Analysis of Guard Cell Viability and Action in Senescing Leaves

Plant Physiol. (1985) 79, 7- 10 0032-0889/85/79/0007/04/$0 1.00/0

Analysis of Guard Cell Viability and Action in Senescing Leaves of Nicotiana glauca (Graham), Tree Tobacco' Received for publication January 25, 1985 and in revised form April 22, 1985

RICHARD OZUNA, RAMON YERA, KIM ORTEGA, AND GARY TALLMAN* Natural Science Division, Pepperdine University, Malibu, California 90265

ABSTRACT not exhibit signs of functionality (24). As a result, Zeiger and Downloaded from https://academic.oup.com/plphys/article/79/1/7/6081518 by guest on 02 October 2021 Schwartz (24) have suggested that yellowing leaves retain sto- In an attempt to determine whether low epidermal conductances to matal control throughout the senescence process. water vapor diffusion of senescing leaves were caused by internal changes These studies raise the question of how stomatal movements in guard cells or by factors external to guard cells, stomatal behavior was are controlled in senescing leaves. Stomates of senescing leaves examined in intact senescing and nonsenescing leaves ofNicotiana glauca may fail to open fully because properties of guard cells that (Graham), tree tobacco, grown in the field or in an environmental cham- facilitate turgor production are damaged or modified. Alterna- ber. Conductances of senescing leaves were 5 to 10% of the maximum tively, stomatal movements in senescing leaves might be con- conductances of nonsenescing leaves of the same plant, yet guard cell trolled solely by factors external to guard cells. duplexes isolated from epidermal peels of senescing leaves developed full In this paper, we examine the diurnal patterns oftranspiration turgor in the light in solutions containing KCI, and sodium cobaltinitrite and epidermal conductance, and the water relations of senescing staining showed that K+ accumulated as turgor developed. Ninety-five and nonsenescing leaves ofNicotiana glauca, tree tobacco, grow- per cent of the guard cells isolated from senescing leaves concentrated ing in the field. We show that even though stomates of intact neutral red and excluded trypan blue. Intercellular leaf CO2 concentra- senescing leaves do not respond to changing environmental tions of senescing and nonsenescing leaves of chamber-grown plants were conditions, guard cells isolated from all other tissues and cells of not significantly different (about 240 microliters per liter), but the potas- senescing leaves are capable of full turgor production. We con- sium contents ofadaxial and abaxial epidermes of senescing leaves taken clude that the reduced epidermal conductances of senescing from plants grown in the field were less than half those of nonsenescing leaves are not produced by impaired processes within guard cells, leaves. We conclude that guard cells do not undergo the orderly senes- but by factors external to them. cence process that characteristically takes place in mesophyll tissue during whole-leaf senescence and that the reduced conductances of senescing leaves are produced by factors external to guard cells. MATERIALS AND METHODS Plant Material. A single plant of Nicotiana glauca (Graham), tree tobacco, was used for measurements of diurnal epidermal conductance to water vapor diffusion, pressure-volume curve analysis, measurements of leaf water potential, and epidermal peel and impression analyses. The plant was approximately 3 m high and was growing on a western slope on the campus of Leaf senescence may occur as a genetically determined stage Pepperdine University, Malibu, CA. Samples for K+ analysis of plant development, or it may be induced by a variety of were collected at random from plants at two roadside locations environmental conditions including drought, photoperiod, tem- 6 and 9 km east ofthe campus along Malibu Canyon Road. The perature, or shading. Regardless of how leaf senescence is in- soil in which all plants were growing was highly compacted, duced, it is characterized by the orderly, progressive disassembly fragmented sandstone. of mesophyll tissue. In the process, leaf proteins and Chl are Plants for gas exchange analysis were grown from seed col- catabolized, and the released nitrogen and other inorganic ele- lected from the plant growing on the campus. Plants were ger- ments are mobilized to juvenile tissues of the plant before leaf minated and maintained in sterilized Uni-Gro potting soil (L &L death and abscission (17). Nursery Supply, Inc., Chino, CA). Following germination, plants Stomates of senescing leaves generally open significantly less were transferred to 8-L clay pots and were watered daily with than those of nonsenescing leaves (4, 11, 13, 20), but there is no modified Hoagland nutrient solution. Distilled