argenteum) - plant physiology · boundto fixed, negatively charged sites within the cell wall, the...

Plant Physiol. (1990) 94, 1040-10470032-0889/90/94/1 040/08/$01 .00/0

Received for publication March 13, 1990Accepted June 25, 1990

Quantification of Apoplastic Potassium Content by ElutionAnalysis of Leaf Lamina Tissue from Pea

(Pisum sativum L. cv Argenteum)"

Jean M. Long and Irvin E. Widders*Horticulture Department, Michigan State University, East Lansing, Michigan 48824

ABSTRACT

K+ content and concentration within the apoplast of mesophylltissue of pea (Pisum sativum L., cv Argenteum) leaflets weredetermined using an elution procedure. Following removal of theepidermis, a 1 centimeter (inside diameter) glass cylinder wasattached to the exposed mesophyll tissue and filled with 5 milli-molar CaCI2 solution (10C). From time-course curves of cumula-tive K+ diffusion from the tissue, the amount of K+ of extracellularorigin was estimated. Apoplastic K+ contents for leaves fromplants cultured in nutrient solution containing 2 or 10 millimolarK+ were found to range from 1 to 4.5 micromoles per gram freshweight, comprising less than 3% of the total K+ content within thelamina tissue. Assuming an apoplastic solution volume of 0.04 to0.1 milliliters per gram fresh weight and a Donnan cation ex-change capacity of 2.63 micromoles per gram fresh weight (ex-perimentally determined), the K+ concentration within apoplasticsolution was estimated at 2.4 to 11.8 millimolar. Net movementof Rb+ label from the extracellular compartment within mesophylltissue into the symplast was demonstrated by pulse-chase ex-periments. It was concluded that the mesophyll apoplast in peahas a relatively low capacitance as an ion reservoir. ApoplasticK+ content was found to be highly sensitive to changes in xylemsolution concentration.

The apoplast within leaf tissue constitutes a potentiallyimportant pathway for K+ transport between vascular andmesophyll tissues (6, 22, 23) as well as an extracellular ionreservoir influencing absorption and accumulation into indi-vidual cells (8, 25). The apoplastic compartment comprisesmature xylem, due to the lack of a functional plasma mem-brane, and a continuum of interconnecting cell walls withinmesophyll tissue. K+ content within the apoplast is consideredto be highly variable as a result of fluctuations in import andexport rates via the vascular tissues and fluctuations in thedirection and rate of net fluxes into the symplast, the contin-uum of interconnecting protoplasts. Short-term changes in a

plant's environment, therefore, would be expected to affectsignificantly mesophyll apoplastic K+ content. Gradients inK+ concentration are also thought to exist within the apoplast.During periods of rapid xylem import into a leaf, for example,relatively high concentrations of K+ might be expected to

Michigan State Agriculture Experiment Station Paper No. 13035.This project was funded in part by a Michigan State University All-University Research Initiation Grant.

accumulate extracellulary within the vascular bundles,whereas lower concentrations would exist within the cell wallof mesophyll tissue more distant from the minor veins.The specific role(s) ofthe apoplast as related to K+ transport

and accumulation within leaf lamina tissue cannot be fullyelucidated without quantitative analysis of extracellular K+content and concentration (20). Several indirect approacheshave been reported for collection of apoplastic sap and esti-mation of ionic concentrations including analysis of xylemexudates, vacuum perfusion of leaf lamina discs (1), andpressure dehydration of leaves (14). X-ray microanalysis (7,9, 12) and ion-sensitive microelectrodes have been used tomeasure directly extracellular ion concentrations ofepidermalguard cells (2) and the extensor and flexor cells within thepulvinus of legume leaves (26). The primary limitation ofthese procedures is the inability to estimate the ionic contentwithin the apoplastic reservoir of the mesophyll.

Apoplastic ion content is a function of the amount of ionbound to fixed, negatively charged sites within the cell wall,the Donnan free space, and that in solution within the cellwall. Although the cation exchange capacity of leaf cell wallshas been investigated for a variety of plant species (19, 25),the 'functional' volume of apoplastic solution in situ is moredifficult to estimate. The volume within a unit fresh weightof tissue in theory is influenced by the thickness of the cellwalls and the mean size of mesophyll cells. In addition, thevolume of apoplastic solution is highly variable due to fluc-tuations in the hydration status of the leaf, such as occursduring a diurnal period. Determination of net apoplastic ioncontent is critical to gaining an understanding of the physio-logical importance of the apoplast as a reservoir for ions suchas K+.

In the present paper, we report on an elution procedure forestimation of mesophyll apoplastic K+ content in leaflets ofthe Argenteum mutant ofpea (Pisum sativum). The epidermisand associated cuticle, which constitute a primary barrier forinfusion of the mesophyll tissue with solution and diffusionof ions, can be readily detached from Argenteum leaves, dueto the presence of large air spaces between the epidermal layerand the underlying mesophyll tissue. Hoch et al. (13) reportedthat epidermal strips, free of cell wall fragments, could bepeeled from the lamina tissue, suggesting that minimal me-chanical damage is incurred to the mesophyll cells duringpeeling. Minimization of ion leakage due to cell damage is animportant prerequisite to ion analysis of the apoplast.The objectives of the present study were to estimate the K+

1040

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ELUTION ANALYSIS OF APOPLASTIC K IN PEA LEAF TISSUE

content and concentration within the leaf apoplast in orderto understand better the role and importance ofthe apoplasticK+ reservoir relative to the K+ nutrition of leaf mesophylltissue.

