene (e.g. GUS-glucuranidase,
LUC-®re¯y luciferase or GFP-green ¯uorescent protein)
and used for transformation. The transcriptional
strength of the promoter in the transgenic plants can be
determined by analysing the reporter protein: enzymic
activity of GUS and LUC, histochemical staining of
GUS, and ¯uorescence signal by GFP. Histochemical
staining of GUS in tissues provides a direct visualization
of the site of expression, but does not allow a good
quantitative estimation of expression level, especially
between different experiments. However, this can be
remedied by assaying the enzymic activity of GUS
Compartmentation in C4 plant tissues
587
Fig. 3. Light microscopy of GUS activity staining in a mature leaf of transgenic maize transformed with a chimeric gene containing (A) a 0.9 kb maize
PPDK gene promoter and the reporter GUS gene, and (B) 35S cauli¯ower mosaic virus promoter and the reporter GUS gene. Courtesy of Mitsutaka
Taniguchi. Procedure for transformation and in situ staining of GUS activity was described previously (Taniguchi et al., 2000).
directly in protein preparations extracted from different
tissues or isolated cell types (Taniguchi et al., 2000). As
shown in Fig. 3, a 0.9 kb promoter sequence from the
maize PPDK gene directed GUS expression predominantly in the mesophyll chloroplasts of a transgenic maize
leaf. On the other hand, the constitutive 35S cauli¯ower
mosaic virus promoter directed GUS expression in both
cell types. The maize PPDK gene promoter sequences
apparently contain the necessary cis-acting elements for
its cell-speci®c expression. While cytochemical staining
of GUS activity is a simple method for direct, visual
determination of the reporter enzyme protein localization, it does not permit resolution at the intracellular
level. It is, thus, more useful to determine speci®city for
expression in particular cell types. After ®xation of the
tissues, the blue product produced by GUS tends to stick
to organelles, especially chloroplasts, even though the
protein is located in the cytosol. A concomitant immunolabelling will help resolve this problem. Finally, there is
growing interest in transferring C4 genes to C3 plants
(Ku et al., 1999), and methods of studying compartmentation of gene expression and protein accumulation
will be critical to this work (Sheehy, 2000).
Summary
The use of various techniques in studying the cellular
compartmentation of photosynthetic metabolism in C4
plants has been critical in elucidating the mechanism of
C4 photosynthesis. The current understanding has been
dependent on improvement of, or development of, new
techniques over the past several decades. In the future,
cell-speci®c analysis will be required to further the understanding of C4 photosynthesis, and to analyse the
potential of utilizing the genetic information associated
with this process to improve crop productivity. This
includes studies on the taxonomic-based diversity in the
C4 mechanism (anatomy and biochemistry), the further
characterization of compartmentation of enzymes, and of
speci®c transporters in organelles which are required
for C4 photosynthesis, elucidation of signalling processes
which are responsible for the control of the development
of Kranz anatomy and the associated biochemistry in
C4 plants, and determination of the consequences of
transforming C3 plants with genes from C4 plants which
are responsible for Kranz anatomy and C4 photosynthesis.
Acknowledgements
This work was partly supported by NSF Grant IBN-9807916
and Civilian Research and Development Foundation Grant
RB1-264. Thanks to T Kostman and N Tarlyn for assistance in
preparing the ®gures. The microsocopy was done in the WSU
Electron Microscopy Center.
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