H20 was used in evidence that guard cells ever undergo an orderly senescence place of Hoagland solution for every third watering in order to process like that carried out in mesophyll cells. Guard cells of purge the soil of salts. Plants were maintained in a 1.8 x 1.2 x senescing leaves of Vicia faba are capable of responding to 0.8 m growth chamber (Scientific Systems, New Orleans, LA) changes in CO2 concentration and to kinetin (21). Guard cells with a 12-h photoperiod, 25°C day, 20°C night air temperatures, in epidermal peels from senescing leaves of Gingko biloba (24) and 18.7°C day, 12.7°C night dewpoint temperatures. During the and V. faba (21) are capable of developing a certain amount of light period, the concentration of CO2 in the chamber was turgor, but not full turgor, when illuminated in solutions con- maintained at 340 ± 2 AI/L using and ADC Series 225 differential taining KCI. Chloroplasts in guard cells of senescing leaves of G. IR gas analyzer (Analytical Development Co., Hoddesdon, Eng- biloba are green and show typical fluorescence transitions asso- land) connected to an electronic injection valve (Leeds and ciated with electron transport and photophosphorylation at a Northrup, North Wales, PA) that controlled the release of com- time when chloroplasts in mesophyll cells of the same leaf do pressed CO2 from a cylinder. The chamber was equipped with six 400-w mercury multivapor metal halide lamps and six 400- ' Supported by a grant from the John Stauffer Charitable Trust and w high pressure sodium lamps (General Electric, Co., Cleveland, by Pepperdine University. OH). The photosynthetic photon flux density averaged 950 7 8 OZUNA ET AL. Plant Physiol. Vol. 79, 1985 ,umol/m2. s measured on a horizontal surface near the upper Estimation of Net Photosynthesis, Total Leaf Conductance to portion of the canopy with a Li-Cor quantum sensor (Li-Cor, Water Vapor Diffusion, and Concentration of CO2 in the Inter- Inc., Lincoln, NB). cellular Spaces of Senescing and Nonsenescing Leaves. Net Diurnal Measurements of Transpiration and Epidermal Con- photosynthesis, total leaf conductance to water vapor diffusion, ductance to Water Vapor Diffusion. Transpiration, epidermal and intercellular concentrations of CO2 were measured for five conductance to water vapor diffusion, vapor pressure deficits, senescing and five nonsenescing leaves at insertions one through leaftemperatures, and photosynthetic photon flux densities were five from the bottom of chamber-grown plants. Measurements measured for both adaxial and abaxial surfaces ofseven senescing of water vapor and CO2 fluxes were performed simultaneously and seven nonsenescing leaves of the field plant using a Li-Cor with a miniature, clamp-on assimilation chamber connected to 1600 porometer. Leaves were considered to be senescing if they an EG&G Cambridge model 911 dewpoint analyzer (EG&G, showed marked chlorosis compared to nonsenescing green Waltham, MA) and an ADC series 225 IR CO2 gas analyzer. leaves. The degree of chlorosis of senescing leaves was estimated The assimilation chamber and methods ofdata calculation have visually and varied from 65 to 90% of the total leaf surface area. been described (3, 4). Total leaf conductances to water vapor Significant decreases in Chl content from morning to late evening diffusion were defined as the sum of the reciprocal values of the

were clearly observed for some leaves. Selected leaves were of stomatal and cuticular resistances to water vapor for the two Downloaded from https://academic.oup.com/plphys/article/79/1/7/6081518 by guest on 02 October 2021 consistent size (8-10 x 10-12 cm) and free of canopy counter- epidermes added in parallel. Boundary layer resistances were shading. Measurements were taken at 1- and 2-h intervals be- calculated from measurements on moistened Whatman No. 2 tween 0400 and 2000 h on July 13, 1983. hardened filter paper clamped in the cup and subjected to a Measurements of Bulk Leaf Tissue Water Potential and Pres- variety of flow rates. Flow rates were 350 ml/min. For measure- sure-Volume Curve Analysis. Bulk leaf water potentials were ments, the assimilation chamber was clamped to each leaf for 3 measured every 2 h with a Scholander pressure chamber (16) on min in the light and 4 min in the dark. Conductance measure- three senescing and three nonsenescing leaves while diurnal ments taken with the porometer after the assimilation cup had conductance measurements were in progress. At the end of the been removed from the leaf confirmed that no changes in epi- day, stems with senescing and nonsenescing leaves were cut dermal conductance occurred in that time. under water, covered with a plastic bag, and stored overnight in Estimation of Potassium Contents of Senescing and