Plant Physiol. (1987) 85, 1040-10470032-0889/87/85/1040/08/$0 1.00/0

Osmotic Adjustment, Symplast Volume, and NonstomatallyMediated Water Stress Inhibition of Photosynthesis in Wheat1

Received for publication April 21, 1987 and in revised form August 3, 1987

ASHIMA SEN GUPTA AND GERALD A. BERKOWITZ*Department ofHorticulture and Forestry, Cook College, Rutgers University,New Brunswick, New Jersey 08903

ABSTRACT

At low water potential (,), dehydration reduces the symplast volumeof leaf tissue. The effect of this reduction on photosynthetic capacity wasinvestigated. The influence of osmotic adjustment on this relationshipwas also examined. To examine these relationships, comparative studieswere undertaken on two wheat cultivars, one that osmotically adjusts inresponse to water deficits ('Condor'), and one that lacks this capacity('Capelle Desprez'). During a 9-day stress cycle, when water was withheldfrom plants grown in a growth chamber, the relative water content ofleaves declined by 30% in both cultivars. Leaf osmotic potential (*,)declined to a greater degree in Condor plants. Measuring *, at full turgorindicated that osmotic adjustment occurred in stressed Condor, but notin Capelle plants. Two methods were used to examine the degree ofsymplast (i.e. protoplast) volume reduction in tissue rapidly equilibratedto increasingly low *,,. Both techniques gave similar results. With well-watered plants, symplast volume reduction from the maximum (found athigh *i, for each cultivar) was the same for Condor and Capelle. After astress cycle, volume was maintained to a greater degree at low *,, inCondor leaf tissue than in Capelle. Nonstomatally controlled photosyn-thesis was inhibited to the same degree at low I',, in leaf tissue preparedfrom well-watered Condor and Capelle plants. However, photosyntheticcapacity was maintained to a greater degree at low ,, in tissue preparedfrom stressed Condor plants than in tissue from stressed Capelie plants.Net CO2 uptake in attached leaves was monitored using an infrared gasanalyzer. These studies indicated that in water stressed plants, photosyn-thesis was 106.5% higher in Condor than Capelle at ambient 1C021 and21.8% higher at elevated external [CO21. The results presented in thisreport were interpreted as consistent with the hypothesis that there is acausal association between protoplast (and presumably chloroplast) vol-ume reduction at low *,, and low *I, inhibition of photosynthesis. Also,the data indicate that osmotic adjustment allows for maintenance ofrelatively greater volume at low '1,,, thus reducing lowvI,, inhibition ofchloroplast photosynthetic potential.

Recent gas exchange studies (6, 12, 13) have indicated that, ina number of crop species, inhibited chloroplast metabolism cansubstantially contribute to the overall inhibition of photosyn-thesis in plants exposed to water deficits. In vitro studies at thesubcelluar and cellular levels have indicated that stromal volumereduction due to dehydration, and not low *WI2 or I, per se, is

' New Jersey Agricultural Experiment Station, Publication No. 12149-11-87, supported by State and Hatch funds. This material is based uponwork supported by The National Science Foundation under Grant8414769.

2Abbreviations: ,,, water potential; ',, osmotic potential; RWC,relative water content.

associated with inhibition of chloroplast photosynthetic poten-tial. Working with isolated spinach chloroplasts, Berkowitz andGibbs (4) found that lowering the external solution *I' by 1 MPafrom isotonicity with a nonpenetrating solute inhibited photo-synthesis by 60%. Measurements indicate that stromal volumeis reduced by approximately 50% due to this treatment, as thestroma dehydrates until stromal *4' equilibrates with external 1'(3, 8). In contrast, when the external solution *I,. is lowered fromisotonicity by 1 MPa with a solute that freely penetrates thechloroplast membrane, stromal volumne is not reduced as thechloroplast equilibrates to the low *w. Under these conditions,photosynthesis is not significantly inhibited over physiologicalranges of *,, depression (4, 11). These data suggest that, in situ,the degree of water loss, or volume reduction experienced bychloroplasts as leaf *I' drops during a water deficit, may befundamentally related to the degree of inhibition in photosyn-thetic capacity. Recent studies at the cellular level support thisassertion. Kaiser (10) has shown that decreasing leaf I' hadvarying effects on both protoplast (symplast) volume and non-stomatally controlled photosynthetic capacity of leaf tissue pre-pared from a range of plant species. However, when photosyn-thetic capacity was plotted as a function of protoplast volume,all species demonstrated a similar relationship. Kaiser pointedout that in his studies, low *I'w-induced changes in chloroplastvolume likely coincided with reduction in protoplast volume.

