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Journal of Experimental Botany, Vol. 52, No. 356,
Compartmentation Special Issue, pp. 605±614, April 2001
Single-cell dissection and microdroplet chemistry
William H. Outlaw Jr.1 and Shuqiu Zhang2
Department of Biological Science, Florida State University, Tallahassee, Florida 32306-4370, USA
Received 2 June 2000; Accepted 19 September 2000
Abstract
The unique roles of individual cells may be critical
to the physiology of an organism. In such cases,
micromethods are essential to elucidating the
molecular biology, biochemistry and biophysics of
the specialized cells or even subcellular compartments of the important cells. The great proliferation
of micromethods testifies to their value and no single
review can be comprehensive. This review therefore provides only a generalized overview of one
approach, namely dissection that provides a pure
sample for subsequent extraction and analysis by
microdroplet chemistry. As a means of illustrating
the utility of this approach, an applicationÐstudy of
the interaction of cytosolic malate concentration
and guard-cell phosphoenolpyruvate carboxylaseÐis
provided.
Key words: Cellular localization, compartmentation, guard
cells, histochemistry, individual cell, malate, micro,
phosphoenolpyruvate carboxylase, single cell, stomata.
Introduction
Often, tissue heterogeneity confounds an interpretation
of results obtained by organ-level or tissue-level analyses.
For example, guard cells act semi-autonomously in the
leaf surface and control two of the most important
physiological operations of the plant, namely, acquisition
of CO2 and regulation of water loss. Yet these cells
comprise only a tiny fraction of the whole leaf and even
a tissue-level analysis would reveal little about guard cells
per se. In other cases, exempli®ed by C4 photosynthesis,
abundant but disparate cells contribute uniquely to a
single function. Thus, a leaf-level analysis would not
permit the assignment of one or another task to either
1
2
mesophyll cells or bundle-sheath cells. These two
examples suf®ce to illustrate the well-known requirement
to conduct investigations at the cell level in many
instances. Many sensitive methods have therefore been
developed. In all cases, the ®rst decision is to adopt a
strategy for tissue sampling.
Strategies for obtaining single-cell-size samples fall
into one of two general categories. First, in situ methods
(e.g. enzyme histochemistry, RNA hybridization) rely on
localization of an indicator to a cellular or subcellular
region that remains in the context of the tissue. Second,
other methods depend on the removal of the sample from
the tissue context. A well-known example of this basic
strategy is protoplast isolation, but another method is
to remove a cell-size sample for analysis. The cell-size
sample may be taken directly via a capillary (Sims et al.,
1998) or cellular contents may be removed via pipettes
(Karrer et al., 1995; Koroleva et al., 1998). Alternatively,
the tissue may be stabilized and cells or subcellular
samples subsequently dissected out. Regardless, separation of the sample from the tissue before analysis
facilitates simultaneous multiple analyses, such as use of
separation or array technologies. For example, a large
number of mRNA-abundance patterns in the extract of
a single sample can be monitored at once, impossible
by the analogous in situ method. Monitoring a large
number of analytes simultaneously is not only ef®cient,
but permits assignment of new functions to known
molecules (Brent, 2000) or ions.
Simple manual dissection of cells or subcellular samples
is a simple, low-technology and inexpensive means of
obtaining a small specimen for analysis by microdroplet
chemistry. Curiously, this method is rarely used in plantcell biology. The purpose of this article is to suggest a
wider application of hand dissection, which should be
considered as a starting point for single-cell biochemistry
or molecular biology. Application examples are given that
emphasize the value of manual dissection. The focus is on
To whom correspondence should be addressed. Fax: q1 850 644 0481. E-mail: [email protected]
Present address: College of Biological Sciences, China Agricultural University, Beijing, China 100094.
ß Society for Experimental Biology 2001
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Outlaw and Zhang
sample preparation, handling and extraction, all of which
are common to subsequent analyses of various sorts.