MATERIALS AND METHODS

Plant Culture

Pisum sativum L. cv Argenteum seeds were sown in asterile, peat-based growing medium (Baccto Grower's Me-dium, Michigan Peat Co., Houston, MI) in 0.5 L plasticcontainers. Following emergence, plants were watered dailywith a nutrient solution containing 1 g L' of 20N-8.6P-16.6K (Peter's Soluble Plant Fertilizer, W. R. Grace Co.,Fogelsville, PA). Plants were cultured at a constant tempera-ture (15 ± 2°C) with a 15 h photoperiod provided by 400-Wmercury vapor lamps (model Son-T-400; Philips Electronics,Ltd., Bloomfield, NJ). Average irradiance level within thecanopy was 400 ,uE m-2 s-'. Axillary branches were prunedoff. Experiments were conducted when plants reached the 9to 10 node stage of development.For sand culture experiments, seeds were planted into the

0.5 L plastic containers filled with a course white silica sand.Temperature and light conditions were as previously de-scribed. Upon emergence, seedlings were watered with a one-fourth strength modified Hoagland solution (5) containing1.5 mM K+. Ten d after emergence, seedlings were treatedwith a full-strength modified Hoagland solution containingeither 2 or 10 mM K+. The macronutrient composition of thelow K+ solution included 2 mM KNO3, 4 mm Ca (NO3)2, 2mM Na2H2PO4, 1 mM MgSO4, and 3 mM NH4NO3, while thehigh K+ treatment solution contained 6 mM KNO3, 1 mMNH4NO3, and 2 mm K2SO4 in addition to the other salts.Both solutions included a complete complement of micro-nutrients (5).

Elution Procedure

Leaflets were excised from recently expanded mature leavesat nodes 7 or 8 from randomly selected plants, briefly rinsedwith deionized water, and placed on plastic weighing boatsfloating on slushy ice (1°C) for the duration of the elutionperiod. The abaxial epidermis was removed, using sharpenedforceps, from an area near the base of each leaflet that wasapproximately 1.5 cm wide and which extended from themidrib to one margin. Scanning electron micrographs of thespongy mesophyll for the peeled region revealed negligibledamage to those cells due to detachment of the epidermis(Fig. 1).A cylinder, 0.8 cm high cut from glass tubing of 1 cm i.d.

and polished at one end, was attached to a peeled region of aleaflet using a method similar to that of Greene and Bukovac(1 1). The polished end of the cylinder had been rotated in athin layer of 100% silicon rubber sealant (Dow Coming Corp.,Midland, MI) and pressed gently and evenly onto the leaflet.The sealant was not phytotoxic, but did lower the pH of thesolution within the cylinder by <0.5 pH unit due to a releaseof acetic acid upon curing.Within 2 min after peeling, elution solution (0.5 mL)

containing 5 mM CaCl2 (pH 5.5) and preequilibrated at 1°C

Figure 1. Scanning electron micrograph of spongy mesophyll (200x)exposed following detachment of abaxial epidermis from fully ex-panded Argenteum pea leaflets. Tissue was fixed in 4% glutaralde-hyde, dehydrated in a graded ethanol series, critical-point dried, andgold coated prior to examination.

was added to each cylinder. At predetermined intervals (typ-ically 2, 5, 10, 15, 20, 30, 40, and 60 min), a 200 ,L samplewas withdrawn from each cylinder and replaced immediatelywith an equal volume of CaC12 solution. Replacement ofsolution allowed for collection of an unlimited number ofsamples as well as the avoidance of excessive increases in K+concentration within the eluting solution over time. K+ con-centrations in the elution solution fluctuated below 0.1 mm.Each elution sample was transferred to a 4 mL plastic vial

and diluted with CsCl and HCI solution to achieve a finalconcentration of 1000 ,g Cs mL-' and 1% (v/v) HCI. Inexperiments in which Rb+ was used as a tracer, samples werediluted with KCI solution to achieve a final K+ concentrationof 2000 mg L'. K+ and Rb+ were analyzed by AES2 afteradjusting the aspiration rate and integration time to accom-modate the small, <1 mL, sample size.To generate elution curves, the net cumulative amounts of

eluted K+ (,umol g-' fresh weight) were plotted over time.

2Abbreviations: AES, atomic emission spectrophotometry; FW,fresh weight.

1041



Plant Physiol. Vol. 94, 1990

Fresh weight of eluted mesophyll tissue was estimated byweighing a peeled lamina disc (1 cm diameter) from ananalogous position on the opposite half of the leaflet. Elutedtissue was thought to provide erroneous fresh weight estimatesdue to partial infusion of intercellular air spaces with solution.For long-term experiments, >2 h of elution, lamina tissuefresh weight was estimated from tissue taken from the oppositeleaflet on the compound leaf.