Nonse- the dark. Three senescing and three nonsenescing leaves were nescing Leaf Tissues. Whole leaf tissue, and adaxial or abaxial subjected to pressure-volume curve analysis. Data were analyzed epidermes of senescing and nonsenescing leaves collected from by the method of Wilson et al. (22) in order to determine the plants growing in the field were dried, weighed, ashed at 500°C osmotic potentials and turgor loss points of the bulk leaf tissue overnight, and analyzed for K+ content. Sixteen samples ofeach of samples of each leaf type taken at the end of the day. We did type of tissue from both leaf types, each representing pooled not attempt to account for diurnal adjustments in the turgor loss material from several leaves, were prepared for atomic absorption points of the two leaf types, but such adjustments have been analysis by the method of Isaac and Johnson (8) and analyzed shown to be small (0.1-0.2 MPa) (19). using a Perkin-Elmer model 370A atomic absorption spectro- Estimation of Stomatal Densities. Stomatal densities ofadaxial photometer. and abaxial surfaces of leaves used for conductance measurements were estimated using a modification of the method of RESULTS Brown and Rosenberg (2). Leaves were sprayed with Krylon Crystal Clear 1301 acrylic spray coating (Borden, Inc., Colum- Results of diurnal measurements of epidermal conductances bus, OH), and impressions were lifted from leaf surfaces with to water vapor diffusion, transpiration, bulk leafwater potentials, Highland brand transparent tape No. 5910 (3M Co., Minneap- leaftemperatures, and vapor pressure deficits for seven senescing olis, MN). Six estimates from the adaxial surface and six esti- and seven nonsenescing leaves ofa Nicotiana glauca plant grow- mates from the abaxial impressions of the seven senescing and ing in the field are summarized in Figure 1. Epidermal conduct- seven nonsenescing leaves were counted microscopically at x ances ofnonsenescing leaves increased during the morning hours 100 using an eyepiece reticle containing 100 small squares and and reached a maximum value at 1100 (Fig. IA). As leaf tem- representing 1 mm2 ofthe surface of the leaf. peratures and vapor pressure deficits increased over the course Analysis of Viability and Turgor-Producing Capacity of Iso- of the afternoon (Fig. 1C), epidermal conductances of nonse- lated Guard Cell Duplexes. Methods for isolation of guard cell nescing leaves decreased (Fig. 1A) and leaf water potentials duplexes and analysis of guard cell viability and turgor buildup approached the turgor loss point of the bulk leaf tissue, deter- have been described (6). Briefly, epidermal peels from adaxial or mined graphically from pressure-volume curves to be -1.05 ± abaxial surfaces of senescing or nonsenescing leaves of plants 0.02 MPa (SE, n = 3). Epidermal conductances of senescing growing in the field were treated with cellulolytic enzymes so leaves ranged in value between 5 and 10% of the maximum that guard cell duplexes (defined as two guard cells joined at the epidermal conductances of the corresponding surfaces of nonse- ends) were the only remaining cellular structures. Guard cells nescing leaves (Fig. IA) during daylight hours. Because the plant were tested for viability with neutral red (9) and trypan blue (14). was located at the base of a western slope, peak light levels were Only leaves that were completely yellow to the eye were consid- not reached until 1500 h (data not shown). Transpiration from ered -senescent. Absorbances of 80% acetone extracts of such adaxial surfaces of nonsenescing leaves was lower than from leaves were typically less than 15% of those of nonsenescing abaxial surfaces, but both peaked at 1500 h (Fig. IB), coincident leaves when measured at 660 nm. To assess the ability ofisolated with highest light levels, highest leaf temperatures, and vapor guard cell duplexes to generate turgor, cleaned peels were sub- pressure deficits (Fig. IC), and lowest leaf water potentials (Fig. jected to light and dark treatments for 2 h in a solution of salts 1A). Transpiration from adaxial and abaxial surfaces ofsenescing of Murashige and Skoog medium (12) modified by replacing K+ leaves was considerably lower than from the corresponding sur- salts with Na+ salts followed by the addition of KCI to produce faces of nonsenescing leaves (Fig. 1B), even though leaf temper- concentrations ranging from 0 to 50 mM. At the end of the atures and vapor pressure deficits ofsenescing and nonsenescing incubation period, apertures of isolated guard cell duplexes were leaves were nearly identical (Fig. IC). measured as described previously (6). All experiments were per- The turgor loss point of senescing leaves was determined to be formed at 23°C in room air. In some experiments, strips were -0.93 ± 0.03 MPa (SE, n = 3). The osmotic potentials of the stained for potassium content with sodium cobaltinitrite stain bulk leaf tissue at saturation were calculated by extrapolation (1). using linear regression of points along a pressure-volume curve STOMATAL ACTION IN SENESCING LEAVES 9