Tissue preparation
Cryofixation
Rapid freezing of plant samples is adequate to preserve
most metabolites and enzymes. Generally, if liquid nitrogen is cooled from its boiling point to its melting point
by application of a vacuum, a small sample held by the
edge with thin forceps can be plunged into liquid nitrogen
slurry without the formation of insulating air bubbles.
Any experimental design depends on the purpose at
hand, and general procedures may be inadequate. As
an example, ATP concentrations in vivo are affected by
oxygen, and ATP pools turn over rapidly (Oresnik and
Layzell, 1994). Furthermore, ATP is susceptible to hydrolysis during tissue disruption, freezing, and freeze-drying
(Trautschold et al., 1985) and during storage at 20 8C
(Passonneau and Lowry, 1993). In such cases, alternative
coolants or methods that provide ultra-rapid freezing
by high-pressure ¯uid (Craig and Staehelin, 1988) or
pre-cooled metal tongs should be considered.
Stabilization for dissection
Freeze-drying (removal of tissue water by sublimation
at low temperature and pressure, see Passonneau and
Lowry, 1993) and freeze-substitution (removal of tissue
water by dissolution into an organic solvent at low
temperature, see Parthasarathy, 1995) are generally
adequate means of preserving the chemical integrity and
localization of plant analytes. With these methods, even
a cold-labile, light-activated enzyme activity, pyruvate,
orthophosphate dikinase (EC 2.7.9.1), has been preserved
in dissected cells of a C4 plant (Outlaw et al., 1981).
Similarly, early studies (Fisher and Outlaw, 1979) showed
that the intracellular localization of water-soluble metabolites such as 14C-labelled products of photosynthesis
could be maintained. However, de Brock's caution that
freeze-drying and freeze-substitution are `cumbersome
and often give poor results with plant material' merits
consideration (de Brock, 1995). First, in the case of
freeze-drying, high standards (- 35 8C, F10 mm Hg)
must be maintained throughout the process. Second,
in the case of freeze-substitution, rigorous and strictly
anhydrous procedures must be followed. Even under
the best conditions, each procedure must be evaluated
for artefacts. For example, a fraction of the activity of
sucrose synthase (EC 2.4.1.13) was lost during freezedrying (Hite et al., 1993) as judged by comparisons of the
activity in fresh tissue extracts. As another example, 6%
of photosynthetically formed 14C-phosphoglycerate was
hydrolysed to glycerate during freeze-substitution and
embedment (Outlaw and Fisher, 1975b).
Dissection
Tissue stabilization is the ®rst goal of any dissection
protocol. Tissues that are easy to fragment, such as Vicia
faba leaves, can be freeze-dried intact and subsequently
dissected. Compact, cohesive tissues, such as Dianthus
meristem, may be cryosectioned before freeze-drying and
dissection (Croxdale and Outlaw, 1983). If tissue is freezesubstituted and embedded, it must be sectioned before
dissection. Regardless of the method, stabilized tissues
must be handled in low humidity to avoid artefacts of
diffusion or metabolism. Under these controlled conditions after stabilization, no plant metabolite has been
reported to be degraded. The most labile plant enzyme
documented for these conditions is glucose-6-P dehydrogenase (EC 1.1.1.49), which has a half-time of 30 h
(Croxdale and Outlaw, 1983). Thus, although each tissue
and each analyte should be evaluated, no loss of practical
importance has been ascribed to ambient conditions
during dissection.