Lateral Diffusion Measurements

Lateral diffusion of K+ within the lamina tissue duringelution was evaluated using 86Rb+ as a tracer. Cylinders con-taining a 86Rb+ (0.05 MCi mL-') labeled KCI solution, 500 ,ugmL-', were positioned either 0.5 cm from, external to, orwithin cylinders containing 5 mm CaCl2. In experiments inwhich a cylinder was positioned within another cylinder, 0.9mL of solution was added to the outer cylinder (16 mm i.d.),while only 0.3 mL was added to the smaller inner cylinder (6mm i.d.). Aliquots (200 ML) were collected from the cylinderscontaining unlabeled solution and replaced with CaCl2 solu-tion at specific time intervals. The aliquots were diluted with10 mL of 2.5 mm ANDA (7-amino-1,3-naphthalene disul-fonic acid monosodium salt) solution in 20 mL vials andCerenkov radiation was measured using a liquid scintillationcounter (Tri-Carb 1500, Packard).

Excised Leaf Experiments

Petioles of fully expanded leaves were excised, placed im-mediately in deionized water, and recut to a uniform lengthof 3 cm while submerged. Leaves were then transferred toglass test tubes containing 3.5 mL of aerated RbCl plus 0.5mM CaSO4 solution equilibrated at 25°C in a constant tem-perature water bath. Treatment Rb+ concentrations in solu-tion were 1, 10, and 100 mM. Incandescent lights provided 75,uE m-2 s-'. After 0.5 and 3.0 h, leaflets were excised andpeeled, and Rb+ was eluted from the mesophyll tissue.

For pulse-chase experiments, the petioles of excised com-pound leaves were placed into 3.5 mL of 50 mM RbCl plus0.5 mM CaSO4 solution for a period of 6 min after which theywere rinsed and placed in a 50 mM KCl chase solution for upto 180 min. Rb+ was then eluted from the peeled leaflets afterspecific periods of time and analyzed by AES.

Cell Wall Isolation

Cell walls were isolated from Argenteum leaflets followingtissue homogenization and centrifugation using a modifiedmethod of Bernstein (1). After the final washing, the cell wallpreparation was transferred to tared crucibles, dried at 90°Cfor 24 h, and weighed.

Free Space Characterization

For measurement ofK+ and Cl- uptake into the free space,KCI solutions (0.5, 1, 3, 5, 10, and 20 mM) containing 0.1mM CaSO4 were labeled with either 86Rb+ or 36CL- (NewEngland Nuclear) to specific activities of 0.1 and 0.8 iCimL-', respectively. Leaflets of similar age and position as

those used for elution were prepared as above for treatment

at 1°C. One-half mL of labeled solution was placed in eachcylinder and poured off after 5, 10, 20, or 40 min. The areaof lamina tissue covered by the cylinder was cut out using arazor blade, lightly blotted, and placed in a scintillation vial.For extraction of label, tissue was frozen at -20°C overnight.After the addition of 2 mL of deionized H20, each vial washeated in a gently boiling water bath for 20 min. After cooling,36Cl-labeled samples received 14 mL of scintillation cocktail(Safety Solve; Research Products Intl. Corp., Mt. Prospect,IL), while each "6Rb+ sample received 10 mL of 2.5 mMANDA solution as a wavelength shifter for Cerenkov meas-urement. All samples were allowed to sit several hours beforeradioactivity was determined by standard scintillation count-ing procedures. Calculation of free space parameters were asdescribed by Pitman et al. (19).

Xylem Sap Collection

To induce a positive root pressure, plants were coveredwith a plastic bag during the dark period preceding collectionof xylem exudate. At the onset of the subsequent light period,each stem was excised 3 cm above the soil medium, and thecut surface rinsed with deionized water and blotted. A 10 ULaliquot of exudate was collected using a micropipet andanalyzed for K+ by AES following dilution.

Tissue K+ Analysis

Leaflet tissue samples to be analyzed were briefly rinsed indeionized water, dried at 60°C for 48 h, and weighed. Groundsubsamples were placed into 20 mL glass scintillation vials towhich was added 1 mL of concentrated HNO3. After approx-imately 12 h, the acid was gently boiled off and oxidationcompleted by the dropwise addition of H202. Samples wereanalyzed by AES following the addition of CsCl (1000 gg CsmL-' final concentration).

RESULTS

A typical 60 min time course action curve of K+ frompeeled unrinsed Argenteum leaf lamina tissue at 1°C wascharacterized initially by a high rate of K+ loss followed by aslower semisteady-state rate which developed after approxi-mately 20 min (Fig. 2). The initial high elution rate wasthought to reflect free diffusion ofK+ from the mesophyll freespace, which was facilitated by the infusion of the tissue witheluting solution and the displacement of K+ by Ca2+ fromfixed exchange sites in the cell wall. Based on the uptake of36CL- labeled CaCl2 (5 mM) solution, approximately 0.12 mLg-' fresh weight of elution solution was estimated to haveentered the tissue free space during the initial 20 min. The airwithin the intercellular air spaces was thought not to havebeen completely displaced during elution since the laminatissue retained its buoyancy. Infusion of the free space withsolution should significantly reduce the net resistance for iondiffusion out of the mesophyll tissue. Resistances from aDonnan phase within the cell wall and from an unstirredboundary water layer, however, cannot be ignored.