In this study, attempts were made to extend our understandingof the relationship between changes in protoplast volume andphotosynthetic capacity at low *w. In addition, the work reportedhere addressed the hypothesis that an altered volume/*,,, rela-tionship may facilitate acclimation of cell metabolism to waterdeficits, with regard to maintaining photosynthetic potential atlow *".Many crop plant species are known to osmotically adjust in

response to leaf water deficits. Osmotic adjustment has beenshown by Ackerson and Hebert (1) to allow for greater turgorand photosynthesis at low leaf water potentials. Osmotic adjust-ment during a drought, which involves net solute accumulation,alters the RWC (or protoplast volume)/!,w relationship such thatgreater volume (higher RWC) is maintained when adjusted tissueis subsequently exposed to low *'. Therefore, we hypothesizedthat osmotic adjustment capability may allow acclimation ofphotosynthesis to low leaf *,I' by means other than the mainte-nance of leaf conductance. To test this hypothesis, studies wereundertaken on two cultivars of wheat that Morgan (15) previ-ously characterized as greatly different in their osmotic adjust-ment capability in response to water deficits. Condor was shownto be an osmotic adjuster, and Capelle Desprez lacked thiscapability.Matthews and Boyer (13) have shown that acclimation of

photosynthetic activity to low *I' involves an altered response ofchloroplast metabolism to low *',. However, they did not inves-

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WATER STRESS EFFECTS ON PHOTOSYNTHESIS

tigate whether or not leaf osmotic adjustment was involved withthis acclimation. Also, previous research in this laboratory hasdemonstrated that in situ water deficits can result in acclimationof isolated chloroplasts to low *I' (2). Preliminary data suggestthat an altered protoplast volume/*, relationship may mediatethis acclimation (5). Therefore, Capelle and Condor wheat plantswere evaluated for sensitivity of photosynthesis to low leaf *Pand the degree of photosynthetic acclimation that occurred inresponse to in situ water deficits. Attempts were made to associateacclimation of chloroplast photosynthetic capacity at low 1'with changes in the relationship between leaf 'P, and protoplastvolume that occur in tissue undergoing osmotic adjustmentduring water deficits.

MATERIALS AND METHODS

Plant Growth Conditions. Wheat (Triticum aestivum cvs 'Con-dor' and 'Capelle Desprez') seeds were planted in 15-cm plasticpots that contained 500 g of 1:1 (v/v) peat and vermiculite.Plants (thinned to 1/pot) were grown in a growth chamber at 10h light (21°C, 50% RH, 250 ,uE/m2.s) and 14 h dark (18°C, 50%RH). Pots were irrigated to runoff every other day, with com-mercial fertilizer (Peters) added to the irrigation water every 4thd. Experiments were initiated after 5 weeks of growth. Duringwater stress cycles, water was withheld from some pots; controlpots continued to be irrigated (with water only) during thisperiod. In all cases, the youngest, fully expanded (i.e. exposedauricle), nonsenescing leaf present on a tiller or the main culmof a plant was used for experiments.Water Status. Leaf ,t, was monitored with a pressure chamber

(Soil Moisture Equipment Corp.). Leaves were enclosed in plasticwrap, and the pressure chamber walls were lined with wet papertowels during measurements. Leaf I' was determined by meas-uring the *1' of frozen and thawed leaf discs (two/leaf) with aWescor HR33T microvoltmeter (operating in the hygrometricmode) and C-52 leaf chambers. Leaf turgor was calculated as thedifference between measured I,, and l' values. RWC was ascer-tained by measuring the fresh, rehydrated (minimum of 4 hfloating on distilled water at 4C) and dry (80°C for a minimumof 2 d) weights of five 8-mm diameter discs cut from a leaf. Allmeasurements were taken on the same leaf, and a minimum ofthree leaves taken from plants growing in different pots wereused as treatment replicates for all water status measurements.All leaves sampled for water status measurements were wipedwith moist paper towels (to remove any fertilizer salts) prior touse.

Pressure/volume curve analyses (18) were undertaken on re-hydrated leaves. For each treatment, three leaves taken fromdifferent pots were used. Whole leaves were recut twice at thebase under distilled water and left at 4°C in the dark in coveredbeakers for a minimum of 4 h. The leaves were then wrapped inplastic, placed in a pressure chamber lined with wet paper towels,and dehydrated by subjection to a series of overpressures. Xylemexudate was collected by placing plastic vials with absorbentcotton over the cut end of the leaf. Vials were immediatelycapped and weighed during the overpressure series. Leaf turgid(prior to pressure/volume analysis), fresh (immediately after theoverpressure series), and dry weights were also recorded. Totalxylem exudate recovered was typically 90% of the fresh weightloss of the leaf during the overpressure series. The analysis ofTurner (19) was used to calculate Is at 100% RWC (rehydratedleaves were at maximum turgor prior to use). Bound waterfractions were also calculated from the pressure/volume curves(19). These values were subtracted from the calculated RWC ateach balance pressure during the overpressure series, to generatea relationship between symplastic water content and leaf *P.These data are presented in this report as percentage ofreductionfrom the maximum symplastic water content as a function of

leaf 1W.In addition to pressure/volume analysis of Is at 100% RWC,

leaf osmotic adjustment was monitored by thermocouple psy-chrometry. Leaves were excised from plants (four/treatment),recut twice under distilled water, and allowed to rehydrate to fullturgor. Leaf *Is was measured on discs cut from these leaves.

