Plant Physiol. (1971) 47, 361-365

Method for Determining Solutes in the Cell Walls of LeavesReceived for publication July 7, 1970

LEON BERNSTEINUnited States Salinity Laboratory, Soil and Water Conservation Research Division, Agriclultural ResearchService, United States Department of Agriculture, Riverside, California 92502

ABSTRACT

A perfusion method is described whereby large discs ofamphistomatous leaves are vacuum-perfused with water sothat either successive fractions of perfusate may be analyzedfor solutes or the infused water may be displaced and collectedafter equilibration with the leaf cells. With castor bean leaves,estimates of electrolyte concentration in cell wall water by thetwo methods were similar. Total electrolytes in leaf cell wallwater of castor beans (Ricinus communis), sunflower (Hel-ianthus annuus), and cabbage (Brassica oleracea capitata)from nonsaline cultures were about 2, 2, and 10 milliequiva-lents per liter, respectively, increasing to 4, 10, and 30 milli-equivalents per liter under saline conditions. Electrolytes re-covered in successive fractions were similar in composition,and continuous perfusion resulted in a steady release ofsolutes, the concentration in the perfusate varying inverselywith the perfusion rate. Diffusional release of solutes fromcells was less than expected at low perfusion rates, suggestingthat solute reabsorption may increase as solute concentrationin the perfusate increases with decreased perfusion rates. Per-fusate concentration and composition were essentially unaf-fected by temperature (2 and 23 C) or by perfusing with 0.5mM CaSO4 rather than with water. Electrolytes in perfusateson an equivalent basis were Ca2", 30%; Mg2+, 10%; and Na++ K+, 60%, the proportions of sodium increasing from 10 to50% in leaves (cabbage) that accumulated sodium undersaline conditions. Salinity (added NaCl) of the root culturemedium caused a 3- to 5-fold increase in total cell wall elec-trolyte concentration, but this amounted to an increase fromless than 1 or a few per cent to no more than 7% (in cabbage)of the cell sap electrolyte concentrations. Solutes in the cellwall appear to be in dynamic equilibrium with intracellularsolutes.

In a tissue at water equilibrium, the water potential is thesame at all points and there is no net movement of waterfrom any point to another. The components of water potential,however, may vary. The intracellular pressure component(turgor) is absent at air interfaces of cells, and the negativepotential components (solute plus matric) will, therefore, havea smaller absolute magnitude than inside the turgid cell. Aver-age values for the potential components (12) for the regions ofeven a single cell are meaningless in view of the wide latitudethat is possible. For any given region of the cell, the compo-nents of water potential must be specifically determined. This isvery much the case for the cell walls of leaves and other aerialorgans whose total water potential is directly measured when-ever the water potential of the tissue is studied but whose water

potential components, matric and solute, are completely un-known. Data on the relationship of matric potential to watercontent of killed plant tissues (3, 11) are not helpful in de-termining the matric potential in cell walls. Even if the watercontent determination were specific for the cell wall, the watercontent of the cell wall cannot be inferred from water contentof intact tissues, so the matric potential of the cell wall re-mains unknown even when total water potential and tissuewater content are determined. A method is therefore neededfor the direct determination of solute potential in the cellwalls of plant tissues.

Unlike aquatic plants and plant roots for which the normalmedium is, or contains, liquid water, there is no direct aqueouscontact or water bridge between the cell walls of leaves and theexternal environment. Even when the surface of the leaf iswetted, the cuticle and intercellular air space interrupt all butthe most tenuous contact between the surface water and thecell wall surfaces of the leaf interior. Special techniques arerequired to make interior cell wall surfaces accessible for ex-traction and determination of cell wall solutes. Perfusion ofamphistomatous leaves offered promise of increasing the ac-cessibility of leaf cell walls, and a method has been developedthat permits determination of solutes in cell walls of leaves (2).Briefly, the method consists of applying vacuum to one surfaceof the leaf disc, bringing water in through the open stomata ofthe opposite epidermis and out through the stomata of the epi-dermis under vacuum. By collecting successive fractions ofperfusate, it was thought that excess solute in the first fractioncompared to later fractions could serve as the basis for cal-culating the solutes originally present in the cell walls. Then bydetermining the weight of cell walls in the leaf disc and thevolume of water they retained at the given water potential, theconcentration of solutes in the cell walls could be calculated.When leaf cells were found to be leakier than anticipated, anequilibrium technique was developed whereby water infusedinto the leaf was allowed to equilibrate with the leaf cells be-fore it was displaced and its solute content determined.

