Plant Physiol. (1986) 82, 995-9990032-0889/86/82/0995/05/$0 1.00/0

Use of the Pressure Vessel to Measure Concentrations of Solutesin Apoplastic and Membrane-Filtered Symplastic Sap inSunflower Leaves'

Received for publication October 28, 1985 and in revised form July 30, 1986

JOHN J. JACHETTA*2, ARNOLD P. APPLEBY, AND LARRY BOERSMADepartment ofCrop Science (J.J.J., A.P.A.) and Department ofSoil Science (L.B.), Oregon StateUniversity, Corvallis, Oregon 97331

ABSTRACI

A simple, repeatable, and accurate method is described for the collec-tion of apoplastic and membrane-filtered symplastic sap fractions, andfor the determination of the origin of these fractions within the leaf. Theapoplastic distribution patterns of the naturally occurring apoplastic leafsolutes, and the apoplastic dye PTS (trisodium 3-hydroxy-5, 8, 10-pyrenetrisulfonate) were compared. Aliquots of sap were expressed fromdetached sunflower leaves in a pressure chamber over intervals of 0.02to 0.04 megapascal. Three distinct fractions were detected in the ex-pressed sap volume. These were successively released and identified as apetiole-midrib fraction, a minor vein-cell wall fraction, and a mixedfraction consisting of a contribution from the minor vein-cell wall withan increasing proportion of membrane-filtered cell sap.

The ideal method for the collection ofan apoplastic sap samplefrom leaves would collect sap without altering the existing con-centrations of solutes within it. Alterations in the sap concentra-tions could arise from either the addition of a solvent, usuallywater or buffer, or from contamination by intracellular solutesfrom damaged or leaky cells. Generally, samples ofleafapoplasticsap have been obtained by analysis of the washing medium ofsmall leafpieces (10), and larger leaffragments with the epidermisremoved (5), by passing a buffer solution across abraded leafsurfaces (7), by infiltration and centrifugation of leaves (12), orby the expression of sap from leaves under pressure in 3 to 5 barincrements (1). The results of studies involving either the abra-sion of leaf epidermis (7) or the use of small leaf pieces may beconfounded by leakage of solutes from broken cells. Moreover,studies involving the movement of apoplastic sap into largevolumes of washing medium, or with infiltration of the tissueswith water or buffer, preclude the determination of actual con-centrations of apoplast sap solutes as they exist in the leaf. Theanalysis ofsap expressed from leaves during pressure dehydration(1) avoids this type of dilution of apoplastic sap. However, theuse oflarge pressure steps makes it impossible to determine whenthe expressed apoplastic sap begins to be diluted by plasma-lemma-filtered symplastic sap; also, the origin of the expressedsap, i.e. petiole-midrib, minor veins-cell walls and symplast,

'Contribution of the Oregon Agricultural Experiment Station, OregonState University, Corvallis, Technical Paper No. 7702.

2Currently, Sr. Plant Physiologist, Union Carbide Agr. Prod. Co.,P. 0. Box 12014, Research Triangle Park, NC 27709.

cannot be determined.In this study, a method is described that utilizes pressure

dehydration for the collection of samples of apoplastic sap oververy small pressure intervals. The samples are correct with respectto concentration and their origin within the leafcan be deduced.The distribution of naturally occurring apoplastic solutes andthe fluorescent dye PTS3 were used as models for this study. PTSwas chosen for this study because it is known to be confined tothe apoplast of the plant (6, 22), is not phytotoxic at the concen-trations used in this study (about 1-2 mg/g fresh weight) (17),and has been shown not to bind to cell wall materials (16). Thistechnique has been used to study the short-distance transport ofherbicides between the apoplast and symplast; the results havebeen presented in a separate report (9).