Protoplast Volume. The technique developed by Kaiser (10)was also used to monitor cell volume reduction with declining*w. Five 4.6 mm discs were cut from leaves, vacuum-infiltratedtwice (with constant swirling until vigorous bubbling; approxi-mately 15 s), and then incubated for precisely 1 h at roomtemperature. The *,, of the infiltration solution was altered byvarying the sorbitol concentration. The solution also contained3.125 gCi 3H20 and 0.5 ,uCi '4C-sorbitol. After the infiltrationsolution was removed, the discs were rinsed with unlabeledinfiltration solution and then immediately frozen in liquid N2.After the liquid N2 evaporated, 5 ml of 96% ethanol with 10 Nformic acid was added to the tubes, and the label was leachedout of the discs by shaking (220 rpm on a rotary shaker) for 24h. Measurement of 3H20 leached out of the discs represented thesummed apoplastic and symplastic volume ofthe leaf tissue, andthe apoplastic volume was ascertained from the '4C-sorbitolvalue. The difference represented the symplastic (protoplast)volume of the leaf tissue at a given leaf Iw (the leaf *4,, equili-brated with the solution '4). All protoplast volumes reported inthis manuscript are the means of three different sets of discsprepared from three different plants.Leaf Slice Photosynthesis. Nonstomatally controlled photo-

synthetic capacity ('4CO2 fixation) of leaf tissue at a range of Iwwas monitored as described previously (16). Slices (0.75 mmaverage width) were prepared from two 7-mm-diameter leafdiscsand vacuum infiltrated twice briefly in 3.5 ml ofreaction mediumthat contained 20 mM Hepes-NaOH (pH 7.6), 1 mM MgCl2, 1mM MnCl2, 2 mM Na2EDTA, 2 mM NaH2PO4, and varyingsorbitol. After 15 min incubation in the dark, and 3 min in thelight (410 ,uE/m2. s) in a water bath at 25°C, 0.5 ml of reactionmedium containing ['4C]NaHCO3 (0.167 Ci/mol) was added(final NaHCO3 concentration was 15 mM), and the leaf sliceswere incubated for a further 10 min. Acid-stable 14C extractedfrom the leaf tissue was assayed using liquid scintillation spectro-photometry (Beckman LS100 counter). All reported data are themeans of at least two, and usually three, replications fromdifferent plants.

In Situ Gas Exchange. Net photosynthesis, stomatal resist-ance, and internal CO2 concentrations of attached leaves wereascertained as described previously (16) using an ADC (P. K.Morgan Instruments Inc., Andover, MA) portable infrared gasanalysis system. Light (800 ,gE/m2 * s) was provided by a sodiumvapor lamp with a 10-cm water heat filter, and leaf temperaturewas maintained between 19 and 22.5°C by placing the aluminumheat exchanger of the leaf chamber in an ice water bath.

RESULTS AND DISCUSSION

Leaf Water Status during In Situ Stress. The effect of a waterstress cycle on Condor and Capelle wheat plants is shown inFigure 1. During the stress, leafRWC declined similarly in bothcultivars; from near 100% 1 d after watering, to about 70% after9 d. These data indicate that, during the 9-d stress cycle, plantsof the two cultivars experienced similar levels of dehydration.However, leaf *I' and Is during the stress were markedly differ-ent in the two cultivars. In Condor plants, leaf Iw declined by1.27 MPa, from -0.97 MPa down to -2.24 MPa. Capelle leafIw declined from -0.69 MPa down to -1.1 1 MPa. Thus, thedata in Figure 1 indicate that, with Condor, a 30% decrease inRWC was associated with a far greater leaf Iw depression thanwith Capelle. As stated previously, Morgan (14) characterizedCondor plants grown in a growth chamber as capable ofundergo-

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SEN GUPTA AND BERKOWITZ

w

w-J

w

ccl

zw

I-

z0

a.

-J

<1.5zw

0

2.0Condorr

s5.6 C

a.w2.8 _

<0

a.4 Capelle

DAY OF STRESS

FIG. 1. Relative water content (A) of Condor (0) and Capelle (U)plants subjected to a 9-d water stress cycle. Leaf water potential (0, *)and osmotic potential (0, *) ofCondor (B) and Capelle (C) plants duringthe stress cycle is also shown. Plants were last irrigated on d 1. All datapresented are the means of at least three replications. Sample standarderrors (representative of all data points) are included with some datapoints to indicate the range of variation.

ing leaf osmotic adjustment during a 6 to 10 d stress cycle;Capelle plants did not osmotically adjust under similar condi-tions. Data presented in Figure 1 suggest that leaf declineduring the stress was greater in Condor plants, due to leaf Is

depression associated with osmotic adjustment. The decline inCondor leaf Is during the stress cycle was substantially greaterthan that occurring in Capelle, dropping by 0.94 MPa, whileCapelle leaf *' declined by only 0.15 MPa. Leaf decline inCondor resulted in turgor maintenance over most of the stresscycle; after 7 d of stress, turgor was similar to values calculatedfor well watered plants (i.e. compare the difference between Is

and I,, of Condor plants on d 1 and 8 in Fig. 1 B). According tothe data in Figure 1, a similar environmental water stress resultedin a similar level of dehydration in the two cultivars of wheattested. However, leaf response to this dehydration was qualita-tively different in Condor and Capelle in terms of 4s decline andturgor maintenance. This resulted in different IW,/RWC relation-ships in Condor and Capelle plants subjected to the environmen-tal stress.

