Stomatal responses in isolated epidermis of the crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perr.

The optimal conditions for opening of stomata in detached epidermis of the Crassulacean Acid Metabolism (CAM) plant Kalanchoe daigremontiana were determined. Stomatal opening in CO₂–free air was unaffected by light so subsequently all epidermal strips were incubated in the dark and in CO₂–free air. Apertures were maximal after 3 h incubation and were significantly greater at 15° C than 25° C. Thus stomata in isolated epidermis of this species can respond directly to temperature. Stomatal opening was greatest when the incubating buffer contained 17.6 mol m^–3 K⁺, but decreased linearly with increasing K⁺ concentrations between 17.6 and 300 mol m^–3; the decrease in aperture was shown to be associated with increasing osmotic potentials of the solutions. Reasons for this behaviour, which differs from that of many C₃ and C₄ species, are discussed. Stomatal apertures declined linearly upon incubation of epidermis on buffer solutions containing between 10^–11 and 10^–5 mol m^–3 abscisic acid (ABA). Hence stomata on isolated epidermis of K. daigremontiana respond to lower concentrations of ABA than those of any species reported previously.     

The relationship between turgor pressure and titratable acidity in mesophyll cells of intact leaves of a Crassulacean-acid-metabolism plant,Kalanchoe daigremontiana Hamet et Perr

Day/night changes in turgor pressure (P) and titratable acidity content were investigated in the (Crassulacean-acid-metabolism (CAM) plant Kalanchoe daigremontiana. Measurements of P were made on individual mesophyll cells of intact attached leaves using the pressure-probe technique. Under conditions of high relative humidity, when transpiration rates were minimal, changes in P correlated well with changes in the level of titratable acidity. During the standard 12 h light/12 h dark cycle, maximum turgor pressure (0.15 MPa) occurred at the end of the dark period when the level of titratable acidity was highest (about 300 eq H⁺·g^-1 fresh weight). A close relationship between P and titratable acidity was also seen in leaves exposed to perturbations of the standard light/dark cycle. (The dark period was either prolonged, or else only CO₂-free air was supplied in this period). In plants deprived of irrigation for five weeks, diurnal changes in titratable acidity of the leaves were reduced (H=160 eq H⁺·g^-1 fresh weight) and P increased from essentially zero at the end of the light period to 0.02 MPa at the end of the dark period. Following more severe water stress (experiments were made on leaves which had been detached for five weeks), P was zero throughout day and night, yet small diurnal changes in titratable acidity were still measured. These findings are discussed in relation to a hypothesis by Lüttge et al. 1975 (Plant Physiol. 56,613-616) for the role of P in the regulation of acidification/de-acidification cycles of plants exhibiting CAM.Abbreviations CAM crassulacean acid metabolism - FW fresh weight - P turgor pressure     

Gradient in the degree of Crassulacean acid metabolism within leaves of Kalanchoe daigremontiana

Leaves of the Crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perr., about 3.3 mm thick, showed higher rates of net CO₂ exchange through the lower than through the upper surface during day and night, although the lower surface received only a small fraction of the light which was incident on the upper surface. Nocturnal acidification was more pronounced in cells from the lower than from the upper portion of leaves. The lower activity of the exposed side of these long-lived succulent leaves may be related to the potentially adverse effects of excessive light.Abbreviations CAM Crassulacean acid metabolism - PFD photon flux density (400–700 nm)     

Promotion of stomatal opening in the grass Anthephora pubescens nees by a range of natural and synthetic cytokinis

Stomatal opening in detached epidermis of Anthephora pubescens Nees incubated in the light and CO₂-free air was enhanced by each of 6 natural and 4 synthetic cytokinis. Apertures were maximal following incubation with 10 mmol m^-3 cytokinin in PIPES buffer for all except N⁶-[ ²-isopentyl] adenine and N⁶-[ ²-sopentyl] adenosine which both opened stomata maximally at 0.1 mmol m^-3. Experiments which were undertaken to optimise the conditions of incubation showed that opening was maximal after 3 h incubation and that while 10 mmol m^-3 kinetin increased the rate of stomatal opening, it did not affect the duration. Exogenous KCl was not needed for opening and light was saturating even at the low level of 140 mol m^-2 s^-1.     

