Report
Microscale Processes in Single Plant Cells
n living organisms, chemical and biochemical reactions occur on a microscale level in distinct, tiny areas. Of course, nerve cells, liver tissue cells, and erythrocytes are cells with very different "jobs" in a complex organism, and many chemical reactions in these cells are specialized. However, even adjacent cells within one tissue are not identical and perform different functions and thus can differ significantly. This is valid for animal and human as well as for plant tissues. Cells interact and communicate with surrounding cells through a complex system of forces and fluxes. A macroscopic view of the entire organism or whole tissue does not reflect such processes on a fine scale. Analysis of the chemical compounds in tissue samples of common size, containing a mixture of different cells and cell types, gives only integrated values of concentrations, which do not provide information about the cellular level. (We are assuming a cell volume of 100 pL so 1 mg of tissue contains —10 000 cells) Hence an
sample-handling procedures, such as dilutAnalyzing single plant ing, measuring volume, dividing into subsamples, mixing with reagents, and storcells or subcellular ing, a difficult task. In addition, the high ratio causes high evapocompartments yields surface-to-volume ration rates and the risk of contamination. intrinsic problems of ultrasmall volinformation that These umes render sample manipulation much difficult. can't be obtained morePrerequisites for a suitable analytical method for subnanoliter-volume samples from bulk samples. such as single cells are high selectivity and investigation of individual cells is required to reflect the distribution of compounds and dynamic processes within a tissue on this scale. The physicochemical properties of ultrasmall-volume samples make normal K. Bächmann H. Lochmann A. Bazzanella Technische Universitat Darmstadt (Germany)
low absolute detection limits. In cells, analytes occur with hundreds of other compounds, which may be present in much higher concentrations and may be very similar to the analytes of interest. Compared with animal cells, the situation is even more complex in plant tissues. Plant cells show a strong compartmentalization of solutes and metabolites between cytoplasm and vacuole. In this case, vacuolar concentrations must be distinguished from cytoplasmic concentrations to gain detailed knowledge about microscale pro-
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Report cesses. Thus, analyzing single cells or even subcellular compartments with microanalytical methods yields information on a smaller scale, which cannot be obtained from bulk samples. This Report examines the extremely challenging analyses of volumes in the picoliter-or-lower range. Picoliter-volume samples by CE
For the simultaneous determination of different species, the chromatographic or electrophoretic separation of the compounds prior to detection is advantageous. In past years, capillary electrophoresis (CE) has been capable of efficient separations and sensitive detection of compounds in single animal or human cells, and numerous applications have been published in this area. Most of the pioneering work in this field was done by Ewing (1-3), Yeung (4-6), and Jorgen(7). Jorgenson also earlier demonstrated the suitability of open-tubular LC for single-cell analysis (7-9) Additional important approaches have been demonstrated byZare (10 1D Sweedler (12 13) and others (14-16) The cells investigated ranged from giant dopamine snail neurons to single human erythrocytes or individual vesicles. The sampling techniques depend on the accessibility of the particular cell. Cells located in a tissue have been dissected from the tissue, homogenized in a microvial, and separated by CE. Other methods are based on direct extraction of cytoplasmfromthe cell into the separation capillary or injection of the entire cell lysis in the capillary and then separation Detec-
Figure 1. Compartmentalization of a plant cell. 646 A
tion is accomplished by laser-induced fluorescence or amperometric detection. Several excellent reviews on single animal-cell analysis by CE are available (17,18). Compared with animal cells, not much attention has been paid to the analysis of single plant-cell vacuoles by CE. Several other analytical techniques have been used for this purpose, such as direct measurement of ions in single vacuoles by ion-selective microelectrodes (19), energy-dispersive Xray (EDX) analysis (20,21), and microfluorometric enzymatic assays (20,21) of extracted vacuolar sap. These methods, however have intrinsic drawbacks. Ion-selective microelectrodes are limited to determining a small number of inorganic ions and EDX can be used only for elemental analysis. Hence the corresponding chemical species or the soluble and insoluble parts cannot be distinoiiished which limits the tvne of information obtained For microsoectrofluorometric analysis enzymeassavs variety of compounds are available but simultaneous determination of analytes is not possiuie anu cross-sensiuviues can occur. Bec CFi h'trh re 1 ti enaration technique, it is not restricted in these ways, n - 4 . 1 .
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allowing the analysis of plant microsamples (22) and vacuo.ar samples (23,24) in the picohter range. Morphology of a plant cell
Figure 1 is a schematic of a plant celll which is morphologically divided into two major compartments—the cytoplasm, which contains the different organelles (chloroplasts, mitochondria, nucleus, etc.), and the vacuole. Both compartments are separated by the vacuolar membrane. The vacuole, which occupies 70-99% of the total cell volume, is by far the largest organelle of a plant cell. It osmotically regulates the pressure of the cell and stores a partly reversible pool of substances. Solutes and metabolites occur in very different concentrations in the two compartments. Compartmentalization of nutrients and other solutes between the vacuole and cytoplasm is an important part of the plant's response to changes in nutrient availability and its ability to withstand stresses (25). The composition of the cytoplasm is highly regulated, whereas the vacuole accumulates compounds presented to
Analytical Chemistry News & Features, October 1, 1998
Figure 2. Schematic of sample acquisition from a single plant-cell vacuole, creating subsamples, and internal standardization.