Cultivar Differences in Stress Associated Osmotic Adjustment.Turgor maintenance during water deficits is not conclusive evi-dence of osmotic adjustment capability (17). Therefore, cultivar

differences in osmotic adjustment during water deficit wereevaluated by three different methods. Leaf Is at 100% RWC wasestimated using pressure/volume curves and by directly meas-uring the *,' of rehydrated leaves using thermocouple psychro-meters. Additionally, *Is decline during the stress cycle in excessof the degree induced by the solute-concentrating effects ofdehydration was estimated using Morgan's (14) analysis of theRWC/I' relationship in the two cultivars.Data presented in Table I show the calculated *I at 100%

RWC for well watered and stressed plants of the two cultivars.Both methods of analysis indicate that plants ofthe two cultivarsdiffered greatly in net solute accumulation and/or productioninduced by water stress. Pressure/volume and psychrometricanalyses both document osmotic adjustment in Condor duringthe stress cycle, but not in Capelle. With Capelle, psychrometricanalysis indicated no statistically significant difference betweenIs at 100% RWC in the leaves of well watered and stressedplants. According to psychrometric analysis, osmotic adjustmentwas nearly 1 MPa in Condor plants. The results of the pressure/volume analysis were somewhat different: osmotic adjustmentstill occurred in Condor, although to a much lesser extent (0.2MPa). This analysis suggests a net loss in solute level in Capelleleaves during the stress cycle; leaf Is at 100% RWC was estimatedto increase from -1.2 MPa to -0.92 MPa. Osmotic adjustmentwas found to occur in Condor, and not in Capelle, by examiningthe logarithmic transformation of stress-induced changes inRWC and *4s. In the absence of osmotic adjustment, I' at partialcell hydration follows the Van't Hoff relationship (where Vis thecell volume, and the subscript 100 represents the fully hydratedstate) (17): *I = ,I'oo Vloo//V. Substituting RWC for volume andlogarithmic conversion allows for a linear relationship (14): log

= log (*,'00 RWCoo) - log RWC. This analysis indicates thatwhen I' and RWC changes (occurring during dehydration) arerelated in a double logarithmic plot, solute accumulation or lossduring a stress cycle can be seen as deviations from the predictedlinear relationship. Changes in the bound water fraction couldalso influence this relationship (17), thus invalidating the analy-sis. However, pressure/volume curve results indicated that thebound water fraction was approximately 7 to 12% in bothcultivars and did not change in stressed plants (data not shown).This analysis ofthe RWC/IS relationship ofCondor and Capelleplants subjected to a stress cycle is shown in Figure 2. Withdeclining RWC, the slope of the line depicting this relationshipin stressed Condor plants is greater than the line that predictssolute-concentrating effects on Is; in Capelle, the slope is lower.These data indicate that during a stress cycle, Condor underwentosmotic adjustment, and Capelle leaves lost solutes. Morgan (14)has previously found that, during a stress cycle, Capelle leaveslost solutes, and the I' decline was less than should have occurredsolely due to dehydration.The results of the three analyses of stress-induced changes in

leaf *Is of Condor and Capelle all support Morgan's previousdocumentation of Condor as an osmotic adjuster and Capelle asnot. However, data presented in Table I indicate a substantialdifference in the results of the pressure/volume and psychrome-tric analyses. Both techniques are well documented methods ofestimating osmotic adjustment (7, 19). However, the analysispresented in Figure 2 indicates that Capelle leaves lost solutes(i.e. demonstrating negative osmotic adjustment) during thestress cycle and that osmotic adjustment in Condor was no morethan 0.4 MPa at the end of the stress cycle. Therefore, the datapresented in Figure 2 support the results of the pressure/volumecurve study. The psychrometric technique yielded Is values thatwere often less negative than the respective values calculatedfrom pressure/volume curves (Table I). The psychrometric tech-nique might have allowed for water injection into the intercel-lular space ofthe rehydrated leaftissue, which could have resulted

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WATER STRESS EFFECTS ON PHOTOSYNTHESIS

Table I. LeafOsmotic Adjustment Induced by a Water Stress CycleLeaf *s at 100% RWC was determined for control (unstressed) and stress-cycled plants. Leaf 4' at 100%

RWC was evaluated on rehydrated leaves using either pressure/volume analysis or thermocouple psychrometry.Presented data are the means + the SE of four replications for the psychrometric analysis and three replicationsfor the pressure/volume analysis.

ls at 100% RWC

Capelle Condor

Pressure/volume Psychrometric Pressure/volume Psychrometricanalysis analysis analysis analysis

-MPaControl 1.20 ± 0.05 0.54 ± 0.08 1.16 ± 0.10 0.57 ± 0.03Stress 0.92 ± 0.04 0.70 ± 0.12 1.36 ± 0.04 1.53 ± 0.16

2.5

c-EL

-i

zw

0a-

0

Cl)

0

2.01-

1.51-

1.0

100 90 80 70RELATIVE WATER CONTENT (%)

FIG. 2. Values of leaf osmotic potential and relative water contentfrom Figure 1 plotted on a double logarithmic graph. Values for Condorplants are open symbols, and closed symbols represent Capelle plants.The broken lines represent the theoretical decline in osmotic potentialcaused by the solute-concentrating effects of dehydration associated withdeclining relative water content.

in an underestimation of IS at full hydration.Osmotic Adjustment Effects on the 'J/Volume Relationship.