MATERIALS AND METHODS

Plant Culture. Leaves for these experiments were taken fromplants grown singly in 18-liter water cultures in the greenhousewith weekly renewal of culture solutions and maintenance ofpH between 5 and 6. The medium was modified half-strengthHoagland's solution ([1] but with KHYP04 concentration re-duced to 0.16 mm and B and Mn each to 0.25 mg/liter) plus 1meq/liter added NaCl for the control culture or 24-meq/literincrements of NaCl to give salinity increments of -1.1 barsosmotic potential at 25 C to a maximal salinity of -4.4 bars.Cultures were brought to the desired salinity when plants werein the seedling stage and plants were grown at least a monthat the different salinities before fully expanded nonsenescentleaves were sampled.

Leaf Perfusion. To perfuse a leaf and collect perfusate, leaf361

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Plant Physiol. Vol. 47, 1971

/Fluoroscent desk lightc. 500 ft c. at leaf

surface

to Vacuum filtratormonometer

to needle valve---]controlled oirinlet and vocuum pump

Apparatus for collecting successivefractions of leaf perfusate

FIG. 1. Diagram of leaf perfusion apparatus and detail forsealing leaf to filter plate and for preventing water leaks aroundthe leaf disc by means of a lanolin barrier.

discs 97 mm in diameter were cut out of the distal parts oflarge cabbage (Brassica oleracea capitata), castor bean (Ricinuscommunis), or sunflower (Helianthus annuus) leaves. The leafdiscs were rinsed in distilled water, blotted dry, and mountedon a radially grooved brass or plastic ifiter plate (Fig. 1). Toeffect an air-tight seal of the leaf disc to the filter plate, a 2-to 3-mm wide continuous bead of freshly mixed rubber cement(G. E. silicone rubber cement, RTV 11, plus Nuocure 28 cata-lyste: 2 drops catalyst per 3 g of rubber cement; G. E. SiliconeRubber Products, Waterford, N. Y.') was applied along theadaxial margin of the leaf disc which was then centered andpressed adaxial side down on the filter plate. For continuousperfusion, a circle of 100-mesh Monel screen, 79 mm in diam-eter, was placed between the leaf disc and the filter plate toassure uniform pressure differential and flow. After about aminute, more freshly mixed rubber cement was applied aroundthe margin of the leaf disc to cement it securely to the filterplate. A shellacked iron ring, 79 mm in internal diameter, was

centered over the leaf disc and sealed to it with lanolin to serve

as a water reservoir (Fig. 1). Great care is necessary to assurea complete lanolin barrier between the cement at the leaf marginand the water in the reservoir ring (inset, Fig. 1). Water incontact with the rubber cement may leak under the cement andaround the edge of the disc, and such leaks are difficult to de-tect or stop. Water to a depth of about 1 cm was added insidethe ring reservoir, and the leaf disc was illuminated for 10 minwith white fluorescent light of about 500 ft-c at the leaf surfaceto assure open stomata. Vacuum was then applied equivalentto a pressure difference across the leaf disc of 200 to 500 mm

' Prolonged use and exposure of the catalyst to light renders itphytotoxic. No difficulty is experienced with the catalyst properlystored in brown glass.