MATERIALS AND METHODS

Plant Material. Sunflower plants (Helianthus annuus L. cv'894') were grown from seed in 1-L containers for 21 d in amixture of 33% sand, 33% peat moss, and 33% clay loam soil.Plants were watered with Long Ashton's Solution (8) every 2 dfor the first 14 d, and daily thereafter. The plants were maintainedin a growth chamber with a 14-h photoperiod, 27°C day temper-ature, and 21°C night temperature. Photon flux density at theleaf blade level was 800 gmol m 2 s-' (cool-white fluorescentplus incandescent light). Midrib lengths of the first true leaveswere measured daily; all experiments were performed on the firsttrue leaves 2 to 3 d after midrib elongation ceased.

Pressure-Volume Determinations. P-V experiments were con-ducted as described by Scholander et al. (19, 20), Tyree andHammel (25), and Tyree and Dainty (24). Potential sources oferror for these procedures have been discussed by Cheung et al.(4) and by Tyree and Richter (26, 27).

Leaves were sampled before dawn in the growth chamber fromwhole plants that had been kept overnight in a saturated atmos-phere. To minimize both leaftemperature fluctuations and tissuewater loss during pressure alterations (30), the leaf was excisedfrom the plant and immediately placed in a plastic bag whichcontained a small amount of water (<2 ml), and had beenprehumidified by breathing into it until moisture condensed onthe sides; the atmosphere in the plastic bag was allowed toequilibrate with the atmosphere in the room for 2 min prior touse. The bag was then arranged around the leaf such that air wasexpressed and the opening could be sealed by the lower portionof the latex gasket in the pressure vessel lid. The bag containingthe leaf was then placed in a second dry bag in a similar manner

3Abbreviations: PTS, trisodium 3-hydroxy-5, 8, 10-pyrenetrisulfonate;P-V, pressure-volume; RWC, relative water content.

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Plant Physiol. Vol. 82, 1986

and placed in the pressure vessel (Soil Moisture EquipmentCorp., P. 0. Box 30025, Santa Barbara, CA); neither plastic bagprotruded through the stopper of the pressure chamber. Thepetiole length in all experiments was 50 mm. After the leaf wasplaced in the pressure vessel, a small amount of water (<1 ml)was placed at the point where the petiole protruded from thecover and the pressure in the vessel was increased to 0.02 MPa;the latex gasket was then tightened to the point where gas bubblesno longer escaped from around the petiole base. After a seal wasobtained, all water was removed from the pressure vessel lid byblotting with tissue paper. This measure ensured an air-tight sealwith a minimum of mechanical compression of the petiole. Agas mixture of 98% N2 and 2% 02 was used for pressurizing thechamber.For clarification, the term 'balancing pressure' is defined as

the pressure required to bring the water in the leaf back to thesurface of the cut petiole after the leaf was detached from theplant and placed in the pressure chamber. As such, it approxi-mates the total water potential of the leaf (25). Small increasesin pressure after the balance pressure had been attained causethe leaf water potential to become greater than zero, forcingwater to flow out of the living cells, and ultimately out of theleaf, through the cut petiole, in response to a water potentialgradient.

After an initial balance pressure was determined, the pressurein the chamber was increased in steps of 0.1 to 0.2 MPa over theprevious balance pressure. A period of 15 min was sufficient forcollecting a suitable volume of expressed sap, after which a newbalance pressure was established for the leaf by reducing thepressure until the meniscus receded just slightly from the cutsurface; the pressure was then increased in small pressure inter-vals until a new balance pressure was reached. Expressed sap wasabsorbed by a preweighed collecting device that consisted of a10-cm long glass tube filled with absorbent paper. The insidetube diameter was slightly larger than the petiole diameter. Thecollecting device was weighed immediately after each collection.The RWC was computed from the following formula:

Wo-Wd- VeRWC- Wo-Wd (10)

where Wo is the fresh weight of the leaf at full hydration (I =0), obtained by the extrapolation method of Ladiges (1 1), Wd isthe dry weight of the leaf after oven drying at 60°C for 24 h, andVe is the weight of sap expressed from the sample at variousbalance pressures. At the initiation of each experiment, leaf Iwas typically between -0.12 and -0.20 MPa. At the end of eachexperiment, the final weight of the leaf was measured. Whencomplete P-V curves were developed, the total weight of sapexpressed from the leaf, Ve (total), was within 5% of the initialfresh weight ofthe leafminus the final fresh weight; in subsequentexperiments, where sap was expressed to only -0.5 MPa I, thedifference was less than 2%.P-V analysis was performed on one leaf of each of five plants;

data were plotted as 1/* versusRWC (26, 27). A linear regressionwas performed on the four or five points of the P-V curve that,by inspection, appeared to be in the linear region of the graph.From this linear regression, the putative apoplastic water contentof the leaf was determined as the intercept at I/I = 0 on theRWC-axis.