Data presented in Figure 3 indicate that leaves of well wateredCondor and Capelle plants had similar *I',/symplastic volumerelationships. It appears that, with both species, symplastic vol-ume was maximal between 0 and -1.0 MPa leaf *I', and thendeclined rapidly as the leaf tissue was brought to lower waterpotentials. Symplastic volume was calculated to be reduced byapproximately 20% with a further 0.5 MPa *I' decline and wasreduced by 40% when leaf *I', was brought to -2.0 MPa.The ',,/volume relationship of Condor and Capelle wheat

leaves was also determined using an entirely different method-ology. In this case, the protoplast volume was measured directlyusing dual-label vacuum infiltration. This technique was devel-oped by Kaiser (10) to allow the measurement of protoplastvolume of leaf tissue under conditions that also allow measure-ment of nonstomatally controlled photosynthesis. The results ofthis study are shown in Figure 4. These data also show that theprotoplast volume of leaf tissue of well-watered Condor andCapelle plants responds similarly to *I,, changes. As leaf *I' waslowered, the reduction from the maximal protoplast volume wasvirtually identical for both cultivars. The results presented inFigures 3 and 4 demonstrate, then, that prior to exposure to in

w

2 0

0

0

10CD)-Ja-

20

< 30

0

c-

wO0

1 2

LEAF WATER POTENTIAL (-MPa)FIG. 3. Symplastic (i.e. protoplast) volume at decreasing leaf water

potential ofleaves from well watered Condor (open symbols) and Capelle(closed symbols) plants. These data are compiled from pressure/volumeanalyses. The apoplastic water content of these leaves (from well wateredplants) and the leaves from stressed plants (Fig. 5) ranged between 7 and12%. No significant differences were found between cultivars and stressprehistory, with regard to the calculated apoplastic water fraction.

situ water deficits, Condor and Capelle leaves have a similar I'/

volume relationship. As pointed out by Kaiser (10) and Berkow-itz and Kroll (5), the similar response of both cultivars shouldresult in a similar degree of inhibition of nonstomatally con-trolled photosynthesis at low *I',.

Pressure/volume analysis and dual-label vacuum infiltrationwere used to determine the ',,,/volume relationship in leaves ofCapelle and Condor plants that had been subjected to the in situstress cycle characterized in Figure 1. These data are presentedin Figures 5 and 6. The results of both experiments demonstrateclearly that after exposure to an in situ stress cycle, Condor andCapelle leaves have different ',,,/volume relationships. Withdecreasing *1', protoplast volume is maintained to a greaterdegree in leaf tissue of prestressed Condor plants. Data presentedin Figure 5 indicate that, with Capelle tissue, near maximalvolume is maintained between 0 and -0.8 MPa. When leaf *I'drops below -0.8 MPa, volume decreases rapidly. With Condortissue, at leaf water potentials below -0.8 MPa, the decline inprotoplast volume is less pronounced. Data presented in Figure

Condor

Capelle

CONTROL

_

)k_0

)I-

U

1043

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SEN GUPTA AND BERKOWITZ

0ccU-

w

CO

w

cc

I0-

w

-j

0

t6-j

0a:a-

x

LEAF WATER POTENTIAL (-MPa)

FIG. 4. Dual-label, vacuum infiltration analysis of relative protoplastvolume of leaves from well-watered Condor (open symbols) and Capelle(closed symbols) plants at varying leaf water potential. Each data pointis the mean of three replications. The maximum protoplast volume was1.836 ml/dm2 for Condor and 1.750 ml/dm2 for Capelle.

w

-J

CR-J

c-

0-

Co

a:)

0

o

101

201

301

401

LEAF WATER POTENTIAL (-MPa)FIG. 5. Symplastic volume at decreasing leafwater potential of leaves

taken from Condor (open symbols) and Capelle (closed symbols) plantsexposed to a 9-d in situ stress cycle. These data are compiled frompressure/volume analyses.

6 show the same trends as the results shown in Figure 5, althoughthere are some significant differences. With the dual-label, vac-uum infiltration study (Fig. 6), maximal volume in tissue pre-pared from in situ stressed Condor plants occurred at -2.5 MPa.The results of this study also indicate that at low 'K, there is agreater difference between the degree of volume reduction ofCondor and Capelle tissue. Also, the data in Figure 6 indicatethat at water potentials greater than -2.5 MPa (i.e. approaching0 MPa), protoplast volume decreases. We believe this is anartifact of the measurement technique. When leaf tissue is vac-uum infiltrated with hypotonic solutions, the apoplastic spaceshould be no greater than when tissue is equilibrated with isotonicsolutions. Under hypotonicity, the cell wall should restrict theprotoplast volume, and the result should cause only nonphysio-

o~~~~~~~~~~0

Co

cr

Ulwcn)

a:

0R

w

0

11-CO

a.

a:a-

x:

1 2 3 4LEAF WATER POTENTIAL (-MPa)

FIG. 6. Dual-label, vacuum-infiltration analysis of relative protoplastvolume of leaves from Condor (open symbols) and Capelle (closedsymbols) plants exposed to a 9 d in situ stress cycle. Each data point isthe mean of three replications. The maximum protoplast volume was1.511 ml/dm2 for Condor and 2.159 ml/dm2 for Capelle.

logical increases in turgor. However, in these studies, the absolutevalues of the '4C-sorbitol space increased under apparently hy-potonic conditions (data not shown). Possibly, cell damage atthe cut surface or across the full volume of the disc underhypotonicity caused the cell membranes to become leaky, allow-ing for nonphysiological infiltration of the '4C-sorbitol into thesymplasm. This hypotonic effect was seen previously with leaftissue prepared from in situ stressed corn plants (5) and by Kaiser(10) in tissue prepared from some, but not all, plant speciestested in his study.