I Trade names and company names are included for the benefitof the reader and do not imply any endorsement or preferentialtreatment of the product listed by the United States Department ofAgriculture.

of mercury as measured by a single arm manometer. Completeperfusion of the leaf disc occurred almost instantly or at leastwithin a minute if the stomata were open. With sunflower,perfusion was characteristically spotty, indicating a pattern ofclosed and open stomata. If vacuum was applied continuously,perfusate was collected more or less rapidly depending uponrate of perfusion which was controlled by varying the vacuumapplied by means of the needle valve bypass (Fig. 1). Per-fusate was collected without interruption in a series of tubesby rotating the filter disc to move the ifiter stem from tube totube. Four successive fractions were generally collected in1.5-ml tubes (Fig. 1). Alternatively, the vacuum was interruptedimmediately after infusion for any desired interval to permitthe infused water to equilibrate with the tissue. Reapplicationof vacuum then resulted in displacement of the infused water,the electrical conductivity of which was monitored by amicroconductivity cell (model G5Y 116) attached to the stemof the filter. The conductivity cell leads passed through therubber stopper to a conductivity bridge model RC16B2 (celland bridge from Beckman Instruments Co., Cedar Grove,N. J.). The equilibrated perfusate was also collected for analy-sis of total and specific solutes. Monitoring the EC permittedthe determination of the maximal electrolyte concentration inthe perfusate and also the separation of the equilibrated per-fusate from water that usually collected between the leaf discand the filter plate while vacuum was discontinued duringequilibration. Plastic filter plates and stem assemblies wereused when discs were equilibrated. For immediate and con-tinuous collection of perfusate, the brass disc and copper tubeassembly in Figure 1 were used with the Monel screen betweenthe leaf disc and the ifiter plate.

Cell Wall Determinations. The cell walls in the leaf discswere estimated by homogenization, centrifugation, and weigh-ing. Starch-containing leaf discs, such as castor bean, werestored in dark, moist chambers at 25 C for 1 or 2 days untilcomparable material similarly stored gave negative tests forstarch except in major veins. (Cabbage leaves contain nostarch except for minute grains in guard cells.) Major veins(first and second order veins in castor bean leaves) wereexcised and discarded, and the remainder of the disc was cutinto small pieces, wrapped in aluminum foil, and stored at -10C until analyzed. The sample (0.8-0.9 g fresh weight forcastor bean leaf discs) was homogenized at 2 C in a Duallhomogenizer with 3 ml of water, brought to 30 ml, andcentrifuged at 400g for 10 min. The highly colored pellet waswashed twice by suspension in 30 ml of H20 and recentrifuged,all at 2 C. Three additional washings of the pellet with 0.5%sodium dodecyl sulfate at 25 C followed by three washingswith distilled H20 resulted in an essentially clean cell wallpreparation that was only slightly colored and appeared underthe microscope to contain only cell wall fragments. The cellwall material was filtered on discs of low ash paper (Schleicherand Shull No. 576) in tared Gooch crucibles, dried at 100 C,and weighed. The paper and cell wall material were then ashedto determine the weight of glass residue from the tissue homog-enizer. The cell wall weights were corrected for this contami-nant, which in extreme cases equaled the dry cell wall weight.

RESULTS AND DISCUSSION

Perfusates, whether equilibrated or collected by uninter-rupted perfusion, were characteristically clear and colorlessexcept for those from sunflower leaves, which were faintlyyellow.

Early tests were made with cabbage leaves which, becausethey are thicker, contain more cell wall per unit leaf area.The cell wall water of cabbage leaves is less diluted by per-

362 BERNSTEIN

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SOLUTES IN LEAF CELL WALLS 363

120OFIOOo

E

° 800 L

w 600v400 e

a

2 4 6 8 10min/ml

FIG. 2. Electrical conductivity in ,umho/cm at 25 C as afunction of the reciprocal of the perfusion rate, ml/min, throughcabbage leaf discs. Q: increasing flow rate series through one disc;X: decreasing flow rate series through another disc, both from -2.2bar culture.

fusion because cabbage leaves have a low intercellular airspace volume. Intercellular air space was estimated byweighing leaf discs before and after vacuum infiltration withwater. Cabbage leaves have an air space of 24 ml/ 100 g freshleaf weight; for castor bean leaves the value is 55 ml. Despitethese favorable characteristics, no consistent difference insolute concentration between first and later fractions of cab-bage leaf perfusate was found. Electrical conductivity meas-urements did indicate an inverse relationship between perfusionrate and electrolyte concentration (Fig. 2). It was, therefore,decided to infuse leaf discs and to allow the infused water toachieve solute equilibrium with the leaf tissue before displacingit and measuring the maximal solute or electrolyte concentra-tions. Equilibrium data for perfusates are summarized in TableI for the three species studied to date. With cabbage, equili-bration was approached after only 5 min, but with sunflowerand castor bean longer equilibration periods appeared neces-sary and 45 min was routinely used.