After approximately 20 min of elution, the rate of K+diffusion from the peeled lamina tissue stabilized over time

1 042 LONG AND WIDDERS




12 e-eoPeeled leaflets, rinsed

0l6-0o~ 0 3 4 0 6

ELUTION TIME (min)

Figure 2. Cumulative time-course K+ elution at 100 from peeled

Argenteum leaflet discs (1 cm diameter) preninsed for 20 min in 5 mM

08012 solution and from similarly preninsed and unrinsed peeled

lamina tissue from intact leaflets. Steady-state elution rates, 20 to 60

min, from prerinsed and unrinsed peeled lamina tissue were not

significantly different, 0.09 and 0.12 ,umol g-1 fresh wt (FW) minW1,respectively. The elution rate for prerinsed discs was significantly

higher (P = .01), 0.27 Mlmol g-1 FW min1l. Points are means of 6

replications SE.

(Fig. 2). This semisteady-state condition was attributed to a

net efflux of K+ from the mesophyll symplast and potentially

to the diffusion of ions from mesophyll tissue external to the

confines of the cylinder. Continued K+ efflux from cells and

elution from the tissue was promoted by frequent sampling

and replacement of elution solution from the cylinder. K+concentrations within the eluting solution did not exceed 0.1

mM in most experiments, which resulted in the maintenance

of a gradient in extracellular K+ concentration, lower concen-

trations being present in eluting solution than within the free

space ofthe tissue. Ifsampling were discontinued, the external

elution solution ionic concentration increased until an equi-

librium condition was reached (data not presented).

Prerinsing of peeled lamina tissue for 20 min in 5 mM CaCl2

solution prior to elution nearly eliminated the initial rapid

flux of K+ from the tissue and resulted in the establishment

ofa semisteady rate within approximately 5 min (Fig. 2). This

is consistent with the idea that the mesophyll apoplast is the

primary compartment from which K+ diffuses during the

initial 20 min of elution. The similarity in the mean rates of

K+ elution after 20 min between the prerinsed and the un-

rinsed tissue support the interpretation that the eluted K+during this period was of intracellular origin. Once the free

space is infused with solution, the plasmalemma or tonoplast

membrane would most likely constitute the primary resist-

ances limitingK+ diffusion. Mechanical damage to mesophyll

cells, such as by cutting out of lamina discs, resulted in an

increased rate of elution during the period from 20 to 60 min.Radioisotope tracer experiments using 86Rb+ suggest that

diffusion of ions from mesophyll tissue external to that cov-

ered by solution within the cylinder might contribute signifi-

cantly to the net amount of eluted ions measured after ap-

proximately 35 min (Fig. 3). Prior to that time, however, K+

diffusion from surrounding tissue was found to be negligible.The higher experimentally predicted rates for inwardly di-rected ion movement were attributed to the larger amount ofinitial K+ and 86Rb+ as a result of the larger volume (0.9 mL)in the outer cylinder as compared to the volume of labeledsolution placed into the inner cylinder (0.3 mL). The esti-mated time until appearance of ions originating from thesurrounding tissues into the eluting solution, however, shouldbe considered as a conservative estimate. Under normal elu-tion conditions, no solution is applied to the mesophyll tissuesurrounding the cylinder after detachment of the epidermis.Resistances to inward ion diffusion from tissue surroundingthe cylinder, therefore, are considered to be greater than underthe tracer experimental conditions.

Modification of the temperature of the lamina tissue andthe eluting solution significantly affected the rates of K+elution from the tissue (Fig. 4). The disparity in elution ratesincreased with time. Within the first 3 min, the mean netrates of K+ elution from the tissue at 1 and 20°C were notsignificantly different. During the period from 5 to 8 min, thedifference in elution rate between the two temperature treat-ments had increased to 2.4 (x) and by 20 to 50 min hadreached 3.1 (x). The fact that the Qio for free diffusion in anaqueous solution is 1.2 to 1.5 corroborates the conclusionthat the K+ eluted initially from the tissue is of extracellularorigin. With time, membrane-mediated fluxes are thought toconstitute an increasingly larger percentage of the eluted K+.Compartmentation of cations within tissue was also dem-

onstrated by a pulse chase experiment in which the petiole ofexcised leaves was dipped into a 50 mM RbCl solution for 6min and then transferred to a 50 mm KCI chase solution.Through exposure to a pulse, a limited amount of Rb+ wastaken up through the petioles and transported into the leaflets.Elution of peeled leaflets after 10, 30, and 180 min in thechase solution revealed large differences in the rates of Rb+diffusion from the tissue (Fig. 5). The longer the period of

0.060_- Outward Diffusion

v h-- Inward Diffusion0.05-

-LJML 0.04-

60.03-

0%_0.02-

W

-j 0.010-

0.00T_o 20 40 60 80

DIFFUSION PERIOD (min)

Figure 3. Net inward lateral diffusion of 86Rb+-labeled KCI (5 zg K+mL-1) into the eluting cylinder (6 mm i.d.) from surrounding mesophylltissue or outward diffusion of K+ from tissue within the confines ofthe cylinder over time. The direction of diffusion did not significantlyaffect the net amount of 86Rb+-labeled K+ at any time during elution.FW = fresh weight.