Despite the qualitative differences between the two methodsof determining *,.,/volume relationships, we conclude from thedata presented in Figures 3 to 6 that leaves of well wateredCondor and Capelle plants had similar *,,/volume relationshipsand that, after a stress cycle, protoplast volume was reduced atlow I,, less in Condor than in Capelle. This difference in cellularacclimation to low *I', was associated with differences in stressinduced osmotic adjustment capability. Condor plants demon-strated at least 0.2 MPa osmotic adjustment, and Capelle likelyunderwent negative osmotic adjustment (by 0.3 MPa) after ex-posure to a 9-d stress cycle that reduced RWC by 30% in plantsof both cultivars.Osmotic Adjustment-Induced Acclimation of Photosynthesis

to Low Leaf *i.' Data presented in Figure 7 show the responseof nonstomatally controlled photosynthesis of leaf tissue pre-pared from well watered Condor and Capelle plants. Photosyn-thesis for both cultivars was optimal at the highest leaf *I', tested;-0.95 MPa. With decreasing below -0.95 MPa, photosyn-thesis was inhibited. The response to Condor and Capelle leaftissue to low appears similar.A similar study was undertaken after exposure of plants to the

in situ stress cycle characterized in Figure 1. The results areshown in Figure 8. It appears that photosynthetic response tolow leaf in tissue prepared from prestressed Condor andCapelle plants is different. These results indicate that cellularlevel acclimation in Condor results in the maintenance of rela-tively higher chloroplast photosynthetic potential in Condor leaftissue, compared with Capelle. For example, photosynthesis at-2.5 MPa was inhibited by 9% in Condor and 30% in Capelle,compared with the respective controls. Maximal photosyntheticrates with Capelle occurred at -0.95 MPa leaf (the same aswith well watered plants, Fig. 7), while the leaf at whichmaximal photosynthesis occurred shifted down to -1.9 MPa

~~~~~~~STRESS*0

Condor

*Y b

Capelle 00

I I1 2 3 4

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WATER STRESS EFFECTS ON PHOTOSYNTHESIS

wI L + XQCONTROL

0: 10

70*_ Cael

w Z

UI) >- 30

<D

LEAF WATER POTENTIAL (-MPa)

FIG. 7. Nonstomatally controlled photosynthesis of leaf tissue pre-

pared from well watered Condor (open symbols) and Capelle (closed

symbols) plants at varying leafwater potential. Two separate experiments

are shown for Condor. The data are presented as percentage of inhibition

from the maximal rate for each cultivar. Each data point is the means of

three replications. The maximal photosynthetic rates for Condor and

Capelle were 97.8 and 89.6 Mmol C02/mg Chi * h, respectively. As stated

in the "Materials and Methods" section, a similar amount of leaf area

(0.77 cm2) was used for the assay of Condor and Capelle leaves at each

water potential tested. For these well-watered plants, this leaf area rep-

resented 24.1 Mg Chl for Condor and 24.5 MLg Chl for Capelle. Because

the Chl/area was similar for both cultivars, the maximum photosynthetic

rates (calculated on a Chl basis) of the two cultivars can be compared.0-012 ~

l oe adofro ondor

<E Capelle e t O

LEAF WATER POTENTIAL (-MPa)

FIG. 7. Nonstomatally controlled photosynthesis oflafvarissu wate-paredtafom welea watseprared foCondor (open symbols) and Capelle(coe

(closed symbols) plants exposed to a 9-d, in situ stress cycle. The dataare presented as percentage inhibition from the maximal rate for each

cultivar. The maximal photosynthetic rates were 74.2 and 111.49 amolC02/mg Chl- .h for Condor and Capelle, respectively. Each assay had0.77 cm2 leaf material, which contained 22.2ag Chl for Condor and 15.3

0g Chl for Capelle. These Chl values indicate that, in contrast to Condor,

there was substantial Chl loss on a leaf area basis in stressed Capelle

plants (compare these values with those in the legend of Fig 7). Whenthe Chl loss in Capelle tissue is accounted for, the maximal photosyn-

thetic rates of the leaf material prepared from stressed Condor and

Capelle plants were nearly identical (i.e. when calculated on an area

basis). Each data point is the mean of three replications.

with Condor. It should be noted that low *I', inhibition ofphotosynthesis was somewhat less in tissue prepared from pre-stressed Capelle plants than in the control (compare the Capelleleaves in Figs. 7 and 8). Some degree of acclimation occurred inCapelle leaves, despite the absence of osmotic adjustment or analtered protoplast volume/*,,, relationship, in response to in situstress. However, the acclimation was not as great as that occur-ring in Condor.Our interpretation of the data presented in Figures 3 to 8 is

consistent with the hypothesis that water stress-induced osmoticadjustment facilitates cellular level acclimation to low *'. Thisacclimation is likely mediated by the maintenance of relativelyhigher protoplast volumes when the prestressed tissue subse-quently is dehydrated. Volume maintenance at low *I, canreduce inhibition of nonstomatally controlled photosynthesis atlow I'. The differences in osmotic adjustment capability be-tween Capelle and Condor wheat plants (Table I) may be thebasis for the differences in volume maintenance and relativephotosynthetic rates at low *I', displayed by prestressed Condorand Capelle plants (Figs. 5, 6, and 8).Gas Exchange Studies. To ascertain if differences in cellular