Distribution of Apoplastic Solutes. Because it was importantto maintain the natural distribution of solutes in the leaf, anondisruptive technique to sample the midrib and cell wallapoplastic sap from intact detached leaves was developed. TheScholander-Hammel pressure vessel was found suitable for thisuse. The apoplastic concentrations of naturally occurring leafsolutes and PTS were studied by analyzing xylem exudate ex-pressed from the leaves, in very small increments, as they dehy-

drated under pressure.Leaves were excised, prepared, and placed in the pressure

chamber (Fig. 1) in the manner described for the P-V determi-nations. Before a leaf was placed in the pressure chamber, thecut end of the petiole was rinsed for 30 s in distilled H20 andthen blotted dry. After an initial balance pressure was deter-mined, pressure in the chamber was increased in steps of0.02 to0.04 MPa over the previous balance pressure. A period of 3 to 5min was sufficient for collecting a suitable sample of expressedsap, after which a new balance pressure was established for theleaf. In determining the new balance pressure, the chamber mustbe depressurized just to the point where sap has receded fromthe cut xylem surface; this was 0.01 to 0.02 MPa below the newbalance pressure; greater changes caused internal mixing of thexylem sap and precluded an accurate determination of the dis-tribution of the apoplastic solutes. Expressed sap was absorbedon a 0.32-cm2 filter paper disk (Wescor, Inc., Logan, UT), whichhad been placed on the cut end of the petiole protruding fromthe chamber, until the disk appeared saturated when viewedunder x 10 magnification. At saturation, the disk held about 8.0,ul of sap. A small vial packed with saturated tissue paper at thebottom was inverted over the petiole and disk to form a humid-ified chamber and retard evaporation from the filter paper disk.Upon full saturation, the disk was transferred immediately tothe sample chamber of a vapor pressure osmometer (WescorInc., model 51 lOC) for measurement. Slight volumetric errorsin estimating full saturation of the sample disk did not reducemeasurement accuracy (±2 mOs/kg), as the osmometer accom-modated sample volume variations in excess of 10% withoutnoticeable variation in the indicated osmolarity. The distribution

FIG. 1. Pressure vessel apparatus used to obtain sap efflux from leavesunder pressure.

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SAMPLING APOPLASTIC AND MEMBRANE-FILTERED SYMPLASTIC SAP

and concentration of apoplastic solutes with applied pressurewas determined over successive fractions for one leaf of each offive plants, and the results were averaged.

Distribution of FP1S. Plants were grown as previously de-scribed. Before plants were sampled, they were kept in a dark-ened, humidified growth chamber for 12 h at 21°C. At the endof the dark period, plants were removed from the growth cham-ber and all but the fully expanded first true leaves were removed.The plants were then excised under water at the soil surface.Shoots were transferred to flasks containing 100 FM Ca(NO3)2and kept in a dark growth chamber for 90 min to allow recoveryof plant I to approximately -0.15 MPa. Shoots and flasks werethen weighed and placed in a lighted growth chamber (300 ,molm-2 s_' at leaf blade level) for an additional 90 min which wasfound to be sufficient to allow transpiration to reach a steadystate. Shoots were then transferred to 20 ml scintillation vialscontaining 5 ml of 100 .M Ca(NO3)2 plus 200 mg/L PTS. Shootsand vials were weighed, and plants were maintained in the lightedgrowth chamber to allow absorption of the treatment solutionfor 90 min.