Leaves from nonsaline cabbage cultures gave equilibratedsolutions with ECs3 of 700 to 1000 ,umho/cm. Equilibriumperfusate concentrations increased with increasing salinity ofthe culture medium to 1700 to 2000 ,umho/cm for the -2.2bar culture and to 3000 to 3400 ,umho/cm for the -4.4 barNaCl culture. Concentrations of mixed electrolytes in meq/liter approximate 1/100 of the EC in jsmho/cm and this wasconfirmed for the equilibrated perfusates. Thus, the solutionsranged from about 7 to 10 meq/liter (control) to 30 to 34 meq/liter for the most saline culture. Analysis for specific cationsgave approximately 30% of total cation equivalents as Ca2+,10% as Mg2", and 60% Na+ + K+. Potassium predominated(about 50%) in the control and sodium predominated in the-4.4 bar cultures (about 50%) as leaves accumulated moreNa+.

Perfusates of sunflower and castor bean leaves from controlcultures contained only about one-fourth or one-fifth as muchelectrolyte as did the cabbage leaf perfusates; those from salinecultures of these species were also lower than correspondingcabbage leaf perfusates. The range for sunflower was fromabout 2 to 10 meq/liter and for castor bean 2 to 4 meq/liter.Cation composition of castor bean perfusates was similar tothat of cabbage except that Na remained low (about 10%)even under saline conditions, since castor bean leaves did notaccumulate sodium. Limited analyses for anions in cabbage

Abbreviation: EC: electrical conductivitv.

leaf perfusates indicated about 25% NOa-, 25% So,42- 10%Cl-, and 40% organic anions on an equivalent basis.The absence of consistent differences in solute concentra-

tion between the first and later fractions of perfusates fromcabbage leaves was attributed to the extreme leakiness of cab-bage leaf cells. This was not affected by the presence or ab-sence of calcium, since perfusion with 0.5 mm CaSO4 gaveperfusates very similar to those obtained by perfusion withwater (distilled and laboratory-demineralized water at pH 5.5was regularly used in perfusion). When castor bean and sun-flower leaf cells were found to be less leaky, successive frac-tions of continuously perfused castor bean leaves were ex-amined. With this species the first 1.5-ml fraction wasconsistently higher in electrolytes than succeeding fractions.The difference was reasonably consistent provided the per-fusion rate was constant from fraction to fraction. With leavesfrom a -2.2 bar culture, the electrical conductivity of the firstfraction averaged 18 ,umho/cm greater than those of suc-ceeding fractions and for the nonsaline culture only 7.1,umho/cm greater (Table II). Compared to the electrolytelevel in the equilibrated perfusates, these concentrations repre-sented a 22- and 24-fold dilution, respectively (395/18 and

Table I. Electrical Conductivities of Leaf Perfiusates followingEquilibration of Itifiltrated Leaves

Perfusion halted after initial infiltration for indicated periods(equilibration time, min). Vacuum reapplied and EC of displacedequilibrated water monitored dropwise. Leaves from plantscultured on half-strength Hoagland's solution (nonsaline) orwith added NaCl to reduce osmotic potentials (at 25 C) as indi-cated.

Plant Equilibration Time EC

min jsmho/cmCabbageNonsaline 10 740

35 990

-2.2 bars 5 171010 196015 165015 1770

-4.4 bars 15 294015 336022 3200

SunflowerNonsaline 45 240

45 170

-2.2 bars 45 100045 850

-4.4 bars 15 50030 79045 900

Castor beanNonsaline 45 160

45 180

-1.1 bars 45 17045 180

-2.2 bars 45 39045 400

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Plant Physiol. Vol. 47, 19 7

Table II. Electrical Coniduictivity ofSuccessive Fractionis ofCastorBeani Leaf Perfiusates, Collected by Conttinuous Perfusionz

Different leaf discs were used for each perfusion rate.