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0 - I- 0 0 wcoE I to 0 Ix10. 0 10 20 30 40 50 0 KCI (mM) -J FIG. 2. Changes in apertures ofadaxial (A) and abaxial (B) guard cell duplexes isolated from senescing (---) and nonsenescing ( ) leaves of N. glauca (Graham), tree tobacco, and incubated in various concen- Hour of Day (PST) trations of KCl in light (0) and in darkness (0). Each value is the mean FIG. 1. Diurnal patterns of (A) epidermal conductances to water of 120 measurements, 20 on two cleaned epidermal peels from each of vapor diffusion and bulk leaf water potentials, (B) transpiration, and (C) three separate leaves. One SE is contained within the dimensions of each vapor pressure deficits and temperatures of seven senescing (---) and symbol. seven nonsenescing ( ) leaves of N. glauca (Graham), tree tobacco, growing in the field and measured on July 13, 1983. Adaxial (0) and Table I. Net Photosynthesis, Total LeafConductance to Water Vapor abaxial (0, *) leaf surfaces are reported separately. The turgor loss point Diffusion, and Intercellular LeafCO2 Concentrations ofFive Senescing (tlp) of nonsenescing leaves at saturation was determined graphically and Five Nonsenescing Leaves ofNicotiana glauca Grown in an from pressure-volume curves. Values are means and 1 SE. If error bars Environmental Chamber are lacking, I SE is within the dimensions of the symbol. Net Total Leaf Intercellular Conductance to Concentration Sample Photosynthesisnet Water Vapor of to be -0.73 ± 0.01 MPa (SE, n = 3) for senescing leaves and C02 -0.88 ± 0.04 MPa (SE, n = 3) for nonsenescing leaves. Osmotic tUmol/m2.S mmol/m2.S til/L potentials of the two leaf types were significantly different when tested with a t-ratio test (t = 5.53, P < 0.01). Nonsenescing 11.3 (1.1) 784.2 (91.3) 239.2 (5.7) Stomatal densities (per mm2) were 123 ± 1.4 (SE, n = 42) and Senescing 0.4 (0.3)a 7.7 (1.5)' 214.8 (52.1) 113 ± 1.5 (SE, n = 42) for the abaxial and adaxial surfaces of a Significant at 0.05 level when compared to nonsenescing by t ratio. senescing leaves while the corresponding surfaces of nonsenescing leaves contained 120 ± 1.2 (SE, n = 42) and 112 ± 1.5 (SE, n plexes from senescing leaves reached maximum apertures in 10 = 42) stomates. mM KCI in the light while those from nonsenescing leaves There were no apparent differences between cleaned epidermal reached their maximum apertures at a concentration of 30 mM peels of senescing and nonsenescing leaves when examined by (Fig. 2B). The differences in the responses of duplexes isolated light microscopy. Guard cell chloroplasts in cleaned peels from from senescing leaves and nonsenescing leaves may have been senescing leaves were green and appeared intact. Ninety to 95% caused by differences in the permeabilities of membranes of the of guard cells isolated from both leaf types concentrated neutral guard cells of the two types of leaves to ions, but we did not red and excluded trypan blue. Duplexes from both adaxial and attempt to test this hypothesis. abaxial surfaces of senescing and nonsenescing leaves became Results ofgas exchange analyses for senescing and nonsenesc- turgid upon illumination in solutions containing KCI. In all cases ing leaves are summarized in Table I. Total leafconductances to examined, guard cells accumulated K+ as turgor developed, as water vapor diffusion and net rates ofphotosynthesis ofsenescing judged by sodium cobaltinitrite staining. Photographs of du- leaves were 1 to 3% those of nonsenescing leaves. In some plexes given light and dark treatments and stained for K+ have senescing leaves, Chl degradation was uniform and evenly dis- been published (6). Light and dark KCI response curves were tributed, while in others only a localized region of the leaf was virtually identical for cleaned strips from the adaxial surfaces of chlorotic. Regardless of the distribution pattern, leaf conduct- both types of leaves (Fig. 2A), except that dark responses were ances were reduced in all areas of the epidermis of senescing greater in 40 and 50 mm KCI for senescing than nonsenescing leaves, including those over green mesophyll. The concentrations leaf duplexes. The responses of abaxial duplexes from both leaf of CO2 in the intercellular spaces of the two leaf types were not types were similar but not identical (Fig. 1B). Maximum aper- significantly different. tures achieved by duplexes isolated from abaxial surfaces of Results of K+ analyses of whole