Morphological resolution is the second goal of any
dissection protocol. When coherent masses of a single-cell
type, such as palisade parenchyma, are present, relatively
large masses can be dissected easily. In other cases, it is
possible to tease out the desired structure (representative
references in Zhou et al., 2000), but usually, excision is
required. Therefore, laser microdissection was developed
early (Meier-Ruge et al., 1976), but the technology did
not become widely used, probably because of artefacts
associated with heat required to burn the tissue into
sections. However, interest in this general technology
has mushroomed recently (Bonner et al., 1997; Schlindler,
1998; Simone et al., 1998; Suarez-Quian et al., 1999)
with the development of laser capture microdissection. In
essence, laser capture microdissection depends on overlaying a 5 mm thick cryopreserved section with thermoplastic ®lm. An infrared laser is focused on the area of
interest (resolution ;1 mm, Simone et al., 1998), melting
the polymer and embedding the tissue. When the area of
interest is lifted, the adjacent, unembedded tissue shears
away. Thus, laser-capture microdissection is not dissection in the usual sense of the word, and this technique
does not burn through the edges of a tissue section as
previous laser-based methods did. The aggressive marketing of this method has had the positive effect of focusing
attention on the importance of cell-speci®c physiology,
biochemistry and molecular biology, but the positive
attributes of simple manual dissection (Passonneau
and Lowry, 1993) are vastly understated in the market
(http:uuwww.arctur.comufaqs.html). Hand-dissection can
be simply carried out by use of a razor blade fragment
mounted onto a handle (Passonneau and Lowry,
1993). Indeed, manual or assisted dissection of speci®c
animal cells has a long history (Lowry, 1973) and ®nds
current application in medicine and molecular biology
Microanalysis
607
Fig. 1. Nanogram-size cell samples that were hand dissected from freeze-dried Vicia faba lea¯et. (A) Guard-cell pair (;6 ng), dissected as a unit from
the abaxial epidermis. (B) Abaxial epidermal-cell sample. (C) Palisade cell (;12 ng). (D) Spongy cell (;14 ng). The scale bar is 20 mm. Dry cell masses
are from Jones et al. (Jones et al., 1977) and Outlaw and Lowry (Outlaw and Lowry, 1977). (Reproduced with permission of Springer-Verlag; from
Hampp R, Outlaw Jr WH, 1987. Mikroanalytik in der p¯anzlichen Biochemie. Naturwissenschaften 74, 431±438.)
(Cannizzaro, 1996; Macintosh et al., 1998) and biochemistry (Teutsch et al., 1995). In the speci®c case of higher
plants, cells such as palisade cells can be hand dissected
cleanly (Fig. 1), and the general limit of hand dissection
is about 2 mm (Outlaw, 1980). As the examples in the
following sections show, hand dissection permits the
study of intra- and intercellular compartmentation with
picolitre resolution. It is important to note, however, that
many biological studies do not require the resolution
illustrated in this article.
Basis for analyte expression
Overview
Interpretation of the analyte content of microdissected
samples requires a speci®c biological basis for expression.
The appropriate basis, for example, protein, chlorophyll,
dry mass, cell volume, membrane surface area, depends
on the biological question. However, the size of dissected
samples is intrinsically limiting and direct microassays of
the usual bases, for example, protein (Outlaw, 1995), are
destructive and laborious. Fortunately, in some applications, determination on a cell basis is adequate. In other
applications, a simple non-destructive method for determining mass or volume, as described below, is required.
Subsequently, the cell, mass, or volume expression is
converted to the relevant basis.
Determination of mass of microdissected freeze-dried
samples by the quartz fibre `fish pole' balance
Developed in 1939 by Lowry when he was working with
Linderstrom-Lang, the quartz ®bre balance was continually re®ned over the following 40 years when a safe source
of radiation (sealed 241Am) to dispel static electricity was
added (Outlaw Jr WH, unpublished results). As described
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Outlaw and Zhang
in Fig. 2, the balance is ingeniously simple and inexpensive to construct. It is also utterly sensitive, capable of
determining the mass of sub-ng samples (Kato et al.,
1973). The disadvantages are practical. First, although
with care they are relatively easy to use, small balances
that are used for mass determinations of single plant cells
require ®nesse to construct. Second, each balance has a
small useful linear range (say, a 10-fold range of masses).
Third, they are relatively imprecise compared with other
balances.