ECC_____ ____________W t. of

Perfusion Rate Frac- Frac- Frac- Frac- lECi-EC2 Perfusate intion tion t tion Fraction 1

ml/min jOmho/cm 8

A. -2.2 bar NaClculture

1.5 34 25 25 25 9 1.3960.19 72 55 56 57 17 1.3210.067 120 92 96 90 28 1.548

Avg 18 1.422B. Nonsaline cul-

ture0.36 40.8 30.3 ... ... 10.5 1.2060.43 42.5 37.5 ... 5.0 1.3770.33 30.2 24.5 ... ... 5.7 1.526

Avg 7.1 1.370

170/7.1). These values indicate cell wall water volumes in theperfused disc areas of 0.065 ml (1/22 x 1.422) and 0.057 ml(1/24 x 1.370), respectively. Cell wall dry weights in castorbean leaf tissue equivalent to the perfused areas were 40.7 and37.5 mg for the saline and nonsaline cultures, respectively,so that the water content of the walls would appear to be 160%and 152% of the dry weights for the saline and nonsaline discs.These values are somewhat low compared to the more than200% water content reported for various killed plant tissuesat high water potentials (11). The water content of purifiedcabbage leaf cell walls was also about 200% of their dryweight at high water potentials (S. Merrill, unpublished). How-ever, the cell wall determination included vein cells whichprobably contributed relatively little solute to the perfusingwater because of low solute content (e.g., tracheids) and be-cause perfusing water flows around rather than through theveins. These effects cause an overestimation of the cell wallsfrom which the solutes were actually derived. Therefore, thevolume of cell wall water estimated by apparent dilution duringperfusion is in reasonable agreement with the water retentionvalue for leaf cell walls, and the concentration of the equili-brated perfusate appears to be a reliable index of initial cellwall solute concentration.

Early observations on the constancy of the composition ofcabbage leaf perfusates and especially of the initial fractionrepresenting only 1 or 2 air space volumes and later fractionssuggested that the solutes in the cell wall were in equilibriumwith internal cell spaces. As solutes were removed by perfusion,more were released from the cell and in apparently the sameproportions as those initially present in the cell wall. If the re-lease of solutes is strictly diffusional, the concentration ofperfusates should be related to perfusion time according tothe equation for diffusional equilibrium of two spaces, thedonor space with an unlimited supply of the diffusing solutesand the receiving space with a uniform concentration of thediffusing solutes (well stirred):

Ct = C.a (I - e-1t)

where C, and C. are the concentrations in the diffusate at timet and at final equilibrium, respectively.The equilibration data for castor bean in Table I and the

flow rates in Table IIA were used to solve for the constant kin equation 1. Values for t were calculated from the flow rate-

and air space volume of 55% of the 0.92 g fresh weight of an80-mm disc. Values for k decreased with decreasing flow rate(increasing t) from 0.19 for the fastest rate to 0.056 and 0.034for the two slower rates. Similar results were obtained for aseries of observations on cabbage (data in Fig. 2). Flow ratesvaried from 0.1 to 1 ml/min equivalent to t values of 7 and 0.7min for a disc air space of 0.7 ml. When t was greater than 3or 4 min, the average k values were 0.127 but k increased to0.31 at the lowest t of 0.7 min.The variations in k suggest that more than simple diffusion

is involved. The larger k values at shorter times indicate anabnormally great release of solute compared to values observedat longer times. This effect may be due to reabsorption ofleaked solutes, which is relatively unimportant so long asperfusion is rapid and solute concentration does not increaseto levels favoring rapid reabsorption. With decreasing perfusionrates, reabsorption increases in importance as concentrationincreases. Using the lower k values for each species (0.045,the average for castor bean at t = 2.66 and 7.55, and 0.127,the average for cabbage at t > 3.4) the half-times for equilibra-tion were computed as 15 min for castor bean and 5.5 minfor cabbage. These values appear low (4) but not altogetherunreasonable for half-time equilibration values for the cyto-plasmic compartment of leaf cells.To test whether the leakage was primarily a diffusion proc-