1 043




E

bi

0 10 20 30 40 50 60

ELUTION TIME (min)

Figure 4. Effect of elution solution temperature, 20 and 10C, on

cumulative time-course K+ elution from peeled Argenteum leafletlamina tissue. Steady-state elution rates, between 20 and 60 min, at20 and 10C were significantly different at the 1% level, 0.51 and0.018 ,umol g-1 fresh wt (FW) min-', respectively. Estimates ofapoplast K+ (y intercept), 2.3 and 1.5 MAmol g-1 FW were not signifi-cantly different. Points are means of 6 replications ± SE.

time after the pulse, the smaller the fraction of rapidly elutedRb+ and the higher the rate of the slow diffusion component,between 30 and 240 min of elution. Net absorption of Rb+from the apoplast into the mesophyll symplast was thoughtto have occurred during the 10 to 180 chase period.

Elution analysis was found to be highly sensitive to K+concentration within the xylem solution. When excised pe-tioles were placed into 1, 10, or 100 mm RbCl solution (250C)for various periods of time, significant differences in theamount of eluted Rb+ were measured within 30 min of initialuptake. Figure 6 illustrates the Rb+ elution curves after 3 h ofuptake by the leaves. Since xylem sap K+ concentrations have

1.0-

0.8-

E 0.6-

LL

0.4-1

-LJ

.a 0.2- t1w h-- 10 min

.G-e 30 mine-. 180 min

0 50 100 150 200 250

ELUTION TIME (min)

Figure 5. Cumulative time-course Rb+ elution from Argenteum me-

sophyll tissue at three times during a chase period in 50 mm KCI afterhaving dipped the petioles of excised leaves in a 50 mM RbCI solutionfor 6 min. Points are means of 6 replications ± SE. FW = fresh weight.

15-

0~

E

Aio-0

-LJ

LLJ 5-

.0:D

0 1 0 20I I0I

6010 20 30 50

-80

.-N

-60 P'

E3.

-40 c

D-J

-20 Li.0n

ELUTION TIME (min)

Figure 6. Cumulative time-course Rb+ elution from Argenteum me-

sophyll tissue of excised leaves which had been dipped into 1, 10, or

100 mM RbCI solution for 3 h. Points are means of six replications ±SE. Left y-axis scale corresponds to elution from 1 and 10 mm RbCltreated leaves, while right y-axis scale corresponds to elution fromthe 100 mm RbCI treatment. FW = fresh weight.

been reported to fluctuate within the range of 1 to >50 mm,these data suggest that elution analysis is a sensitive analyticaltool for evaluating modulations in K+ transport rate via xyleminto leaf mesophyll tissue.

Apoplastic content of K+ (,gmol g-' fresh weight) was esti-mated from elution curves by fitting a linear regression func-tion through the data from 20 to 60 min (Fig. 7). The K+ ofpresumed intracellular origin was subtracted from the totaleluted by extrapolating the linear function to 0 time, adjustingthe Y-axis intercept to 0, and subtracting the values of theadjusted linear function at the specific elution times fromthose of the nonlinear curve regressed through the originaldata. The result is a difference curve with an asymptoticmaximum which estimates the amount ofK+ of extracellularorigin. A primary assumption in the calculation is that theslow semisteady-state rate observed after 20 min is continuousfrom t = 0. This is probably an overestimate ofK+ efflux rateduring the first 20 min of elution, however, since K+ concen-tration in apoplastic solution in situ prior to epidermal re-moval would be expected to be significantly higher than theresultant equilibrium concentration in the eluting solution,<0.1 mm K+. Potential contamination as a result of K+diffusion from tissue outside the confines of the eluting cyl-inder was not of concern. Due to a delay in arrival (Fig. 3),potential contamination should be a component of the slowrate and thus would be subtracted out in the calculation.For estimation of K+ concentration within the apoplast

solution phase, which is necessary to gain a better understand-ing of the rates and direction of K+ fluxes across mesophyllplasma membranes, the volume of solution in situ within thecell wall and the concentration of fixed negative charges, theDonnan phase, within mesophyll tissue were determined. Drycell walls isolated from Argenteum leaflets comprised about10% of the leaflet fresh weight. Assuming cell walls hold 75to 100% of their dry weight in free water (10, 17), the volumeofapoplastic solution might approach 0.1 mL g-' fresh weight.Alternatively, if one assumes that apoplastic water constitutes

IlO0 mM RbCl

10 mM RbCI

1 mM RbCIr~ ~~~ ~ ~ _ 0

zu

^_~~~~~~~~I0 nI ^V)T - '1% I





O4-

E

-2_-J

0 1 0 20 30 40 50 60

ELUTION TIME (min)

Figure 7. A cumulative time-course K+ elution curve from Argenteummesophyll tissue at 1 0C and a difference curve estimating K+ diffusionfrom the apoplast. The difference curve was obtained by regressinga linear function through the net amounts of eluted K+ between 20and 60 min, extrapolating the function to 0 time, adjusting the Yintercept to 0, and subtracting the values of the adjusted linearfunction from the original data. The asymptotic maximum of thedifference curve estimates apoplastic K+ content. FW = fresh weight.

a minimum of 5% of the total water in a fully turgid leaf (3),and since Argenteum leaflets are approximately 84% water, aminimum volume estimate might be 0.045 mL g-' freshweight.K+ binding to the fixed anions in the Donnan phase (Kd)

over a range of K+ concentrations ([KJ]) and described byEquation 1 was determined experimentally after the methodof Pitman et al. (19).