Following the 90-min PTS exposure, shoots and vials werereweighed to determine the amount of PTS uptake, sealed inprehumidified plastic bags, and placed in a dark growth chamberto retard transpiration and further uptake. After 1, 4, 7, or 10 h,shoots and vials were removed from the plastic bags, one leafwas excised, and sap was expressed and collected in the mannerdescribed for the apoplastic solute study. The remaining leaf wasimmediately mounted on blotter paper and frozen between sheetsof dry ice. Xylem sap from the excised leaf was expressed forapproximately 5 min; each successive balance pressure was 0.02to 0.04 MPa greater than the previous balance pressure. Xylemsap was collected on a filter paper disk under a humidifiedchamber as described previously, after which the filter paper wassealed in a scintillation vial. To determine Ve, the filter paperdisk and vial were weighed before and immediately after collec-tion of xylem sap. After sap was expressed, in increments, to 0.5MPa, the pressure in the chamber was released, the petiole wasquickly excised, and weights of the petiole and leaf blade wererecorded. Leaf parts were then mounted on blotter paper andfrozen between sheets of dry ice. Plant parts were subsequentlylyophilized to prevent redistribution ofthe PTS, and photographsof the leaves were made under UV light.PTS in the expressed xylem sap was extracted from the filter

paper disks in a 20 ml scintillation vial with 2 ml water, and theconcentration of PTS was measured at 404 nm using a VarianCary 219 spectrophotometer.

RESULTS AND DISCUSSION

Pressure-Volume Analysis. A typical P-V curve for a fullyexpanded sunflower leaf is shown in Figure 2. Such curves showthe relationship between the inverse of the balance pressure (1/I) and the cumulative weight of sap expressed (Ve) or the RWCof the leaf (25). Curves of this type typically exhibit an initialrapid linear decline, followed by a curvilinear section, and thena second, but much slower linear decline. Assuming no changesin cell wall elasticity, the initiation of the curved portion of theP-V relationship, following the initial rapid linear decline, is thepoint where some of the cells in the leaf begin to reach zeroturgor; the curve begins a slow linear decline after all the cells inthe leaf have lost their turgidity (4).

Initiation of the curvilinear portion of the P-V relationship forsunflower leaves, grown under the conditions described, occurredat -0.476 ± 0.041 MPa (point A, Fig. 2). Extrapolating thesecond linear portion of the P-V curve to the abscissa (point B,Fig. 2) estimates the volume which would be expressed at infinitebalance pressure. This point has been interpreted as the apoplas-tic water volume (3, 25). For the first fully expanded true leaves

of sunflowers, the putative apoplastic water content was foundtobe 11.16 ±0.93% RWC.The determination of apoplastic volume by this method has

not been completely free of criticism (26, 27). A number offactors can affect the linearity of the P-V relationship and theaccurate evaluation of apoplastic water volume. These factorsinclude: (a) the potential development of negative turgor in cellsduring pressure dehydration, (b) compression of the apoplastunder pressure, (c) nonideality of the osmotic reactions of thecell sap, and (d) random errors in I measurements. Tyree andRichter (27) have evaluated these factors as they may affect theaccuracy of apoplastic water volume determinations, and haveconcluded that estimations of apoplastic volume made in thismanner may not be highly accurate, though these potentialsources of error may not be serious provided that the measure-ments are carried out with suitable care. Since our values forapoplastic volume are comparable to published values computedfrom measurements taken from photomicrographs (2), we feelthat this analysis estimates a reasonable and useful value for thisparameter in our system.

Apoplastic Solute Distribution. The concentration of apoplas-tic solutes was determined for exudate samples which had beencollected on filter paper disks at balance pressures increasing insteps of 0.02 to 0.04 MPa as leaves dehydrated under pressure.The concentration of solutes in efflux showed two distinct pla-teaus, with respect to applied pressure (Fig. 3). The first plateauoccurred at 20.0 ± 3.7 mOs/kg and extended from the initialbalance pressure to 0.24 ± 0.02 MPa of applied pressure. Thevolume of sap expressed from the petiole alone after it had beenexcised with two simultaneous cuts from the stem and leaf andsubjected to a pressure of 1 bar, accounted for about 20% of thetotal volume of the first fraction expressed. The first plateau wasfollowed by a decline in osmolality to a second plateau at 11.3± 3.4 mOs/kg, and which extended from 0.33 ± 0.02 to 0.42 ±0.02 MPa applied pressure. The concentration of sap expressedfollowing the second plateau decreased with each increment ofapplied pressure to a value close to that of deionized H20 (3.3 ±2.0 mOs/kg).The major pathway of water movement through mesophyll