Determination of volume of freeze-substituted,
embedded microdissected samples by fluorescence
Outlaw and Fisher devised a ¯uorometric assay for
the volume of microdissected samples of methacrylateembedded tissue (Outlaw and Fisher, 1975a). In brief, the
basis of the assay is inclusion of the ¯uor BBOT ((2,5-bistert-butylbenzoxazolyl)-thiophene) in the methacrylate
monomers. Following polymerization, the BBOT is uniformly distributed throughout the interior of the methacrylate block. The sample is sectioned and microdissected;
then, the ¯uorescence of the sample is assayed (excitation:
338 nm; emission: 475 nm). The ¯uorescence of the sample
is compared with the ¯uorescences of larger reference
samples of the same methacrylate block. The masses of
the reference samples (quartz-®bre balance, above) and
the density of methacrylate permit the conversion of
sample ¯uorescence to sample volume.
Fig. 2. The quartz-®bre `®sh pole' balance, used for weighing nanogramsize tissue samples. A thin, quartz ®bre mounted horizontally at one
end inside a reversed glass syringe (A) is the basic component of the
balance. The syringe protects against air currents and contains a disc of
241
Am that dispels static. The tissue sample is transferred to the balance
®bre from a sample holder (B), which is visualized through an
horizontally mounted dissecting microscope (C). The mass-induced
de¯ection of the balance ®bre toward gravity (D) is measured with the
same microscope (E). The balance housing and the microscope are
mounted onto the same vibration-dampened slab of stone. (Revised and
reproduced with permission of Humana Press; from Passonneau JV,
Lowry OH, 1993. Enzymatic analysis. A practical guide. Totowa,
Humana Press.)
Extraction
Analysis of microdissected samples requires the use
of small volumes in order to diminish the effect of
contamination in reagents (`blank') and to increase the
concentration of the analyte. However, the use of small
volumes introduces the problem of reagent evaporation
as well as some assay-speci®c problems associated with
reagent surface area. Fortunately, in protocols requiring
)20 nl, evaporation is eliminated by working under oil.
It is also possible with extra precautions to control
evaporation in much smaller droplets by working under
oil or in a humid chamber.
Figure 3 describes the oil-well technique (Passonneau
and Lowry, 1993) the basic component of which is a small
slender pipette used to deliver reagents into oil-®lled
holes. Experience with plant applications (down to
1.5 nl, Outlaw and Kennedy, 1978) has been with handfabricated quartz pipettes (Passonneau and Lowry, 1993),
but several automated pipette-construction protocols
(Quinton, 1976) have been published. Overall, however,
manipulation of nanolitre droplets is tedious and labourintensive and should be replaced by automated systems.
Micro¯uidics (aliquoting, transporting and merging
microdroplets) is a big challenge, but biology should
be able to draw from other sciences (e.g. piezoelectric
`ink-jet' systems for the delivery of small volumes).
Current work (Washizu, 1998; Cooper, 1999; Jones TB,
personal communication) demonstrates both progress
and dif®culties in the development of the appropriate
technologies. As an example, Jones (TB Jones, personal
Fig. 3. The `oil-well' technique, used for analysing nanogram-size tissue
samples. A series of cylindrical 3 mm holes, drilled into a 5 mm
thick Te¯on block, are partially ®lled with oil. Extraction and some
subsequent assay steps are conducted under oil (see text), which
eliminates or reduce evaporation of the reagent. A slender, pointed,
constriction pipette delivers an aliquot of extracting solution (A).
A tissue sample is pushed through the oil into contact with the extracting
solution (B). Subsequently (C), aliquots of other solutions may be added
or removed from the oil well, as appropriate for the particular assay.
The volumes shown are for perspective and depend on assay design.
(Revised and reproduced with permission of Humana Press; from
Passonneau JV, Lowry OH. 1993. Enzymatic analysis. A practical guide.
Totowa, Humana Press.)
Microanalysis
communications) has been able to produce and merge
7 nl droplets using electrostatic forces, which are adaptable to microprocessor automation. When perfected,
this level of miniaturization should be adequate for most
purposes.