Kd = b, (1 - e (b2 [KJ] + b3 [Ks]2)) (1)

The curve, describing asymptotic experimental saturation,indicated a maximum cation exchange capacity (b,) of 2.63,umol g-' fresh weight at an extracellular K' concentration of20 mm (Fig. 8). The effective concentration of fixed anionswas calculated to be 360 mmol L-'.

Equation 2 defines the difference between the total apo-plastic K+ content (KJ) and K' bound to the Donnan phaserelative to the volume of the apoplastic solution (v).

[K5] = (Kt - Kd)/v (2)By solving Equations 1 and 2 simultaneously, the K+ concen-tration in apoplastic solution ([K5]) was estimated.

In fully expanded leaves from pea, apoplastic solution K+concentration ranged from approximately 5 to 12 mm assum-ing a volume of 0.1 ml g-' fresh weight (Table I). K+ concen-tration within the culture solution, 2 and 10 mM, significantlyaffected both the net apoplastic content and the concentrationestimates. A large fraction of net apoplastic content, 74 to66%, was bound to the Donnan phase. Xylem solution K+concentrations, 5.4 and 14.2 mM K+, approximated the apo-plast concentrations under the cultural conditions used in thisexperiment and the assumed apoplast solution volume. Al-though net apoplastic content was found to be 2.3 to 3.6 ,umol

;c

z-

cit

g-' fresh weight, it only constituted 2.4 to 1.8% of the totalK+ content within the lamina tissue.

DISCUSSION

Freudling et al. (8) proposed that the apoplast of planttissues can serve as a physiologically important cation reser-voir if the number of fixed negative charges in the wall matrixis high and if protoplasts have the capability of maintainingionic concentrations within the extracellular compartment.In pea leaf mesophyll tissue, the K+ content within the apo-plast reservoir was relatively low, <4 ymol g-' fresh weighton the average. Total apoplastic K+ content within the pul-vinus of Phaseolus species was reported to be significantlyhigher, 15 to 75 ,umol g-' fresh weight assuming an 83%moisture content (8). The effective concentration of fixedanions within the mesophyll, 360 mmol L-', which is ameasure of the cation capacitance of the apoplast reservoir,was intermediate relative to other plants: barley, 280 mmolL' (19); tomato, 550 mmol L-' (25); and within pulvinartissue, 200 to 650 mmol L' (8).The ability of the Donnan phase to buffer against changes

in apoplastic solution K+ concentration is a function of netextracellular K+ content and the amount of fixed anionswithin the mesophyll apoplast. This can be readily demon-stratedby a simulation model (Fig. 9). Ifapoplastic K+ contentis relatively low, <3 ,mol g-' fresh weight, moderate fluctua-tions in content would be expected to have little effect on theconcentration within the solution phase ofthe apoplast. How-ever, as sorption of K+ to fixed anions approaches saturation,any additional increase in extracellular K+ content will resultin a relatively large but proportional increase in apoplastsolution K+ concentration. In addition, changes in theamount of fixed negatively charged sites will alter the apo-plastic K+ content at which the buffering capacity of theDonnan phase is lost. As noted by Freudling et al. (8) and

3.0- Y = 2.63(1 - 0(124x 0)

2.0-

O./OA/

0.//. . . . . .1.0^4e

U 5 10 15 20 25

KCL CONCENTRATION (mM)

Figure 8. Exchangeable K+ binding to fixed anions within the Donnanphase of the mesophyll apoplast of Argenteum leaves as a functionof KCI concentration. Determinations were made using peeled laminatissue and evaluating the uptake of tracer 86Rb+ and 36CI- at 00Cplus 0.1 mm CaSO4 over a range of KCI concentrations (21). FW =

fresh weight.

1 045




Table I. Effect of Nutrient Solution Culture K Concentration on Leaf K+ StatusArgenteum pea plants were cultured in nutrient culture solution containing 2 or 10 mM K+. K+ elution

experiments using mature leaflets were conducted as described in "Materials and Methods." Apoplasticsolution K+ concentration and the amount of K+ bound to the Donnan free space were estimatedassuming an apoplastic solution volume of 0.1 mL g-1 fresh wt (FW) and a Donnan cation exchangecapacity of 2.63 mol g-1 FW. Values are means of six replications ± standard errors.

Nutrient Total Steady-State Net Apoplastic ApoplasticSolution Lamina K+ K+ Elution Rate K+Content Solution K+ Donnan K+ Xylem [K+]

[K+]

m inol g Amol g ' FW. MmoIg' FW mM MinoIg' FW mM

2 98±8 2.3±0.5 2.3±0.3 5.9±0.8 1.7±0.2 5.4±0.510 199±9 4.5±0.8 3.6±0.4 11.8±1.4 2.4±0.4 14.2±1.6F testa ** ** * *

a Means significantly different at 1% level (**), 5% level (*).