leaf tissue is thought to be through the cell walls (14, 23, 28, 29).While this may be the predominant pathway during transpiringconditions, the path ofwater movement in the pressure chambermay be different. Cheung et al. (4) and Strochine et al. (21) havesuggested that, after a pressure increment, water driven out ofliving cells would likely flow from cell to cell, across the mem-branes and the thin part of the common cell walls, to the nearestvascular bundle, and then out of the leaf through the vascularelements. This would have the effect of flushing solutes out ofthe cell walls adjacent to the minor veins with water forced outof living cells under pressure. Assuming that the plasmalemmahas a reflection coefficient for cell solutes which is close to unity,the cell membrane should act as a near-perfect semipermeablebarrier ( 17), thus water forced out of living cells should have thecomposition of deionized H20. As water is moved from themesophyll cells and into the xylem elements of the minor veins,the sap in the minor veins would be displaced and forced intoincreasingly larger vascular elements and ultimately out of thepetiole to the atmosphere, following a path of decreasing resist-ance. Stroshine et al. (21), modeling water movement out ofwheat leaves under pressure, showed that the larger vascularbundles represent paths of low resistance through which watercan travel rapidly, while the smaller intermediate veins have alarger resistance. Based on calculations of the expected velocitiesneeded to express sap from xylem at observed experimental rates,Stroshine et al. (21) calculated a Reynolds number for flowthrough the xylem to be 0.068. This value is well within therange of nonturbulent or laminar flow, thus very little mixing

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Plant Physiol. Vol. 82, 1986

FIG. 2. A typical P-V curve for asingle fully expanded sunflowerleaf. Point A estimates the initia-tion of zero turgor. Point B esti-mates the apoplastic water volumeon a percent basis. Regression for-mula refers to the second linearportion ofthe curve.

5 0

concentration, which results in a decrease in the solute concen-tration in the expressed sap, approximately that of deionizedH20. These expected results have been observed in this study,and support the concept of differential permeability of the plas-malemma to water and solutes (18).These data are consistent with the observations of Scholander

et al. (19) who measured the freezing point depression of ex-pressed sap in 26 species of flowering plants. They noted that"The first portion that comes out is contaminated with normalxylem sap." A similar observation was made by Ackerson (1),studying ABA movement in leaves. He noted that the initial

1080

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0 0.1 0.2 0.3 0.4 0.5 0.6

APPLIED PRESSURE (MPa)FIG. 3. Natural apoplastic solutes in exudates of sunflower leaves

subjected to pressure dehydration. The pressure in the chamber wasincreased in steps of 0.02 to 0.04 MPa over the previous balance pressureto obtain exudates. Samples were collected over a period of 3 to 5 min.Data for one representative replication.

between expressed volumes should occur as sap is expressedunder pressure. This model of water movement out of a leafunder pressure suggests that the first fraction of sap expressedfrom sunflower leaves, to approximately 0.24 MPa applied pres-sure in this study, contains solutes from the petiole and midribof the leaf. This would be followed by a fraction of water fromthe minor veins and cell walls. The concentration of this sapfraction is given by the second plateau of the apoplastic soluteefflux curve (Fig. 3). The sap that is expressed at still higherpressures must come from living cells and pass plasmalemmamembranes. Thus, this sap is expected to have a very low solute

I-

540

360

180

APPLIED PRESSURE (MPe)FIG. 4. Trisodium 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS) in ex-

udates of sunflower leaves subjected to pressure dehydration. The pres-sure in the chamber was increased in steps of 0.02 to 0.04 MPa over theprevious balance pressure. Samples were collected over a period of 5

min. Data from one representative replication.