ess, cabbage leaves were perfused with water at 2 and 23 C.Perfusates were similar in composition and concentration atcomparable perfusion rates (0.4 to 0.5 ml/min), suggesting adiffusional loss. With slower perfusion rates, it may be possi-ble to demonstrate a temperature effect, which would indicatea metabolic requirement for solute reabsorption.

CONCLUSIONS

Electrolyte concentrations of 2 to 10 meq/liter in the cellwalls of control culture leaves (about 0.6 and 3.6% of cell sapconcentrations of castor bean and cabbage, respectively) arenot too surprising in view of the large leaching losses that mayoccur from normal leaves even through the cuticle (5, 9). Thegreater cell wall concentrations and diffusional loss rates forcabbage than for castor bean and sunflower leaves may be off-set so far as leaching losses are concerned by the exceptionalcuticular and waxy protection of the cabbage leaf. The 2- to3-fold increase in cell wall electrolyte concentration with in-creasing salinity of the culture medium is quite out of propor-tion to the increased electrolyte content of the leaves which isabout 30 and 60% for cabbage and castor bean, respectively.Salinity appears to increase the permeability of leaf cells tointernal electrolytes (rate of leakage) as well as to decrease theefficiency for reabsorption of electrolyte (higher equilibriumconcentrations). The leakiness of leaf cells and solute reab-sorption are apparently responsible for the similarity of com-position of solutes initially present in the cell walls and thosediffusing out of cells with continued perfusion.The apparent dynamic equilibrium between cell wall solutes

and intracellular solutes inferred above suggests the need forextreme caution in assessing absorption of ions or other solutesalready present in leaf cells by radioisotope techniques. Whenas much as 1 meq/liter or more of K+ may normally exist incell walls in equilibrium with inner cell space, the uptake oflabeled K, for example, may measure influx, provided specificradioactivity is not too altered by efflux, but would not meas-ure net flux or accumulation (7, 8).The inferred condition of dynamic equilibrium also indicates

that solutes in the cell wall even under saline conditions do notrepresent an accumulation of excess unabsorbable solutes (6)but rather solutes that are in dynamic equilibrium with solutes inintracellular space. The concentration of electrolytes and prob-

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SOLUTES IN LEAF CELL WALLS

ably of total solutes in leaf cell walls, moreover, is only a smallfraction of the intracellular concentrations.

If only data from equilibrated leaf discs were available, thequestion might be properly raised as to the effect of prolongedtreatment on cellular integrity. However, since the results fromimmediate rapid perfusion are consistent with those fromequilibration, prolonged infusion appears to be noninjurious.Anaerobiosis is probably not a factor, since with continuousillumination infused leaves can presumably photosynthesize atrates up to the compensation point.

That vacuum perfusion per se does not induce solute leak-age to give the observed results is also deducible from theagreement between concentrations in the equilibrated per-fusates and those calculated from the excess solute in the firstof several successive perfusate fractions. Artificially inducedleakage would affect solute concentrations during steady stateperfusion and possibly at equilibration, but could hardly ac-count for the excess solute found in the first of several suc-cessive perfusate fractions. Not only were the quantities ofexcess solute in the first fractions in close agreement with thoseexpected on the basis of the equilibrium perfusate concentra-tions, but these first fractions were collected within 1 or a fewminutes of the start of perfusion or only a small fraction of the15-min half-time for equilibration. An induced leakiness couldnot, therefore, have affected the initial cell wall solute con-centrations to the degree observed. The validity of the findingsis also attested to by good reproducibility, hardly consistentwith leaf damage, even when perfusion conditions (degree ofvacuum) varied, and the absence of any visible symptoms ofinjury following perfusion, transpiration of intercellular water,and storage of leaf discs for 1 or more days.