Example applications
Overview: technical approaches
Two complementary applications will illustrate use of
hand dissection followed by microdroplet chemistry. The
®rst uses freeze-drying (for a whole-cell sample) and the
second, freeze-substitution (for a subcellular sample).
Fortuitously, both rely on measurement of oxidation of
NADH either by real-time microdroplet ¯uorometry
(®rst example, an enzyme assay) or by indirect end-point
analysis by enzymatic cycling (second example, a metabolite assay). As NAD(P) can be coupled directly or
indirectly to most enzymatic reactions, generic methods
of measuring the oxidation of NAD(P)H or reduction
of NAD(P)q can be adapted for varied purposes.
Overview: biochemical context of analyses
Post-translational regulation by reversible protein phosphorylation of phosphoenolpyruvate carboxylase (PEPC,
EC 4.1.1.31) isoforms in C4 and CAM cells has long
been known (Chollet et al., 1996; Vidal and Chollet,
1997; Nimmo, 2000). This regulatory phosphorylation is
manifested kinetically as reduced sensitivity to malate
inhibition under suboptimum assay conditions that are
presumed to mimic the physiological milieu. PEPC also
plays a central role in stomatal movements (Outlaw, 1990;
Asai et al., 2000), and guard cells contain a speci®c
isoform (Schulz et al., 1992; Wang et al., 1994; Nast and
MuÈller-RoÈber, 1996). In contrast to the photosynthetic
isoforms, speci®c regulatory phosphorylation of guardcell PEPC was not detected in guard-cell protoplasts
(Schnabl et al., 1992). Moreover, a direct measurement
of cytosolic malate concentration in undisturbed plant
tissue was lacking (but see Steingraber and Hampp, 1987;
Chang and Roberts, 1989). Thus, the example analyses
below focus, ®rst, on in vitro inhibition of guard-cell
PEPC by malate and, second, on malate concentration in
plant cytosol. Information obtained from both investigations is necessary to an understanding of how the
accumulation of malate in guard cells during stomatal
opening is regulated.
Activation state of guard-cell phosphoenolpyruvate
carboxylase in relation to the physiological status
of the leaf
This section describes a study of enzyme kinetics in dissected cells using NADH ¯uorescence in microdroplets
609
as the reaction indicator. Other uses of micro¯uorometry
having cellular and subcellular resolution that do not rely
on hand dissection will not be discussed. Valuable and
widespread, these other methods include quantitative
in situ auto¯uorescence kinetics analysis of chlorophyll
(Vaughn and Outlaw, 1983; Oxborough and Baker, 1997;
Baker et al., 2001) and pyridine nucleotides (Grif®ths
et al., 1998) as well as analysis of ion-sensitive ¯uorescence of xenobiotics (McAinsh and Hetherington, 1998).
It is also noted that many arti®cial ¯uorogenic substrates
have been developed that provide alternative methods
and extend the scope of enzymatic reactions that can
be studied quantitatively with small dissected samples
(Haugland, 1995; Gee et al., 1999).
Fluorescence is a more speci®c means of measuring
reduced NAD(P) than absorbance is. In addition,
¯uorescence is a measurement of absolute light and is
thus inherently more sensitive than absorbance, which
is calculated from diminution of transmitted light.
Finally, the ¯uorescence signal can be increased within
limits because ¯uorescence is proportional to excitation.
Although there are disadvantages to measuring NAD(P)H
¯uorometrically (e.g. temperature dependence), it is an
attractive method when sensitivity is an important aspect
of analytical design. Thus, ¯uorometric methods for
measuring pyridine nucleotides and other substances in
microdroplets were developed (Rutili et al., 1976; Mroz
and Lechene, 1980; de Josselin de Jong et al., 1980, and
references therein) as instrumentation became available.
Building on these earlier methods, Outlaw et al. increased
the sensitivity ;100 3 through optimization of the optical
system and dedicated software (Outlaw et al., 1985a, b),
making it possible to measure single-cell enzyme activities
in real time using natural substrates in solution. Although
the analysis described in the following paragraph was
produced with custom-fabricated equipment, currently
available turnkey systems (Deutsch et al., 2000) would
appear to be easily adapted to freeze-dried dissected
plant cells.