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SAMPLING APOPLASTIC AND MEMBRANE-FILTERED SYMPLASTIC SAP

volume of expressed sap contained approximately 10 times asmuch ABA as sap obtained during later stages of dehydration,and that the osmotic potential of the expressed sap depended onthe balance pressure at which it was collected.The solute composition of the xylem sap is largely made up of

mineral ions, though many other substances occur (13). Figure3 indicates that the concentration of solutes in the leaf is highestin the petiole-midrib fraction and that the concentration in theminor vein-cell wall fraction is significantly less. This is thedistribution of ions which would be expected if the xylem streamwere depleted by active and facilitated removal of ions from theapoplast by the living cells ( 15). This view of ionic distributionin the apoplast is supported by the observation that if sunflowerplants remained in conditions which allowed no transpirationfor 48 h (100% humidity in the dark) prior to sampling, theconcentration of the first plateau was reduced to approximately12 mOs/kg and the second plateau did not occur (data notshown). This indicates that under conditions of no light andgreatly reduced transpiration, the leaf cells can deplete the cellwall portion of the apoplast of solutes almost entirely.PTS Distribution. Shoots were xylem-fed 200 mg/L PTS for

90 min and allowed to equilibrate in the dark at 100% humidityfor 1, 4, 7, and 10 h. Photographs of the nonexpressed leaves(not shown) which were opposite those used for subsequentxylem sap analysis were taken under UV light and indicated thatPTS was distributed throughout the leaf. This pattern of distri-bution did not change over the course of the experiment.Two concentration plateaus were observed in the efflux analy-

sis of PTS in exudates collected during the dehydration ofsunflower leaves under pressure (Fig. 4). The first plateau was at215 ± 16 mg/L and extended from the initial balance pressureto 0.22 ± 0.04 MPa of applied pressure. The second plateauextended from 0.28 ± 0.03 to 0.33 ± 0.3 MPa applied pressureand averaged 906 ± 267 mg/L.While the PTS efflux curves had plateaus at approximately the

same positions as the apoplastic solute efflux curve, the patternof PTS efflux from the leaf was quite different. The first fractionexpressed was approximately the same concentration as theoriginal experimental solution taken up by the excised shoots.This indicates that none of the PTS was removed from thetranspiration stream as the treatment solution moved into theleaves. This is consistent with the work of Strugger (22) andDybing and Currier (6), who showed that PTS is completelyexcluded from the symplast.The second fraction expressed was approximately 5 times more

concentrated than the first. As more sap was expressed, theconcentration of PTS in the xylem sap was successively reducedwith each increment of applied pressure to a low value. Therewere no significant differences in efflux patterns or plateauconcentrations between shoots allowed to equilibrate in the darkfor 1, 4, 7, or 10 h.These results suggest an accumulation of PTS in the minor

vein-cell wall fraction due to the nonvolatility of this materialand to its inability to penetrate the plasmalemma as water waslost through transpiration during the 90 min treatment period.The rapid dilution following the second plateau is interpreted asthe result of plasmalemma-filtered cell sap displacing the minorvein-cell wall solution as sap is expressed from the leaf.

Thus, the technique of pressure dehydration using small pres-sure increments can be used to collect three distinct fractions ofexpressed sap, i.e. an initial petiole-midrib fraction, followed bya minor vein-cell wall fraction, and a third mixed fraction whichis comprised of a decreasing minor vein-cell wall component and

an increasing component of membrane-filtered symplastic sap.This method should prove extremely useful in studying thedistribution, concentration, and movement of a large variety ofsubstances that translocate via the apoplastic route.