Since nonelectrolytes may contribute significantly to thetotal cell wall solute concentration, the osmotic potential of thecell wall cannot be inferred solely from electrolyte content.Some direct determinations of osmotic potentials by the freez-ing point method (1) were made on equilibrium sunflowerperfusates. The control leaf perfusates averaged 180 ,umho/cm.For mixed electrolytes, the expected osmotic potential at 25 Cwould be -0.39 X 180 x 10-' or -0.07 bar (10). Actualosmotic potential was -0.32 bar, indicating that only 22%of the osmotic potential was due to electrolytes. For equilib-rium perfusates from saline cultures, the average EC was 750.tmho/cm, the calculated osmotic potential, -0.29 bar, and theobserved osmotic potential, -0.65 bar, indicating about 55%of the osmotic potential was due to electrolytes. A significantproportion of nonelectrolyte was also observed for cabbageleaf perfusates by comparing the electrical conductivity andtotal dissolved solutes. Electrolytes appeared to contribute 40%of total dissolved solutes for leaf perfusates from nonsaline

cultures and 60% for leaf perfusates of saline cultures. Totalwater potentials for sunflower and presumably castor bean cellwall water are thus > -1 bar at all salinities but for cabbagemay well be around -1 and -2 bars for nonsaline and salinecultures, respectively. Osmotic potential is therefore a minorcomponent in well hydrated leaves of sunflower and probablycastor bean but a major component of total water potential inwell hydrated cabbage leaves. Additional direct determinationsof osmotic potential are needed.

Because stomata must be open to permit perfusion by themethod described, the effects of extreme leaf water deficits oncell wall solute concentrations cannot be directly observed. Instudies with cabbage, wilted leaf samples collected during mid-day were compared with turgid ones collected early in themorning. No noteworthy difference in perfusate concentrationor composition was noted, but leaf discs did recover someturgor before perfusion was initiated, and cell wall solute con-centrations may have changed. An alternative method for per-fusing wilted leaf samples through cut leaf margins with solu-tions of nonabsorbable osmotica having the same waterpotential as the wilted leaf is being developed. By this methodthe cell wall solutes in wilted leaves should be ascertainable.

Acknowledgments-I deeply appreciate the help provided by Messrs. G. Akin whoanalyzed the leaf perfusates, Stephen D. Merrill who developed the method for cellwall determination, and Robert A. Clark who cultured many plants for these experi-ments. I am also indebted to Dr. Stephen L. Rawlins for helpful discussions and adviceduring the course of this research.

LITERATURE CITED

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2. BERNSTEIN, L. AND G. AKIN. 1969. A method for determining solutes in the cellwalls of leaves. XI Int. Bot. Congr. Abst. p. 14.

3. Boyer, J. S. 1967. Matric potentials of leaves. Plant Physiol. 42: 213-217.4. MAcRoBBIE, E. A. C. AND J. DAINTY. 1958. Ion transport in Nitellopsis obtusa.

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plant foliage. Plant Physiol. 39: 590-593.6. OERTLU, J. J. 1968. Extracellular salt accumulation, a possible mechanism of salt

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and first findings with excised leaf tissue of corn. Plant Physiol. 39: 338-341.8. SMITH, R. C. AND E. EPSTEIN. 1964. Ion absorption by shoot tissue: Kinetics of

K and Rb absorption by corn leaf tissue. Plant Physiol. 39: 992-996.9. TUKEY, H. B., JR., H. B. TUKEY, AND S. H. WITrWER. 1958. Loss of nutrients by

foliar leaching as determined by radioisotopes. Proc. Amer. Soc. Hort. Sci.71: 496-506.

10. U. S. Salinity Laboratory Staff. 1954. Diagnosis and improvement of saline andalkali soils. U. S. Dept. Agr. Hdbook 60.

11. WIEBE, H. H. 1966. Matric potential of several plant tissues and biocolloids.Plant Physiol. 41: 1439-1442.

12. WILSON, J. W. 1967. The components ot leaf water potential. I. Osmotic andmatric potentials. Aust. J. Biol. Sci. 20: 329-347.

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