The speci®c application example concerns the kinetics
state of guard-cell PEPC. Aware that enzyme activities in
guard-cell protoplasts can be labile (Hite and Outlaw,
1993) and that the phosphorylation domain of PEPC
is easily proteolysed (Chollet et al., 1996), Zhang et al.
designed a PEPC assay based on microdroplet ¯uorometry suitable for analysis of single guard-cell pairs dissected from freeze-dried leaf tissue (Zhang et al., 1994).
The principle of the assay is real-time measurement of the
PEPC product, OAA, by coupling to malate dehydrogenase. Thus, PEPC activity is indicated by a decline in
NADH ¯uorescence following the addition of the tissue
sample to assay cocktail. This assay is chosen for illustration because it is more dif®cult to perform than the
ordinary single-cell assay (contrast with Tarczynski and
Outlaw, 1990). The dif®culty stems from the requirement
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Outlaw and Zhang
for low NADH concentration (because the assay is conducted under suboptimum conditions) and the need to
consume much of the NADH over a short time-course
(to prevent a protein dephosphorylation artefact). An
additional complication was introduced by the need for
high malate concentration in the presence of endogenous
and analytical malate dehydrogenase (cf. Outlaw and
Manchester, 1980). Despite these assay constraints, it was
easily demonstrated that PEPC in guard cells of opening
stomata was insensitive to malate, whereas this negative
allosteric effector inhibited PEPC in guard cells of
closed stomata (Fig. 4). This alteration in kinetics, which
corresponded to a physiological state of the tissue, led to
the demonstration that guard-cell PEPC is reversibly
phosphorylated when stomata are stimulated to open
(Du et al., 1997; Cotelle et al., 1999).
ATPuADP, GTPuGDP, GSSGu2GSH, and Co AuAcetyl
Co A. Currently, various new cycling procedures for
pyridine nucleotides (Obon et al., 1999) and other substances (Sakakibara et al., 1999) are being developed,
attesting to the currency and importance of this approach.
The cycling systems based on NAD and NADP are the
most important for two reasons. First, NAD(P) redox
reactions, as mentioned, can be coupled directly or
indirectly to many enzymes and metabolites. Second,
the oxidized and reduced forms of pyridine nucleotides
can be selectively destroyed. (For other general information about enzymatic cycling, consult Lowry, 1973;
Outlaw, 1980; Passonneau and Lowry, 1993.)
The principle of enzymatic cycling is simple (Fig. 5),
as further explained as part of the following generalized
procedure for a metabolite:
Quantification of cytoplasmic malate concentration
. In a ®rst or extraction step, the tissue is pushed onto a
microdroplet (Fig. 3A, B), which is then heated to destroy
endogenous enzymes (and co-factors in some cases).
This section describes a study of subcellular metabolite
concentrations in dissected cells using enzymatic cycling.
Enzymatic cycling as a means of providing chemical
ampli®cation can be traced to Warburg in the 1930s
(Lowry, 1990), but it was through OH Lowry's knack and
persistence that laboratory protocols became routine. By
1980 (Lowry, 1980), 19 different cycling protocols had
been published for NADquNADH, NADPquNADPH,
. In a second or speci®c step, the metabolite is coupled
enzymatically to a co-factor by addition of a second
microdroplet (Fig. 3C). The simplest case broadly outlined here involves an analytical dehydrogenase that is
speci®c for the metabolite of interest and the cofactor is
NADH. After this step, NADq is equal to the amount of
metabolite originally present in the extract.
