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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 606 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 608 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 610 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.) 612 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. References Asai N, Nakajima N, Tamaoki M, Kamada H, Kondo N. 2000. Role of malate synthesis mediated by phosphoenolpyruvate carboxylase in guard cells in the regulation of stomatal movement. Plant and Cell Physiology 41, 10±15. BaÈchmann K, Lochmann H, Bazzanella A. 1998. Microscale processes in single plant cells. Analytical Chemistry 70, 645A±649A. Baker NR, Oxborough K, Lawson T, Morison JIL. 2001. High resolution imaging of photosynthetic activities of tissues, cells and chloroplasts in leaves. Journal of Experimental Botany 52, 615±621. 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. Bonner RF, Emmert-Buck M, Cole K, Pohida T, Chuaqui R, Goldstein S, Liotta LA. 1997. Laser capture microdissection: molecular analysis of tissue. Science 278, 1481±1483. Brent R. 2000. Genomic biology. Cell 100, 169±183. Cannizzaro LA. 1996. Chromosome microdissection: a brief overview. Cytogenetics and Cell Genetics 74, 157±160. Chang K, Roberts JKM. 1989. Observation of cytoplasmic and vacuolar malate in maize root tips by 13C NMR spectroscopy. Plant Physiology 89, 197±203. Chollet R, Vidal J, O'Leary MH. 1996. Phosphoenolpyruvate carboxylase: a ubiquitous, highly regulated enzyme in plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 273±298. Cooper JM. 1999. Towards electronic Petri dishes and picolitrescale single-cell technologies. Trends in Biotechnology 17, 226±230. Cotelle V, Pierre JN, Vavasseur A. 1999. Potential strong regulation of guard cell phosphoenolpryuvate carboxylase through phosphorylation. Journal of Experimental Botany 50, 777±783. Craig S, Staehelin LA. 1988. High pressure freezing of intact plant tissues. Evaluation and characterization of novel features of the endoplasmic reticulum and associated membrane systems. European Journal of Cell Biology 46, 80±93. Croxdale JG, Outlaw Jr WH. 1983. Glucose-6-phosphatedehydrogenase activity in the shoot apical meristem, leaf primordia and leaf tissues of Dianthus chinensis L. Planta 157, 289±297. de Brock M. 1995. In situ histochemistry on plastic embedded plant material. Methods in Cell Biology 49, 153±163. de Josselin de Jong JE, Jongkind JF, Ywema HR. 1980. A scanning inverted micro¯uorometer with electronic shutter control for automatic measurements in micro-test plates. Analytical Biochemistry 102, 120±125. Deutsch M, Kaufman M, Shapiro H, Zurgil N. 2000. Analysis of enzyme kinetics in individual living cells utilizing ¯uorescence intensity and polarization measurements. Cytometry 39, 36±44. Du Z, Aghoram K, Outlaw Jr WH. 1997. In vivo phosphorylation of phosphoenolpyruvate carboxylase in guard cells of Vicia faba L. is enhanced by fusicoccin and suppressed by abscisic acid. Archives of Biochemistry and Biophysics 337, 345±350. Microanalysis Ewert MS, Outlaw Jr WH, Zhang SQ, Aghoram K, Riddle KA. 2000. Accumulation of an apoplastic solute in the guard-cell wall is suf®cient to exert a signi®cant effect on transpiration in Vicia faba lea¯ets. Plant, Cell and Environment 23, 195±203. Fisher DB, Outlaw Jr WH. 1979. Sucrose compartmentation in the palisade parenchyma of Vicia faba L. Plant Physiology 64, 481±483. Freeman TC, Lee K, Richardson PJ. 1999. Analysis of gene expression in single cells. Current Opinion in Biotechnology 10, 579±582. Gee KR, Sun WC, Bhalgat MK, Upson RH, Klaubert DH, Latham KA, Haugland RP. 1999. Fluorogenic substrates based on ¯uorinated umbelliferones for continuous assays of phosphatases and b-galactosidases. Analytical Biochemistry 273, 41±48. Gerhardt R, Heldt HW. 1984. Measurement of subcellular metabolite levels in leaves by fractionation of freeze-stopped material in non-aqueous media. Plant Physiology 75, 542±547. Grif®ths EJ, Lin H, Suleiman MS. 1998. NADH ¯uorescence in isolated guinea-pig and rat cardiomyocytes exposed to low or high stimulation rates and effect of metabolic inhibition with cyanide. Biochemical Pharmacology 56, 173±179. Hampp R, Outlaw Jr WH. 1987. Mikroanalytik in der p¯anzlichen Biochemie. Naturwissenschaften 74, 431±438. Haugland R. 1995. Detecting enzymatic activity in cells using ¯uorogenic substrates. Biotechnic and Histochemistry 70, 243±251. Hite DRC, Outlaw Jr WH. 1993. Evaluation