Leaf anatomy and ultrastructure of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana

Light-microscopic analysis of leaf clearings of the obligate Crassulacean-acid-metabolism (CAM) species Kalanchoe daigremontiana Hamet et Perr. has shown the existence of unusual and highly irregular venation patterns. Fifth-order veins exhibit a three-dimensional random orientation with respect to the mesophyll. Minor veins were often observed crossing over or under each other and over and under major veins in the mesophyll. Paraffin sections of mature leaves show tannin cells scattered throughout the mesophyll rather evenly spaced, and a distinct layer of tannin cells below the abaxial epidermis. Scanning electron microscopy showed that bundle-sheath cells are distinct from the surrounding mesophyll in veins of all orders. Transmission electron microscopy demonstrated developing sieve-tube elements in expanded leaves. Cytosolic vesicles produced by dictyosomes undergo a diurnal variation in number and were often observed in association with the chloroplasts. These vesicles are an interesting feature of cell ultrastructure of CAM cells and may serve a regulatory role in the diurnal malic-acid fluctuations in this species.Abbreviations CAM Crassulacean acid metabolism - SEM scanning-electron microscopy - TEM transmission-electron microscopy     

Plasmalemma- and tonoplast-ATPase activity in mesophyll protoplasts,vacuoles and microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana

Adenosine-triphosphatase activity on the plasmalemma and tonoplast of isolated mesophyll protoplasts, isolated vacuoles and tonoplast-derived microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana Hamet et Perr., was localized by a cytochemical procedure using lead citrate. Enzyme activity was detected on the cytoplasmic surfaces of the plasmalemma and tonoplast. The identity of the enzymes was confirmed by various treatments differentiating the enzymes by their sensitivity to inhibitors of plasmalemma and tonoplast H⁺-ATPase. Isolated vacuoles and microsomes prepared from isolated vacuoles clearly exhibited single-sided deposition on membrane surfaces.Abbveviations CAM Crassulacean acid metabolism - H⁺-ATPase proton-translocating ATPase     

Development of the cuticular wax during growth of Kalanchoe daigremontiana (Hamet et Perr. de la Bathie) leaves

The goal of the present study was to monitor cuticular wax accumulation during leaf development of Kalanchoe daigremontiana . Leaves expanded linearly until they were 40–60 d old. Wax coverages of leaves on the third node increased steadily during initial leaf development, from 6.5  µ g·cm⁻² on day 22 to 15.3  µ g·cm⁻² on day 53, and then levelled off. Triterpenoids dominated the wax mixture throughout leaf development, but decreased from 74 to 40–45% in mature leaves, while very long-chain fatty acid (VLCFA) derivatives increased from 19 to 39–44%. The major VLCFA derivatives were alkanes, accompanied by fatty acids, primary alcohols, aldehydes and alkyl esters. In all compound classes, either C₃₄ or C₃₃ homologs predominated during leaf development. Eight different triterpenoids were identified, with glutinol constituting 70% of the fraction, and friedelin (20%) and germanicol (10%) as further major components of the young leaf wax. The glutinol percentage decreased, while the relative amounts of epifriedelanol and glutanol increased during development. Various leaf pairs upwards from the third node showed similar growth patterns and developmental time courses of cuticular wax amounts and composition. Based on these surface chemical analyses, the relative activities of biosynthetic pathways leading to various wax components can be assessed.     

Changes in oxidative properties of Kalanchoe blossfeldiana leaf mitochondria during development of Crassulacean acid metabolism