. In a third or destruction step, analytical enzyme(s) and
unreacted co-factor remaining from the speci®c step are
destroyed. Thus, this example would call for addition of
acid, which destroys NADH (but not NADq). After this
Fig. 4. Phosphoenolpyruvate carboxylase (PEPC) activity in individually dissected guard-cell pairs of Vicia faba (see Fig. 1). PEPC was
assayed ¯uorometrically in real time (Outlaw et al., 1985a) under
suboptimum conditions of pH and limiting substrate in a 17 nl droplet.
Each trace is an average of seven individual time-courses; for a singleassay trace under optimum conditions (see Tarczynski and Outlaw,
1990). PEPC in guard cells of closed stomata (left panel) is inhibited by
400 mM malate whereas PEPC in guard cells of opening stomata (right
panel) is insensitive to 400 mM malate. These physiological states
correspond to the non-phosphorylated and phosphorylated forms of
guard-cell PEPC, respectively (Du et al., 1997). (Reproduced with
permission of Elsevier; from Zhang SQ, Outlaw Jr WH, Chollet R, 1994.
Lessened malate inhibition of guard-cell phosphoenolpyruvate carboxylase velocity during stomatal opening. FEBS Letters 352, 45±48.)
Fig. 5. The principle of enzymatic cycling. The molecule to be ampli®ed,
X, is added to reagent containing excess A and an enzyme that couples
the conversion of A to B with the conversion of X to Y. In the same
reagent, Y is reconverted to X, which, again, is converted to Y. For each
turn of the cycle, B and D accumulate. After hundreds or thousands of
cycles, B and D accumulate suf®ciently to be measured by conventional
means. NADquNADH and NADPquNADPH cycles are the most
popular because these co-factors can be coupled stoichiometrically in
a preceding step to a variety of analytes or enzymes. Many other cycling
protocols (e.g. for ATPuADP) are also available and extends the utility
of the approach.
Microanalysis
q
third step, the total NAD present is the NAD formed
stoichiometrically with the reduction of the metabolite in
the speci®c step.
. In a fourth or cycling step (Fig. 5), the co-factor
is ampli®ed. This and the following reactions are
genericÐthey provide a means of measuring total
co-factor, in this case, NAD. (The principles of cycling
are the same for other co-factors, such as NADP or ATP.)
As explained (Fig. 5), NADq initiates a cyclic reaction,
resulting in the accumulation of products B and D. As
the co-factor is added in low concentration, well below
the Km, product accumulation is linear with the amount
of co-factor added.
. In a ®fth or indicator step, the accumulated product
(B or D, Fig. 5) is assayed by a conventional procedure
in which the assay volume is in the millilitre range.
Fig. 6. Emerging root hair of Raphanus sativus. The tissue was quickly
frozen, freeze-substituted at
80 8C, embedded in anhydrous methacrylate, and sectioned. (A) Light micrograph, demonstrating a dense
cytoplasmic cap and a contrasting clear vacuole. Such clear visualization
tests the limits of hand dissection of distinct regions of a cell. The scale
bar is 10 mm. (B) Electron micrograph of the cytoplasmic cap. The
scale bar is 1 mm. (Reproduced with permission of the Histochemical
Society; from Bodson MJ, Outlaw Jr WH, Silvers SH, 1991. Malate
content of picoliter samples of Raphanus sativus cytoplasm. Journal of
Histochemistry and Cytochemistry 39, 435±440.)
611
The speci®c application example concerns the cytosolic
concentration of malate, a negative effector of PEPC.
Aware that plant-tissue malate concentration is heterogeneous, subject to environmental conditions (Gerhardt
and Heldt, 1984), and as high as the millimolar range
(Steingraber and Hampp, 1987; Chang and Roberts,
1989), Bodson et al. designed a malate assay suitable for
use on undisturbed subcellular tissue samples in which
PEPC activity and whole-tissue malate accumulation
could be co-ordinately determined (Bodson et al., 1991).