Starrach et al. (21), the cation exchange capacity of theDonnan phase is not static, but is influenced by the pH andthe K+ concentration in the apoplast solution and by thelaying down of additional cell wall during leaf ontogeny.A primary limitation in the use of the elution method for

quantification of apoplastic solution phase ion concentrationis the measurement or estimation of the effective volume.The relative volume ofapoplastic solution within a tissue (mLg_' fresh weight) should not be only a function ofthe anatom-ical characteristics of the tissue, the thickness and density ofthe cell wall and the cellular dimensions, but also of thehydration status of the tissue. Transpirational water loss, forexample, is thought to concentrate extracellular solutes dueto a reduction in effective volume (4, 15). The ultimate effectofvolume on the K+ concentration within the solution phase,however, will be a function of the net apoplastic K+ contentrelative to the cation exchange capacity of the cell wall (Fig.9). If apoplastic K+ content is sufficiently high to saturate thefixed anion exchange sites, a reduction in effective solutionvolume will significantly increase K+ concentration withinthat phase, at least in the short term until a new equilibriumis established between the apoplast and symplast (4). Atrelatively low cation contents, fluctuations in volume wouldbe of lesser consequence (Fig. 9).Although significant differences in apoplast solution K+

concentration, 5.9 to 11.8 mm (Table I), resulted from mod-ification of K+ levels in the nutrient culture solution, a highdegree of plant to plant variability was noted in pea. Consid-ering the relatively low cation sorption capacity of fixedanions within the mesophyll apoplast and the low volume ofthis compartment, fluctuations in apoplast solution phase ionconcentration are not surprising. These fluctuations mightresult from changes in the rates of ion transport processeswithin leaves, including xylem and phloem import-exportrates and flux kinetics into the symplast. For example, anincrease in the concentration of RbCl from 1 to 10 and 100mm taken up through the xylem for a 3-h period (Fig. 6)resulted in large increases in apoplastic solution K+ concen-trations, 4, 120, and 1100 mM Rb+, respectively, as deter-mined by elution analysis. Even though phloem transport wasblocked as a result of excising the leaf, the increase in Rb+concentration was of such high magnitude to suggest thatrelatively moderate fluctuations in xylem K+ concentration

0-J0

E

I-l0

-J

0

CL)

r-i

I-0cn

CL

zo-0-0 v-0.04mL g FW. Dm2.U63 -gm FW.-a v-0.04mLn'4 FPW. Dm3.60 -g" FW

150-

100-

50-

200-40.-ol. - 0.10 mL g. FW.ol. - 0.04 mL g. FW B

150-

100-

50f

0-0 2 4 6 8 10 12

TOTAL APOPLASTIC K+ (jsmol g-' FW)

Figure 9. Relationship of apoplastic K+ concentration within thesolution phase to net apoplastic K+ content as determined by acomputer simulation model. The top curves (A) illustrate the effect ofcation exchange capacity of the Donnan phase, 2.63 or 3.6 jlmol g-1fresh wt (FW), on the concentration of K+ within the solution phaseof the mesophyll apoplast assuming a volume of 0.04 mL g-' FW.The lower curves (B) illustrate the effect of volume of the solutionphase, 0.1 or 0.04 mL g-1 FW, on the K+ concentration within themesophyll apoplastic solution assuming a cation exchange capacityof 2.63 MAmol g9' FW.





(10 mm or less), such as might occur during a diurnal period,might produce significant changes in apoplast K+ content andconcentration.

Previously reported estimates of leaf apoplast K+ concen-

tration range from a low of 1 to 5 mm in barley (1), to 13 to17 mm in halophytic species (12) and a high of 100 to 200mm in guard cell walls (2) or within the pulvinar tissueapoplast oflegume species (8, 16). Komor et al. (15) indirectlyestimated an apoplastic concentration of 15 mm in matureleaves of Ricinus communis on the premise that sieve tubesolute concentration could be used as an indicator of apo-plastic solute supply.

In response to an increase in extracellular K+ concentration,a net influx of K+ into the mesophyll symplast will occur

until electrochemical equilibrium is reached (4, 24, 25). Thequestion that needs to be addressed is how rapidly a new

dynamic equilibrium can be established between K+ withinthe symplast and apoplast in situ. To answer this question, itis necessary to identify and quantify all the ion transportprocesses within leaf tissue which are influencing K+ contentand partitioning between these two compartments. Pitman(18) proposed that ion import and export rates via the vasculartissues into leaf lamina tissue, the sites, mechanisms, andkinetics for uptake into the symplast, and the capacity for cellto cell movement within the symplast would all play a role inthe spatial and time-course regulation of apoplastic ioniclevels. To the authors' knowledge, simulation models havenot been developed which integrate these ion transport vari-ables in order to gain a better understanding of the regulationof ion movement and accumulation within the various com-partments in leaf tissue. If indeed apoplastic solution phaseK+ concentrations are highly variable within the short-termand spatially within the mesophyll tissue, it is important todetermine what effects such fluctuations might have on phys-iological processes within mesophyll cells.