Kalanchoe blossfeldiana plants grown under long days (16 h light) exhibit a C₃-type photosynthetic metabolism. Switching to short days (9 h light) leads to a gradual development of Crassulacean acid metabolism (CAM). Under the latter conditions, dark CO₂ fixation produces large amounts of malate. During the first hours of the day, malate is rapidly decarboxylated into pyruvate through the action of a cytosolic NADP⁺-or a mitochondrial NAD⁺-dependent malic enzyme. Mitochondria were isolated from leaves of plants grown under long days or after treatment by an increasing number of short days. Tricarboxylic acid cycle intermediates as well as exogenous NADH and NADPH were readily oxidized by mitochondria isolated from the two types of plants. Glycine, known to be oxidized by C₃-plant mitochondria, was still oxidized after CAM establishment. The experiments showed a marked parallelism in the increase of CAM level and the increase in substrate-oxidation capacity of the isolated mitochondria, particularly the capacity to oxidize malate in the presence of cyanide. These simultaneous variations in CAM level and in mitochondrial properties indicate that the mitochondrial NAD⁺-malic enzyme could account at least for a part of the oxidation of malate. The studies of whole-leaf respiration establish that mitochondria are implicated in malate degradation in vivo. Moreover, an increase in cyanide resistance of the leaf respiration has been observed during the first daylight hours, when malate was oxidized to pyruvate by cytosolic and mitochondrial malic enzymes.Abbreviations CAM Crassulacean acid metabolism - MDH malate dehydrogenase - ME malic enzyme     

Malate metabolism in isolated epidermis of Commelina communis L. in relation to stomatal functioning

Epidermal strips with closed stomata were exposed to malic acid labelled with ¹⁴C either uniformly or in 4-C only. During incubation with [U-¹⁴C]malate, radioactivity appeared in products of the tricarboxylic-acid cycle and in transamination products within 10 min, in sugars after 2 h. Hardly any radioactivity was found in sugars if [4-¹⁴C]malate had been offered. This difference in the degree of labelling of sugars indicates that gluconeogenesis can occur in epidermal tissue, involving the decarboxylation of malate. Epidermis incubated with labelled malate was hydrolyzed after extraction with aqueous ethanol. The hydrolysate contained glucose as the only radioactive product, indicating that starch had been formed from malate. Microautoradiograms were black above stomatal complexes, showing that the latter were sites of starch formation. In order to follow the fate of malate during stomatal closure, malate was labelled in guard cells by exposing epidermes with open stomata to ¹⁴CO₂ and then initiating stomatal closure. Of the radioactive fixation products of CO₂ only malate was released into the water on which the epidermal samples floated; the epidermal strips retained some of the malate and all of its metabolites. In the case of rapid stomatal closure initiated by abscisic acid and completed within 5 min, 63% of the radioactivity was in the malate released, 22% in the malate retained, the remainder in aspartate, glutamate, and citrate. We conclude that during stomatal closing guard cells can dispose of malate by release, gluconeogenesis, and consumption in the tricarboxylic-acid cycle.Abbreviations ABA abscisic acid - NAD nicotinamide adenine dinucleotide - NADP nicotinamide adenine dinucleotide phosphate - PEP phosphoenolpyruvate     

Effects of auxins and cytokinins on epigenetic instability of callus-propagated Kalanchoe blossfeldiana Poelln.

A main problem in the vegetative propagation of ornamental plants in vitro is the epigenetic instability of cells removed from their organized environment. With calluses of leaf explants of Kalanchoe blossfeldiana Poelln., cv. Yucatan, the role of plant growth regulators (PGRs) in the occurrence of fasciation was studied.In various combinations of auxins and cytokinins, the auxin 2,4-D (2,4-dichlorophenoxyacetic acid) gave only deformed, inseparable shoot primordia. The most rapid callus induction with regeneration of well-developed sprouts was obtained with the natural IAA (indoleacetic acid) and Z (zeatin).As a first symptom of fasciation, aberration in decussate phyllotaxis can be observed. At increasing concentrations of IAA + Z, this symptom gradually decreased but fasciation proper increased. The optimum concentration was at 1 M for both PGRs. Reduction of exposure to the PGRs from six to three weeks reduced the epigenetic instability.     

Different endogenous cytokinins between male and female Mercurialis annua L.