The purpose was to establish whether malate concentration is suf®cient to inhibit PEPC as predicted from in vitro
kinetics and, if so, whether the in vivo rate is indeed
inhibited. The model was Raphanus sativus root hairs
(Fig. 6), which were freeze-substituted. The dense cytoplasmic `cap' permitted the dissection of picolitre volumes
of cytoplasm, which was analysed for malate content
(Fig. 7). The conclusion was quite clear: high cytosolic
malate concentration did not inhibit PEPC in vivo,
indicating that PEPC existed in an altered kinetic state
in vivo. Although this model was not pursued further,
enzyme phosphorylation (see previous section) and
Fig. 7. The malate content eluted from samples that were hand dissected
out of 6 mm sections of methacrylate-embedded R. sativus root hairs
(Fig. 6). The x-axis is the sample volume determined from the ¯uorescence of a ¯uor (BBOT, see text) that was incorporated into the
embedding matrix. The closed symbols are from samples of cytoplasmic
cap whereas the open symbols are from heterogeneous samples that
contained vacuole (61%) and cytoplasm (39%). Results of two experiments, indicated by the different symbols, are shown. The concentration
of malate in the cytoplasmic cap was 8.4 mM (or as much as 10 mM, if
the volume of the Golgi-derived vesicles is excluded). The concentration
of malate in the vacuole was 54 mM. (Reproduced with permission of
the Histochemical Society; from Bodson MJ, Outlaw Jr WH, Silvers SH,
1991. Malate content of picoliter samples of Raphanus sativus cytoplasm.
Journal of Histochemistry and Cytochemistry 39, 435±440.)
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Outlaw and Zhang
presence of allosteric effectors (particularly glc 6-P) would
be the current interpretation of the altered state.
The malate analysis above demonstrates the potential
utility of dissection and microdroplet chemistry to study
metabolite compartmentation between the cytoplasm
and the vacuole. In other cases, dissection has permitted
the measurement of cell-wall substances, such as mannitol (by radioactive assay, Ewert et al., 2000), sucrose
(by enzymatic cycling, Lu et al., 1997) and ABA (by
ultrasensitive immunoassay, Zhang and Outlaw, 2001).
Flexibility and accessibility of microanalysis
The preceding emphasis on sensitivity limits and morphological resolution dampens equally important messages of this article, assay ¯exibility and accessibility, so
a brief postscript to consider adaptability to a typical
laboratory situation is in order. Consider an analysis for
the sucrose content of a single palisade cell (Fig. 1), which
contains approximately 2 pmol sucrose (Jones et al.,
1977): (1) The initial extracting cocktail, 2 ml, is delivered
into the bottom of a silanized 6 mm borosilicate tube
with a standard commercial repeating pipette, which can
deliver volumes as small as 0.1 ml. (2) A palisade cell is
pushed onto the 2 ml droplet by means of a small quartz
®bre mounted onto a handle. (3) The droplet is covered
by oil (40% hexadecaneq60% USP light mineral oil) and
heated to destroy endogenous enzymes and pre-existing
interfering substances. (4) Additional steps of the assay
(to couple sucrose hydrolysis to reduction of NADPq)
are conducted by addition of reagents through the oil
onto the droplet. (5) An aliquot, 5 ml, is removed for
ampli®cation and ®nal measurement in 1 ml. As this
outline shows, a microassay can be very simple. Indeed,
the only specialized facility for the outlined microassay
is an environment suitable for dissecting.
Conclusion
Plants are successful because biological processes are
compartmented at the cellular and subcellular levels. In
many instances, these processes can only be understood
by investigation of speci®c compartments. A proven,
powerful means of isolating the compartment for biochemical and physiological studies is by dissection
followed by microdroplet chemistry. This sampling
approach should lend itself equally well to single-cell
gene expression analysis by differential display (Renner
et al., 1998) or DNA microarrays (Freeman et al., 1999)
and to microcolumn separation methods (Kennedy et al.,
1989; Valaskovic et al., 1996; BaÈchmann et al., 1998;
WH Outlaw Jr, H Lochmann, K BaÈchmann, unpublished
results).
Acknowledgement
The US Department of Energy supported the work in the
laboratory during the preparation of this review.
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