leaf tissue, adaxial epidermes, senescing leaves and given light treatment were about 90% of and abaxial epidermes of senescing and nonsenescing leaves are those isolated from nonsenescing leaves. However, abaxial du- shown in Table II. The K+ content of whole leaf tissue of 10 OZUNA ET AL. Plant Physiol. Vol. 79, 1985 Table II. Potassium Contents of Whole LeafTissue, Adaxial it clear whether guard cells of senescing leaves have exactly the Epidermis, and Abaxial Epidermis ofSenescing and Nonsenescing same metabolic and physiological properties as those of nonse- Leaves ofN. glauca nescing leaves. To our knowledge, no studies have been published Values are means and SE calculated from 16 measurements of each which separate the effects ofinternal metabolic and physiological treatment. changes that accompany guard cell aging from the effects of factors that develop external to guard cells and affect guard cell Potassium Sample action as leaves approach senescence. It is clear from our studies Nonsenescing Senescing and others (4) that gas exchange is regulated in both types of leaves so that their internal leafCO2 concentrations are the same. mg/g dry wt Whether gas exchange is regulated in senescing leaves for the Whole leaf 12.23 (1.04) 9.06 (0.94)a same reasons that it is carefully controlled in nonsenescing leaves Adaxial epidermis 16.05 (1.60) 7.41 (0.48)a remains to be determined (5, 15). Abaxial epidermis 14.75 (1.34) 6.75 (0.80)a Acknowledgments-We thank P. Hoekstra, C. Paravanno, E. Gosselin, K. a Significant at 0.05 level when compared to nonsenescing by t ratio. Perrin, M. Moyher, P. Weldon, and P. Sachs for their technical assistance and editorial enhancement of the manuscript. We also thank Stephen Davis and senescing leaves was approximately 75% that of nonsenescing Eduardo Zeiger for their helpful discussions and comments. Downloaded from https://academic.oup.com/plphys/article/79/1/7/6081518 by guest on 02 October 2021 leaves. The K+ contents ofepidermes from senescing leaves were slightly less than half those from senescing leaves. LITERATURE CITED 1. ALLAWAY WG, TC HSAIO 1973 Preparation of rolled epidermis of Viciafaba DISCUSSION L. so that stomata are the only viable cells: analysis of guard cell potassium content by flame photometry. Aust J Biol Sci 26: 309-318 The stomates of senescing leaves of plants growing in the field 2. BROWN KW, NJ ROSENBERG 1970 Influence of leaf age, illumination, and in upper and lower surface differences on stomatal resistance of sugar beet do not open response to light; those of nonsenescing leaves do (Beta vulgaris) leaves. Agron J 62: 20-24 (Fig. 1). Yet guard cell duplexes isolated from senescing leaves 3. DAVIS SD 1974 Effect of leafage on stomatal resistance and its significance for ofplants growing in the field respond to light by increasing pore carbon dioxide assimilation in bush bean (Phaseolus vulgaris L.). PhD thesis. apertures to values equal to those measured in duplexes isolated Texas A & M University, College Station 4. DAVIS SD, KJ MCCREE 1978 Photosynthetic rate and diffusion conductance from nonsenescing leaves. We conclude that the low epidermal as a function ofage in leaves of the bush bean (Phaseolus vulgaris L.). Crop conductances observed in senescing leaves of plants growing in Sci 18: 280-282 the field are produced by factors external to guard cells them- 5. FIELD C, HA MOONEY 1983 Allocating leaf nitrogen for maximization of selves. carbon gain: leaf age as a control on the allocation problem. Oecologia 56: 341-347 The results of our gas exchange analysis for senescing and 6. HUDSON S, J TRAIL, D SIMMONS, L BALDWIN, R MYERS, B TOM, J MOHR, G nonsenescing leaves are consistent with the hypotheses that epi- TALLMAN 1983 Preparation of enzymatically cleaned leaf epidermal strips dermal conductance and photosynthesis are either coupled or ofNicotiana glauca. Plant Sci Lett 32: 1-8 7. HUMBLE GD, K RASCHKE 1971 Stomatal opening quantitatively related to act in concert in both senescing and nonsenescing leaves; hence, potassium transport: evidence from electron microprobe analysis. Plant the high correlation between assimilation rates and total leaf Physiol 48: 447-453 conductances (Table I). Our data also support the hypotheses of 8. ISAAC RA, WC JOHNSON 1975 Collaborative study of wet and dry techniques Davis and McCree (4) and Wong et al. (23) that throughout the for the elemental analysis of plant tissue by atomic absorption spectropho- tometry. J Assoc Off Agr Chem 58: 436-440 entire process of leaf aging, stomates respond in such a way that 9. JOHANSEN DA 1940 Plant Microtechnique, Ed 1. McGraw Hill, New York intercellular leaf CO2 concentrations remain constant. We find 10. 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