The endogenous pool of cytokinin metabolites during sexual differentiation of Mercurialis annua L. was studied with a computerized gas-chromatography-mass spectrometry system. Certain metabolites were common to both sexes: ribosides (isopentenyl-adenosine, ribosylzeatin) and the nucleotide of I₆-Ade. Zeatin could be detected only in females while its nucleotide was present in males. The results were obtained with differentiating apices and whole plants. The high Z concentration and the low level of its nucleotide are related to the absence of two dominant complementary genes, determining maleness. Study of the regulation of cytokinin metabolism now seems possible.Abbreviations IPA isopentenyl adenosine - I₆-Ade isopentenyl adenine - Z zeatin - RZ ribosylzeatin     

Inhibition of stomatal opening during uptake of carbohydrates by guard cells in isolated epidermal tissues

The uptake of glucose and other carbohydrates into the guard cells of Commelina communis L. was found to inhibit the opening of the stomata. The concentration of glucose necessary to achieve about 50% inhibition was of the same order of magnitude as the potassium concentration required for opening; the uptake systems for potassium and glucose appear to be competitive and to exhibit the same degree of affinity. It is suggested that the uptake of glucose occurs via a proton cotransport, which, depolarizing the membrane potential, slows down the electrogenic import of potassium ions. The process of stomatal closure, in contrast, appears not to be affected by carbohydrate uptake. In guard cells of Tulipa gesneriana L. and Vicia faba L., which do not possess subsidiary cells, import of glucose or other carbohydrates did not interfere with the regulation of stomatal movements.     

Metabolic energy for stomatal opening. Roles of photophosphorylation and oxidative phosphorylation

The supply of energy for stomatal opening was investigated with epidermal peels of Commelina communis L. and Vicia faba L., under white, blue and red irradiation or in darkness. Fluencerate response curves of stomatal opening under blue and red light were consistent with the operation of two photosystems, one dependent on photosynthetic active radiation (PAR) and the other on blue light, in the guard cells. The PAR-dependent system was 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-sensitive and KCN-resistant and showed a relatively high threshold irradiance for its activation; its activity was most prominent at moderate to high irradiances. The blue-light-dependent photosystem was KCN-sensitive, was active at low irradiances, and interacted with the PAR-dependent photosystem at high blue irradiances. Stomatal opening in darkness, caused by CO₂-free air, fusicoccin or high KCl concentrations, was KCN-sensitive and DCMU-resistant. These data indicate that stomatal opening in darkness depends on oxidative phosphorylation for the supply of high-energy equivalents driving proton extrusion. Light-dependent stomatal opening appears to require photophosphorylation from guard-cell chloroplasts and the activation of the blue-light photosystem which could rely either on oxidative phosphorylation or a specific, membrane-bound electron-transport carrier.Abbreviations DCMU 3(3,4-dichlorophenyl)-1-1-dimethylurea - FC fusicoccin - KCN potassium cyanide - PAR photosynthetic active radiation - WL white light     

Changes in the isozymic pattern of phosphoenolpyruvate

Two major isofunctional forms of phosphoenolpyruvate carboxylase (EC 4.1.1.31) have been separated from the leaves of Kalanchoe blossfeldiana Poelln. Tom Thumb by acrylamide gel electrophoresis and diethylaminoethyl cellulose techniques: one of the forms prevails under long-day treatment (low crassulacean acid metabolism level), the other develops under short-day treatment (high Crassulacean acid metabolism level). Molecular weights are significantly different: 175·10³ and 186·10³, respectively. These results indicate that two populations of phosphoenolyruvate carboxylase are present in the plant, one of which is responsible for Crassulacean acid metabolism activity under the control of photoperiod.The Crassulacean acid metabolism appears to depend on the same endogenous clock that governs other photoperiodically controlled events (e.g. flowering). The metabolic and energetic significance of this feature is discussed. It is suggested that modification in isozymic composition could be an early step in the response to photoperiodism at the metabolic level.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - DEAE diethylaminoethyl - DTT dithiothreitol - LD long day - SD short day - BSA bovine serum albumin     

Daily rhythm of phosphoenolpyruvate carboxylase in Crassulacean acid metabolism plants

Immunotitration of phosphoenolpyruvate carboxylase (EC 4.1.1.31) extracted from leaves of Kalanchoe blossfeldiana v. Poelln. cv. Tom Thumb. It was established that at different times of the day-night cycle the daily rhythm of enzyme capacity does not result from a rhythm in protein synthesis, but rather from changes in the specific activity of the enzyme.Abbreviations CAM Crassulacean acid metabolism - IgG immunoglobulin G - PEP phosphoenolpyruvate To whom correspondence should be addressed     

Evidence for phytochrome involvement in light-mediated stomatal movement in Phaseolus vulgaris L.