of two approaches to the quantitative histochemical localization of sucrose-P synthase in leaves. Histochemical Journal 25, 872±875. Hite DRC, Outlaw Jr WH, Tarczynski MC. 1993. Elevated levels of both sucrose-phosphate synthase and sucrose synthase in Vicia guard cells indicate cell-speci®c carbohydrate interconversions. Plant Physiology 101, 1217±1221. Jones MGK, Outlaw Jr WH, Lowry OH. 1977. Enzymic assay of 10 7 to 10 14 moles of sucrose in plant tissues. Plant Physiology 60, 379±383. Karrer EE, Lincoln JE, Hogenhout S, Bennett AB, Bostock RM, Martineau B, Lucas WJ, Gilchrist DG, Alexander D. 1995. In situ isolation of mRNA from individual plant cells: creation of cell-speci®c cDNA libraries. Proceedings of the National Academy of Sciences, USA 92, 3814±3818. Kato T, Berger SJ, Carter JA, Lowry OH. 1973. An enzymatic cycling method for nicotinamide-adenine dinucleotide with malic and alcohol dehydrogenases. Analytical Biochemistry 53, 86±97. Kennedy RT, Oates MD, Cooper BR, Nickerson B, Jorgenson JW. 1989. Microcolumn separations and the analysis of single cells. Science 246, 57±63. Koroleva OA, Farrar JF, Tomos AD, Pollock CJ. 1998. Carbohydrates in individual cells of epidermis, mesophyll and bundle sheath in barley leaves with changed export or photosynthetic rate. Plant Physiology 118, 1525±1532. Lowry OH. 1973. An unlimited microanalytical system. Accounts of Chemical Research 6, 289±293. Lowry OH. 1980. Ampli®cation by enzymatic cycling. Molecular and Cellular Biochemistry 32, 135±146. Lowry OH. 1990. How to succeed in research without being a genius. Annual Review of Biochemistry 59, 1±27. Lu P, Outlaw Jr WH, Smith BG, Freed GA. 1997. A new mechanism for the regulation of stomatal aperture size in intact leaves. Accumulation of mesophyll-derived sucrose in the guard-cell wall of Vicia faba. Plant Physiology 114, 109±118. 613 Macintosh CA, Stower M, Reid N, Maitland NJ. 1998. Precise microdissection of human prostate cancers reveals genotypic heterogeneity. Cancer Research 58, 23±28. McAinsh MR, Hetherington AM. 1998. Encoding speci®city in Ca2q signaling systems. Trends in Plant Science 3, 32±36. Meier-Ruge W, Bielser W, Remy E, Hillenkemp F, Nitsche R, Unsold R. 1976. The laser in the Lowry technique for microdissection of freeze-dried tissue slices. Histochemistry Journal 8, 387±401. Mroz EA, Lechene C. 1980. Fluorescence analysis of picoliter samples. Analytical Biochemistry 102, 90±96. Nast G, MuÈller-RoÈber B. 1996. Molecular characterization of potato fumarate hydratase and functional expression in Escherichia coli. Plant Physiology 112, 1219±1227. Nimmo HG. 2000. The regulation of phosphoenolpyruvate carboxylase in CAM plants. Trends in Plant Science 5, 75±80. Obon JM, Buendia B, Canovas M, Iborra JL. 1999. Enzymatic cycling assay for D-carnitine determination. Analytical Biochemistry 274, 34±39. Oresnik IJ, Layzell DB. 1994. Composition and distribution of adenylates in soybean (Glycine max L.) nodule tissue. Plant Physiology 104, 217±225. Outlaw Jr WH. 1980. A descriptive evaluation of quantitative histochemical methods based on pyridine nucleotides. Annual Review of Plant Physiology 31, 299±311. Outlaw Jr WH. 1990. Kinetic properties of guard-cell phosphoenolpyruvate carboxylase. Biochemie und Physiologie der P¯anzen 186, 317±325. Outlaw Jr WH. 1995. Extraction and assay of protein from single plant cells. Methods in Cell Biology 50, 41±49. Outlaw Jr WH, Fisher DB. 1975a. Compartmentation in Vicia faba leaves. I. Kinetics of 14C in the tissues following pulse labelling. Plant Physiology 55, 699±703. Outlaw Jr WH, Fisher DB. 1975b. Compartmentation in Vicia faba leaves. III. Photosynthesis in the spongy and palisade parenchyma. Australian Journal of Plant Physiology 2, 435±439. Outlaw Jr WH, Kennedy J. 1978. Enzymic and substrate basis for the anaplerotic step in guard cells. Plant Physiology 62, 648±652. Outlaw Jr WH, Lowry OH. 1977. Organic acid and potassium accumulation in guard cells during stomatal opening. Proceedings of the National Academy of Sciences, USA 74, 4434±4438. Outlaw Jr WH, Manchester J. 1980. Conceptual error in determination of NADq-malic enzyme in extracts containing NADq-malic dehydrogenase. Plant Physiology 65, 1136±1138. Outlaw Jr WH, Manchester J, Brown PH. 1981. High levels of malic enzyme activities in Vicia faba L. epidermal tissue. Plant Physiology 68, 1047±1051. Outlaw Jr WH, Springer SA, Tarczynski MC. 1985a. Histochemical technique. A general method for quantitative enzyme assays of single cell extracts with a time resolution of seconds and a reading precision of femtomoles. Plant Physiology 77, 659±666. Outlaw Jr WH, Springer SA, Tarczynski MC. 1985b. Enzyme assays at the single-cell level: real-time, quantitative, and using natural substrate in solution. In: Heath RL, Preiss J, eds, Regulation of carbon partitioning in photosynthetic tissue. Rockville: American Society of Plant Physiologists, 161±179. Oxborough K, Baker NR. 1997. An instrument capable of imaging chlorophyll a ¯uorescence from intact leaves at very low irradiance and at cellular and subcellular levels of organization. Plant, Cell and Environment 20, 1473±1483. 