the plant in high concentrations. Consequently, toxic compounds that lead to enzyme dysfunctions are stored in the vacuole as well. Hence, the vacuole is the cell compartment with the highest ecophysiologic importance. Reaction of the cell to environmental factors often directly results in changing vacuolar concentrations. Taking vacuolar samples
Unlike animal cells, injecting and analyzing an entire plant cell is not possible because the cells are surrounded by a stable and more-or-lessrigidcell wall, which does not allow transfer into the capillary. The plantcell wall can be digested with enzymes such as pectinase (26), leaving the protoplast. Unfortunately, the important informa-
tion (where the particular cell was positioned within the ttssue) gets lost, and an osmotic medium is required to stabilize the isolated protoplasts, leading to totally different solute and metabolite compositions compared with in vivo conditions. Apart from that situation, the very different concentrations in the vacuole and cytoplasm (not even considering the other cell organelles) would again restrict the information obtained from analyses of protoplasts. Thus, analysis of the pure vacuolar sap is required rather than that of the entire cell. Acquiring vacuolar sap from individual plant cells is very different compared with the cytoplasmic injections from animal cells (23) because of the intrinsic osmotic pressure of plant cells, called turgor. The turgor pressure in a plant cell is of similar magnitude to the pressure in a car tire, in the range 105-106 Pa. Injuring the cell wall and membrane leads to an immediate collapse of cell turgor. Consequently, water will be drawn in osmotically from the surrounding tissue and all concentrations dramatically change. Thus, in vivo sampling of vacuolar sap is essential. Sampling can be performed in living tissue by directly inserting an oilfilled microcapillary into a aacuole ender r microscope. However, controlled extraction of a certain volume of vacuolar sap is not possible this way. As the microcapillary tip penetrates the vacuole, the turgor collapses and an unknown amount of vacuolar sap (between 10 and 100 pL, depending on the cell size and turgor) immediately enters the capillary tip. The capillary must then be rapidly removed from the cell to avoid dilution from water pouring into the cell If this sample is directly transferred into the CE capillary quantification of comis not possible because the sample volume is unknown Consequently, a procedure is needed to allow determination of the sample volume. Figure 2 shows a method of sample acquisition and treatment of vacuolar samples based on micromanipulation (23). The tip of an oil-filled microcapillary is inserted into a vacuole under a microscope, the turgor pressure drops, and vacuolar sap enters the capillary. The sample droplet (10100 pL) is ejected onto the bottom of a Petri dish filled with oil, where it is totally
surrounded by the oil and protected from the environment. A protective oil layer is an effective method for preventing evaporation of picoliter to femtoliter volumes in microtitrations (27-29) and in the analysis of single fog droplets (30,31). Then, a second microcapillary with a constricted inner diameter (constriction capillary) is used to create subsamples of identical volume (—10 pL) from the vacuolar sample. Volume measurement with constriction capillaries is very accurate and reliable (23). The identical subsamples are transferred into small water droplets in the nanoliter range. The same procedure is repeated with an internal standard using the same constriction capillary. Both water and internal standard droplets are prepared in the original Petri; thus all sample preparation steps 3.re done in the SJ1TTIP
dish der the protective oil layer The water droplets containing the identical volumes of the vacuolar subsample and the internal standard are subsequently injected by manipulating the anodic capil-
lary orifice toward the drop with a micromanipulator and sucking it in. Separation is performed, and the concentrations of compounds are calculated using the signal of the internal standard. As several subsamples are created from a vacuolar sample, subsequent analyses for different compounds are possible in the sap of an individual vacuole. Hence, many compounds can be determined in one vacuole. No contact with the ambient air takes place in the entire sample-handling procedure, including injection into the CE capillary. Determining solutes and metabolites
Using the injection technique described above, inorganic cations, anions, and carboxylic acids were measured with indirect UV detection. Figures 3(a) and 3(b) show electropherograms for the cation and anion determination in subsamples from the same vacuole of a wheat epidermal celll The corresponding vacuolar sample, —60 pL, was divided into subsamples of —10 pL.
Figure 3. CE of a wheat cell vacuole. (a) Inorganic cations: 1, cesium (internal standard); 2, ammonium; 3, potassium; 4, sodium; 5, calcium; 6, magnesium, (b) Inorganic anions: 1, bromide (internal standard); 2, chloride; 3, sulfate; 4, nitrate; 5, unknown; 6, malate. (c) Sugars: 1, sucrose; 2, glucose; 3, fructose, (d) Amino acids: 1, Gin; 2, Ser; 4, Ala; 5, Gly; 6, Val; 8, Met; 10, Leu; 11, Glu; 13, Asp; 3, 7, 9, and 12, unknown, (a), (b), and (d) are vacuolar subsamples of wheat epidermal cells. The same cell was used for cation and anion analyses. (c) is a vacuolar subsample of a wheat mesophyll cell. (Figures 3c and 3d adapted with permission from Ref. 23.) Analytical Chemistry News & Features, October 1, 1998 647 A
Report
Figure 4. Scanning electron micrograph of cell types in t h e upper epidermis of a cereal leaf. (Adapted w i t h permission from Ref. 23.)
Three subsamples were used for repeated injections into the cation and anion system, resulting in high reliability of the data. CsBr was used as the internal standard for cation and anion analyses. Analyzing sugars in the vacuole, mainly glucose, fructose, and sucrose, is possible without derivatization by dynamic labeling based on chelation of Cu(II) {32). This method provides separation of the electrophoretically migrating chelates and direct UV detection by way of an inner-ligand band arising from the bonds between Cu(II) and the hydroxy groups of the sugars. Figure 3(c) shows the determination of sugars in the vacuole of a wheat mesophyll cell. Sample acquisition is performed by inserting the sampling capillary through one of the stomata to reach the mesophvll tissue below the epidermis The constriction capillary used for the subsamples in Fioiires 3(a) and 3(b) was used for this analysw T h e internal standard