Observations made with primary leaves of Phaseolus vulgaris L. demonstrated that phytochrome modulates light-induced stomatal movement. Removal of the far-red-absorbing form of the pigment (Pfr) with far-red (FR) radiation decreased the time required by the stomata to reach maximal opening following a dark-to-light transition; this effect of FR was fully reversible with red. Removal of Pfr with FR also decreased the time required to reach maximal closure following a light-to-dark transition, and the rate of closure was dependent on the final irradiation treatment before darkness. No evidence was found for phytochrome involvement in determining stomatal aperture under constant conditions of either darkness of light.Abbreviations and symbols Chl chlorophyll - D darkness - FR far-red - phytochrome photostationary state - Pfr, Pr FR- and R-absorbing forms of phytochrome, respectively - R red     

Inhibition of stomatal opening in sunflower leaves by carbon monoxide,and reversal of inhibition by light

When leaves of Helianthus annuus, whose stomates had been opened in the dark in the absence of CO₂, were exposed to 25% carbon monoxide (CO), stomatal conductivity for water vapor decreased from about 0.4 to 0.2 cm·s^-1. The CO effect on stomatal aperture required a CO/O₂ ratio of about 25. As this ratio was decreased the stomata opened, indicating that inhibitio of cytochrome-c oxidase by CO is competitive in respect to O₂. Photosynthetically active red light was unable to reverse CO-induced stomatal closure even at high irradiances, when CO₂ was absent. When it was present, stomatal opening was occasionally, but not consistently observed. Carbon monoxide did not inhibit photosynthetic carbon reduction in leaves of Helianthus.In contrast to red light, very weak blue light (405 nm) increased the stomatal aperture in the presence of CO. It also increased leaf ATP/ADP ratios which had been decreased in the presence of CO. The blue-light effect was not related to photosynthesis. Neither could it be explained by photodissociation of the cytochrome a ₃-CO complex which has an absorption maximum at 430 nm. The data indicate that ATP derived from mitochondrial oxidative phosphorylation provides energy for stomatal opening in sunflower leaves in the dark as well as in the light. Indirect transfer of ATP from chloroplasts to the cytosol via the triose phosphate/phosphoglycerate exchange which is mediated by the phosphate translocator of the chloroplast envelope can support stomatal opening only if metabolite concentrations are high enough for efficient shuttle transfer of ATP. Blue light causes stomatal opening in the presence of CO by stimulating ATP synthesis.     

Separation and purification of the tonoplast ATPase and pyrophosphatase from plants with constitutive and inducible Crassulacean acid metabolism

Tonoplast vesicles were isolated from Kalanchoe daigremontiana Hamet et Pierrer de la Bâthie and Mesembryanthemum crystallinum L., exhibiting constitutive and inducible crassulacean acid metabolism (CAM), respectively. Membrane-bound proteins were detergent-solubilized with 2% of Triton X-100. During CAM induction in M. crystallinum, ATPase activity increases four-fold, whereas pyrophosphatase activity decreases somewhat. With all plants, ATPase and pyrophosphatase could be separated by size-exclusion chromatography (SEC, Sephacryl S 400), and the ATPase was further purified by diethylaminoethyl-ion-exchange chromatography. Sodium-dodecyl-sulfate electrophoresis of the SEC fractions from K. daigremontiana containing maximum ATPase activity separates several protein bands, indicating subunits of 72, 56, 48, 42, 28, and 16 kDa. Purified ATPase from M. crystallinum in the C₃ and CAM states shows a somewhat different protein pattern. With M. crystallinum, an increase in ATP-hydrolysis and changes in the subunit composition of the native enzyme indicate that the change from the C₃ to the CAM state is accompanied by de-novo synthesis and by structural changes of the tonoplast ATPase.Abbreviations CAM Crassulacean acid metabolism - DTT dithiothreitol - kDa kilodalton - PAGE polyacrylamide gel electrophoresis - PPiase pyrophosphatase - SEC size exclusion chromatography - SDS sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Vacuolar solutes in the upper epidermis of barley leaves