614 Outlaw and Zhang Parthasarathy MV. 1995. Freeze-substitution. Methods in Cell Biology 49, 57±69. Passonneau JV, Lowry OH. 1993. Enzymatic analysis, a practical guide. Totowa, Humana Press. Quinton PM. 1976. Construction of picoliter-nanoliter self®lling volumetric pipettes. Journal of Applied Physiology 40, 260±262. Renner C, TruÈmper L, P®tzenmeier J-P, Loftin U, Gerlach K, Stehle I, Wadle A, Pfreundschuh M. 1998. Differential mMRA display at the single-cell level. Biotechniques 24, 720±724. Rutili G, Arfors KE, Ulfendahl HR. 1976. Fluorescence measurements in nanoliter samples. Analytical Biochemistry 72, 539±545. Sakakibara T, Murakami S, Eisaki N, Nakajima M, Imai K. 1999. An enzymatic cycling method using pyruvate orthophosphate dikinase and ®re¯y luciferase for the simultaneous determination of ATP and AMP (RNA). Analytical Biochemistry 268, 94±101. Schlindler M. 1998. Select, microdissect and eject. Nature Biotechnology 16, 719±720. Schnabl H, Denecke M, Schulz M. 1992. In vitro and in vivo phosphorylation of stomatal phosphoenolpyruvate carboxylase from Vicia faba. Botanica Acta 105, 367±369. Schulz M, Hunte C, Schnabl H. 1992. Multiple forms of phosphoenolpryuvate carboxylase in mesophyll, epidermal and guard cells of Vicia faba. Physiologia Plantarum 86, 315±321. Simone NL, Bonner RF, Gillespie JW, Emmert-Buck MR, Liotta LA. 1998. Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Trends in Genetics 14, 272±276. Sims CE, Meredith GD, Krasieva TB, Berns MW, Tromberg BJ, Allbritton NL. 1998. Laser-micropipet combination for singlecell analysis. Analytical Chemistry 70, 4570±4577. Steingraber M, Hampp R. 1987. Vacuolar and cytosolic metabolite pools by comparative fractionation of vacuolate and evacuolate protoplasts. In: Marin B, ed. Plant vacuoles. New York: Plenum Press, 417±423. Suarez-Quian CA, Goldstein SR, Pohida T, Smith PD, Peterson JI, Wellner E, Ghany M, Bonner RF. 1999. Laser capture microdissection of single cells from complex tissue. Biotechniques 26, 328±335. Tarczynski MC, Outlaw Jr WH. 1990. Partial characterization of guard-cell phosphoenolpyruvate carboxylase: kinetic datum collection in real time from single-cell activities. Archives of Biochemistry and Biophysics 280, 153±158. Teutsch HF, Goellner A, Mueller-Klieser W. 1995. Glucose levels and succinate and lactate dehydrogenase activity in EMT6uRo tumor spheroids. European Journal of Cell Biology 66, 302±307. Trautschold I, Lamprecht W, Schweitzer G. 1985. UV-method with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, ed. Methods of enzymatic analysis, 3rd edn. Weinheim: Verlagsgesellschaft mbH VII, 346±357. Valaskovic GA, Kelleher NL, McLafferty FW. 1996. Attomole protein characterization by capillary electrophoresis-mass spectrometry. Science 273, 1199±1202. Vaughn KC, Outlaw Jr WH. 1983. Cytochemical and cyto¯uorometric evidence for guard cell photosystems. Plant Physiology 71, 420±424. Vidal J, Chollet R. 1997. Regulatory phosphorylation of C4 PEP carboxylase. Trends in Plant Science 2, 230±237. Wang X-C, Outlaw Jr WH, De Bedout JA, Du Z. 1994. Kinetic characterization of phosphoenolpyruvate carboxylase extracted from whole-leaf and from guard-cell protoplasts of Vicia faba L. (C3 plant) with respect to tissue pre-illumination. Histochemical Journal 26, 152±160. Washizu M. 1998. Electrostatic actuation of liquid droplets for microreactor applications. IEEE Transactions on Industry Applications 34, 732±737. Zhang SQ, Outlaw Jr WH. 2001. The guard-cell apoplast as a site of abscisic acid redistribution in Vicia faba L. Plant, Cell and Environment 24, 347±356. 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. Zhou Y, Wang H, Wei J, Cui L, Deng X, Wang X, Chen Z. 2000. Comparison of two PCR techniques used in ampli®cation of microdissected plant chromosomes from rice and wheat. Biotechniques 28, 766±774.