acclimation to low 'I' in Condor and Capelle plants resulted indifferent in situ photosynthetic rates of water stressed plants, gasexchange studies were undertaken. The photosynthetic rates, leafresistance, and internal leaf CO2 concentrations of well watered(control) and water stressed Condor and Capelle plants are shownin Table II. These studies were undertaken at ambient (389 ppm)and high (680 ppm) CO2 concentrations. At ambient C02, pho-tosynthesis was 22.8% higher in Condor plants. This differencecould be due to the somewhat lower leaf resistance of controlCondor plants compared with control Capelle. When rates underwater stress are compared, the percentage increase of Condorover Capelle rises to 106.5%. This increase in the differencebetween the photosynthetic rate of Condor, as compared withCapelle when plants are stressed, cannot be attributed to stomataleffects. Leaf resistances of stressed Capelle and Condor plantswere similar, and internal CO2 concentrations were even lowerin Condor than Capelle under stress. The studies undertaken athigh [CO2] also indicate that, under water stress, Condor plantshave relatively greater photosynthetic capacity than Capelle, andthat this difference is not mediated by differences in stomatalresponse between the cultivars. At high [CO2], there was nostatistically significant difference between the photosyntheticrates of well watered Condor and Capelle plants. However, atthe end of a stress cycle, the Condor plants demonstrated asignificant 21.8% increase in photosynthetic rates compared withstressed Capelle plants. This increase in water stressed Condorplant photosynthesis, compared with stressed Capelle plants,occurred despite a slight decrease in the internal [CO2] in Condorplants (545 ppm) compared with Capelle (592 ppm). It shouldbe noted that the photosynthetic rates of both Condor andCapelle plants under stress at both high and ambient [CO2] weresubstantially lower than the respective controls. Therefore, theresults of this study should be interpreted with caution. Althoughin situ photosynthesis was found to be higher in water stressedCondor than in Capelle, and the factors mediating this effect areconcluded to be nonstomatal, there obviously were physiologicalconditions existing in the stressed leaves of both cultivars thatresulted in inhibited photosynthetic capacity. In a study byMatthews and Boyer (13), which documented nonstomatallymediated acclimation of photosynthesis to water stress in sun-flower plants, photosynthesis of prestressed plants at low leaf 'I,was still substantially inhibited from the photosynthetic rates ofcontrol plants at similar internal CO2 concentrations. In theirstudy, acclimation of photosynthesis of prestressed plants to lowleaf *I' was evidenced during a second in situ water stress cycle.Perhaps, if the stressed plants were rewatered and subjected to a

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SEN GUPTA AND BERKOWITZ

Table II. Photosynthesis (net CO2 Uptake), LeafResistance, and Internal Leaf[C02] ofAttached Leaves ofWater Stressed and Control (Unstressed) Condor and Capelle Plants under Ambient and High External CO2

All data are the means ± SE of four replications.

Capelle

Control Stress

Condor

Control Stress

Leaf resistance (s/cm)High C02 1.25 ±.11 16.2 ± 2.3 0.92 ± .09 24.2 ± 5.4Ambient CO2 1.09 ± .06 13.7 ± 3.0 0.65 ± .10 15.2 ± 2.7

Internal [CO2] (ppm)High CO2 526 ± 16 592 ± 7 545 ± 34 545 ± 32Ambient CO2 299 ± 9 359 ± 7 301 ± 10 339 ± 5

Photosynthesis (IAmoI/m2 _ s)High C02 16.84 ± 0.64 2.51 ± 0.11 17.25 ± 0.47 3.06 ± 0.36AmbientCO2 12.08 ± 0.58 1.23 ± 0.06 14.83 ± 1.36 2.54 ± 0.33

Stress Control% difference, Condor vs CapelleHigh CO2 + 21.8 + 2.4Ambient CO2 +106.5 +22.8

second stress cycle, the cellular level acclimation that occurredin Condor, as characterized in vitro (Figs. 3-8), would haveresulted in an even greater degree ofphotosynthetic rate enhance-ment in Condor as compared to Capelle. Nonetheless, we inter-pret the data presented in Table II as indicating that cellular levelacclimation in Condor plants undergoing osmotic adjustmentcan result in relatively higher in situ photosynthetic rates underwater stress compared with stressed Capelle plants.

CONCLUSION

The results presented in this report clearly identify an associ-ation between osmotic adjustment, altered *I'/protoplast volumerelationship, and maintenance of photosynthetic capacity at low'I'. It seems likely that the differences demonstrated betweenCondor and Capelle plants with regard to these parameters arenot coincidental. However, the occurrence of osmotic adjust-ment, maintenance of protoplast volume at low I', and areduction in low *I' inhibition of photosynthesis of stress-accli-mated Condor plants, and not in Capelle, is evidence of only anassociative relationship. The data presented in this report do notprove a causal relationship.There may be many lesions that affect chloroplast metabolism

under water stress, but we believe that at least some of theseinhibitory mechanisms are related to stromal volume reduction.To the extent that these volume-related lesions inhibit photosyn-thesis, osmotic adjustment-related volume maintenance at low*I', can mediate acclimation to low *,. This assertion does notpreclude cellular level acclimation to stress that may be mediatedby other factors. Matthews and Boyer (13) attributed the factormediating photosynthetic acclimation to low I,, in their studyto changes at the thylakoid level. Enhanced electron flow at lowI,v was demonstrated when naked thylakoids were prepared fromprestressed plants. Their results can be interpreted, then, asprecluding involvement of an altered *I',/protoplast volume inintact cells as mediating the acclimation they demonstrated invitro. Flower and Ludlow (7) found that osmotic adjustment-induced alterations in the *I'/RWC relationship of pigeonpearesulted in a reduction in the leaf ',v at which cell death occurred,although leaves that had osmotically adjusted to different degreesall died at a similar RWC. Berkowitz (2) found that low *'4inhibited photosynthesis to a lesser extent in chloroplasts isolatedfrom in situ stressed plants that had undergone osmotic adjust-ment than in plastids isolated from well watered plants. The datareported here support the hypothesis developed in this previousstudy that an altered stromal volume/*,, relationship mediated