The concentrations of vacuolar solutes in different cells of the upper epidermis of the third leaf of barley (Hordeum vulgare L.) were studied in leaves of different ages grown under different irradiances (120 or 400 mol photons·m^–2·s^–1). Vacuolar saps were extracted from individual cells located at various positions between adjacent veins and were analysed for their osmolality and the concentrations of K⁺, Ca²⁺, Cl^–, NO ₃ ^– and malate. Each ion showed a cell-specific distribution within the epidermis that was both quantitatively and qualitatively dependent on the leaf developmental stage and on the light level. During leaf ageing, Ca²⁺ accumulated preferentially in interstomatal cells (i.e. those located between longitudinally adjacent stomata) at concentrations up to 180 mM. Under low light conditions, this was accompanied by a more or less equal decrease in K⁺ concentration. Epidermal malate was found only in plants grown continuously or transiently under the high irradiance and reached highest concentrations in trough and interstomatal cells (60 to 150mM). Chloride concentration was highest in cells overlying the veins (designated as ridge cells) and lowest in cells located between the veins (trough cells), while NO ₃ ^– exhibited the reverse distribution, although the precise patterns were age-dependent. Epidermal osmolality increased with age, but the intercellular differences in the osmolalities were small compared to differences in vacuolar solute composition. A cell-to-cell analysis of the region surrounding the stomata showed that the steepest changes in the vacuolar solute composition of epidermal cells occurred at the boundary between ridge or trough cells and the adjacent near-stomatal cells.Abbreviations EDX analysis energy dispersive X-ray analysis We wish to thank Andrew Davies and Alison Bell (Bangor) for their technical advice. This work was financed as an Agricultural and Food Research Council Linked Research Group project between Bangor and Rothamsted (grants LR5/187 and 521).     

Vanadate inhibition of stomatal opening in epidermal peels of Commelina communis

An H⁺ ATPase at the plasma-membrane of guard cells is thought to establish an electrochemical gradient that drives K⁺ and Cl^– uptake, resulting in osmotic swelling of the guard cells and stomatal opening. There are, however, conflicting results regarding the effectiveness of the plasma-membrane H⁺-ATPase inhibitor, vanadate, in inhibiting both H⁺ extrusion from guard cells and stomatal opening. We found that 1 mM vanadate inhibited light-stimulated stomatal opening in epidermal peels of Commelina communis L. only at KCl concentrations lower than 50 mM. When impermeant n-methylglucamine and HCl (pH 7.2) were substituted for KCl, vanadate inhibition was still not observed at total salt concentrations50 mM. In contrast, in the absence of Cl^–, when V₂O₅ was used to buffer KOH, vanadate inhibition of stomatal opening occurred at K⁺ concentrations as high as 70 mM. Partial vanadate inhibition was observed in the presence of the impermeant anion, iminodiacetic acid (100 mM KHN(CH₂CO₂H)₂). These results indicate that high concentrations of permeant anions prevent vanadate uptake and consequently prevent its inhibitory effect. In support of this hypothesis, an inhibitor of anion uptake, anthracene-9-carboxylic acid, partially prevented vanadate inhibition of stomatal opening. Other anion-uptake inhibitors (1 mM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 1 mM 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid, 200 M Zn²⁺) were not effective. Decreased vanadate inhibition at high Cl^–/vanadate ratios may result from competition between vanadate and Cl^– for uptake. Unlike metabolic inhibitors, vanadate did not affect the extent of stomatal closure stimulated by darkness, further indicating that the observed action of vanadate represents a specific inhibition of the guard-cell H⁺ ATPase.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - FC fusicoccin - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid We thank Drs. R.T. Leonard (University of California, Riverside, USA) and K.A, Rubinson (Yellow Springs, Oh., USA) for helpful comments on the research, Janet Sherwood (Harvard University) for excellent plant care, and Angela Ciamarra, Anne Gershenson, Gustavo Lara (Harvard University) and Orit Tal (Hebrew University) for valuable technical assistance. This research was supported by a grant from the National Science Foundation (DCB-8904041) to S.M.A.