In summary, elution analysis using Argenteum pea was

found to be an effective and sensitive procedure for quantifi-cation of apoplastic K+ content and concentration withinmesophyll tissue. The apoplast in pea leaf tissue, however,has a relatively low capacity as an ion reservoir due to a

moderately low solution phase volume and a relatively lowconcentration of fixed negative charges within the Donnanphase. Apoplastic solution phase K+ concentration was foundto be responsive to changes in xylem sap concentration.

LITERATURE CITED

1. Bernstein L (1971) Method for determining solutes in cell wallsof leaves. Plant Physiol 47: 361-365

2. Bowling DJF (1987) Measurement of the apoplastic activity ofK+ and Cl- in the leaf epidermis of Commelina communis inrelation to to stomatal activity. J Exp Bot 38: 1351-1355

3. Cheung YNS, Tyree MT, Dainty J (1975) Water relations param-eters on single leaves obtained in a pressure bomb and someecological interpretations. Can J Bot 53: 1342-1346

4. Cosgrove DJ, Cleland RE (1983) Solutes in the free space ofgrowing stem tissue. Plant Physiol 72: 326-331

5. Epstein E (1972) Mineral Nutrition of Plants: Principles andPerspectives. John Wiley and Sons, New York, pp 36-39

6. Erwee MG, Goodwin PB, Van Bel AJE (1985) Cell-cell com-munication in leaves of Commelina cyania and other plants.Plant Cell Environ 8: 173-178

7. Flowers TJ, Liuchli A (1983) Sodium vs. potassium: substitutionand compartmentation. In A Uiuchli, RL Bieleski, eds, Inor-ganic Plant Nutrition. Encyclopedia of Plant Physiology (NewSeries), Vol 15B. Springer-Verlag, Berlin, pp 651-681

8. Freudling C, Starrach N, Flach D, Gradmann D, Mayer WE(1988) Cell walls as reservoirs of potassium ions for reversiblevolume changes of pulvinar motor cells during rhythmic leafmovements. Planta 175: 193-203

9. Fromm J, Eschrich W (1989) Correlation of ionic movementswith phloem unloading and loading in barley leaves. PlantPhysiol Biochem 27: 577-585

10. Gaff DF, Carr DJ (1961) The quantity of water in the cell walland its significance. Aust J Biol Sci 14: 299-311

11. Greene DW, Bukovac MJ (1971) Factors influencing the pene-tration of naphthaleneacetamide into leaves of pear (Pyruscommunis L.). J Am Soc Hortic Sci 96: 240-246

12. Harvey DMW, Hall JL, Flowers TJ, Kent B (1981) Quantitativeion localization within Suaeda maritima leaf cells. Planta 151:555-560

13. Hoch HC, Pratt C, Marx GA (1980) Subepidermal air spaces:basis for the phenotypic expression of the Argenteum mutantof Pisum. Amer J Bot 27: 905-911

14. Jachetta JJ, Appleby AP, Boersma L (1986) Use of the pressurevessel to measure concentrations of solutes in apoplastic andmembrane-filtered symplastic sap in sunflower leaves. PlantPhysiol 82: 995-999

15. Komor E, Kallarackal J, Schobert C, Orlich G (1989) Compari-son of solute transport in the phloem ofthe Racinus communisseedling and the adult plant. Plant Physiol Biochem 27: 545-550

16. Kumon K, Tsurumi S (1984) Ion efflux from pulvinar cells duringslow downward movement of the petiole ofMimosa pudica L.induced by photostimulation. J Plant Physiol 115: 439-443

17. Muhlethaler K (1967) Ultrastructure and formation of plant cellwalls. Annu Rev Plant Physiol 18: 1-24

18. Pitman MG (1975) Whole plants. In DA Baker, JL Hall, eds,Ion Transport in Plant Cells and Tissues. Elsevier, New York,pp 267-308

19. Pitman MG, Luttge U, Kramer D, Ball E (1974) Free spacecharacteristics of barley leaf slices. Aust J Plant Physiol 1: 65-75

20. Robinson JB (1971) Salinity and the whole plant. In T Talsma,RJ Philip, eds, Salinity and Water Use. John Wiley and Sons,New York, pp 193-206

21. Starrach N, Flach D, MayerWE (1985) Activity offixed negativecharges of isolated extensor cell walls of the laminar pulvinusof primary leaves of Phaseolus. J Plant Physiol 120: 441-455

22. Tanton TW, Crowdy SH (1972) Water pathways in higher plants.III. The transpiration stream within leaves. J Exp Bot 23: 619-625

23. Thompson WW, Platt KA, Campbell N (1973) The use of lan-thanum to delineate the apoplastic continuum in plants. Cy-tobios 8: 57-62

24. Widders IE, Lorenz OA (1983) Effects of leaf age and positionon the shoot axis on potassium absorption by tomato leafslices. Ann Bot 52: 489-498

25. Widders IE, Lorenz OA (1983) Effect of leaf age on potassiumefflux and net flux in tomato leaf slices. Ann Bot 52: 499-506

26. Zucker CE, Satter RL (1989) Light-promoted changes in apo-plastic K+ activity in the Samanea saman pulvinus, monitoredwith liquid membrane microelectrodes. Planta 179: 421-427

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