chloroplast acclimation to low *,. This assertion assumes thatvolume changes in the chloroplast of Condor and Capelle leavesfollowed the changes in protoplast volumes monitored in thisstudy. Although theoretical (10, 15) and experimental evidence(2) exists to support this contention, we only speculate that thisoccurred here. An examination of this relationship in waterstressed leaves is an obvious goal of future research in this area.The results of these recent studies of water stress effects on

photosynthesis, in addition to the previously discussed work ofKaiser (10) and Berkowitz and Kroll (5), which delineated aclose association between protoplast volume and photosynthesis,and the results reported in this study suggest that a reappraise-ment of factors mediating water stress inhibition of photosyn-thesis may be in order. For over a decade, the much-cited reviewby Hsiao et al. (9) stood as a major analysis of the relativeimportance of factors contributing to the overall water stressinhibition of photosynthesis in plants. At the time, the majorityof the pertinent research reports indicated that low ,'-inducedstomatal closure was responsible for water stress inhibition ofphotosynthesis in most crop plants. In this light, Hsiao et al.concluded that the major effect of osmotic adjustment on pho-tosynthesis was mediated by the maintenance of leaf turgornecessary for stomates to remain open. They also concluded thathydration state (i.e. protoplast volume reduction) at low *4,, waslikely not involved in inhibited cell metabolism under waterdeficits. The results presented in this report suggest that volumechanges occurring during leaf water deficits may significantlyaffect photosynthesis. Also, the data presented here suggest thatosmotic adjustment capability may impact photosynthetic poten-tial at low *I'm in ways other than those currently established inthe literature.

Acknowledgment-We gratefully thank Dr. John Moseman of the United StatesDepartment of Agriculture at Beltsville for supplying the 'Condor' and 'CapelleDesprez' wheat seeds.

LITERATURE CITED

1. ACKERSON RC, RR HEBERT 1981 Osmoregulation in cotton in response towater stress. I. Alteration in photosynthesis, leaf conductance, translocation,and ultrastructure. Plant Physiol 67: 484-488

2. BERKOWITZ GA 1987 Chloroplast acclimation to low osmotic potentials. PlantCell Rep 6: 208-21 1

3. BERKOWITZ GA, M GIBBS 1983 Reduced osmotic potential effects on photo-synthesis. Identification of stromal acidification as a mediating factor. PlantPhysiol 71: 905-911

4. BERKOWITZ GA, M GiBBs 1983 Reduced osmotic potential inhibition ofphotosynthesis. Site specific effects of osmotically induced stromal acidifi-cation. Plant Physiol 72: 1100-1109

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WATER STRESS EFFECTS ON PHOTOSYNTHESIS

5. BERKOWITZ GA, KS KROLL 1986 Water stress induced osmotic adjustmentfacilitates resistance of non-stomatal controlled photosynthesis and alteredprotoplast volume response to low water potentials. Plant Physiol 80S: 552

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7. FLOWER DJ, MM LUDLOW 1986 Contribution of osmotic adjustment to thedehydration tolerance of water-stressed pigeonpea (Cajanus cajan (L.) millsp.) leaves. Plant Cell Environ 9: 33-40

8. HELDT HW, F SAUER 1971 The inner membrane of the chloroplast envelopeas the site of specific metabolite transport. Biochim Biophys Acta 234: 83-91

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10. KAISER WM 1982 Correlation between changes in photosynthetic activity andchanges in total protoplast volume in leaf tissue from hygro-, meso- andxerophytes under osmotic stress. Planta 154: 538-545

11. KAISER WM, G KAISER, PK PRACHAUB, SC WILDMAN, U HEBER 1981 Pho-tosynthesis under osmotic stress. Inhibition of photosynthesis of intactchloroplasts, protoplasts, and leaf slices at high osmotic potentials. Planta

153: 416-42212. KRIEG DR, RB HUTMACHER 1986 Photosynthetic rate control in sorghum:

stomatal and nonstomatal factors. Crop Sci 26: 112-11713. MATTHEWS MA, JS BOYER 1984 Acclimation of photosynthesis to low leaf

water potentials. Plant Physiol 74: 161-16614. MORGAN JM 1980 Osmotic adjustment in the spikelets and leaves of wheat. J

Exp Bot 31: 655-66515. MORGAN JM 1984 Osmoregulation and water stress in higher plants. Annu

Rev Plant Physiol 35: 299-31916. PIER PA, GA BERKOWITZ 1988 Characterization of leaf K' modulation of

nonstomatal mediated water stress inhibition of photosynthesis. Plant Phys-iol. In press

17. RADIN JW 1983 Physiological consequences of cellular water deficits: osmoticadjustment. In HM Taylor, WRT Jordan, TR Sinclair, eds, Limitations toEfficient Water Use in Crop Production. American Society of Agronomy,Madison, WI pp 267-276

18. RICHTER H 1978 A diagram for the description of water relations in plant cellsand organs. J Exp Bot 29: 1197-1203

19. TURNER NC 1981 Techniques and experimental approaches for the measure-

ment of plant water stress. Plant Soil 58: 339-366

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