LITERATURE CITED

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2. BOYER JS 1967 Matric potential of leaves. Plant Physiol 42: 213-2173. CAMPBELL GS, RI PAPENDICK, E RABIE, AJ SHAYO-NGOWI 1979 A comparison

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4. CHEUNG YNS, MT TYREE, J DAINTY 1976 Some possible sources of error indetermining bulk elastic moduli and other parameters from pressure volumecurves of shoots and leaves. Can J Bot 53: 1345-1346

5. DELROT S, M FAUCHER, J-L BONNEMAIN, J BONMORT 1983 Nyethemeralchanges in intracellular and apoplastic sugars in Vicia faba leaves. PhysiolVeg 21:459-467

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7. GEIGER DR, SA SOVONICK, TL SHOCK, RJ FELLOWS 1974 Role of free spacein translocation in sugar beets. Plant Physiol 54: 892-898

8. HEWITr EJ 1966 Sand and water culture methods used in the study of plantNutrition. Tech. Comm. No. 22, Ed 2. Common Agric Bureaus, London

9. JACHETTA JJ, AP APPLEBY, L BOERSMA 1986 Apoplastic and symplastic path-ways of atrazine and glyphosate transport in shoots of seedling sunflower.Plant Physiol 82: 1000-1007

10. KURSANov AL, MI BROVCHENKO 1970 Sugars in the free space of leaf plates:their origin and possible involvement in transport. Can J Bot 48: 1243-1250

11. LADIGES PY 1975 Some aspects of tissue water relations in three populationsof Eucalyptus viminalis Labill. New Phytol 75: 53-62

12. MADORE M, JA WEBB 1981 Leaf free space analysis and vein loading inCucurbita pepo. Can J Bot 59: 2550-2587

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14. MOLz FT, IKENBERRY El 1974 Water transport through plant cells and cellwalls: theoretical development. Proc Soil Sci Soc Am 38: 699-704

15. PATE JS 1975 Exchange of solutes between phloem and xylem and circulationin the whole plant. In MH Zimmerman, JA Milburn, eds, Encyclopedia ofPlant Physiology. Transport in Plant I: Phloem Transport. Springer-Verlag,New York, pp 451-468

16. PETERSON CA, LV EDGINGTON 1976 Entry ofpesticides into the pl nt symplastas measured by their loss from an ambient solution. Pestic Sci 7 483-491

17. PETERSON CA, M GRIFFITH, NPA HUNER 1985 Permeability of the suberizedmestome sheath in winter rye. Plant Physiol 77: 157-161

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20. SCHOLANDER PF, HT HAMMEL, EA HEMMINGSEN, ED BRADSTREET 1964Hydrostatic pressure and osmotic potentials in leaves of mangroves andsome other plants. Proc Natl Acad Sci USA 51: 119-125

21. STROSHINE RL, JR COOKE, RH RAND, JM CUTLER, JF CHABOT 1976 Mathe-matical analysis of pressure chamber efflux curves. Proc Am Soc Agric EngPaper No 794585, Ithaca, NY

22. STRUGGER S 1949 Praktikum der Zell-und Gewebephysiology der Pflanze.Springer-Verlag, Berlin

23. TANTON TW, SH CROWDY 1972 Water pathways in higher plants. III. Thetranspiration stream within leaves. J Exp Bot 23: 619-625

24. TYREE MT, J DAINTY 1973 The water relations ofhemlock (Tsuga canadensis).II. The kinetics of water exchange between the symplast and apoplast. CanJ Bot 51: 1481-1489

25. TYREE MT, HT HAMMEL 1972 The measurement of turgor pressure and thewater relations of plants by the pressure bomb technique. J Exp Bot 23:267-282

26. TYREE MT, H RICHTER 1981 Alternate methods of analyzing water potentialisotherms: some cautions and clarifications. I. The impact of nonidealityand of some experimental errors. J Exp Bot 32: 643-653

27. TYREE MT, H RICHTER 1982 Alternate methods of analyzing water potentialisotherms: some cautions and clarifications. II. Curvilinearity in water po-tential isotherms Can J Bot 60: 911-916

28. WEATHERLY PE 1963 The pathway of water movement across the root cortexand leaf mesophyll of transpiring plants. In AJ Rutter, ed, Symposium ofthe British Ecological Society. John Wiley & Sons, New York

29. WEATHERLY PE 1965 The state and movement of water in the leaf. Symp SocExpBiol 10: 157-184

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