chloroplast osmotic adjustment and water stress effects on … · water stress effects on...

Plant Physiol. (1988) 88, 200-2060032-0889/88/88/0200/07/$01.00/0

Chloroplast Osmotic Adjustment and Water Stress Effects onPhotosynthesis'

Received for publication September 10, 1987 and in revised form April 19, 1988

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

ABSTRACT

Previous studies have suggested that chloroplast stromal volume re-duction may mediate the inhibition of photosynthesis under water stress.In this study, the effects of spinach (Spinacia okracea, var 'WinterBloomsdale') plant water deficits on chloroplast photosynthetic capacity,solute concentrations in chloroplasts, and chloroplast volume were stud-ied. In situ (gas exchange) and in vitro measurements indicated thatchloroplast photosynthetic capacity was maintained during initial leafwater potential (I,,) and relative water content (RWC) decline. Duringthe latter part of the stress period, photosynthesis dropped precipitously.Chloroplast stromal volume apparently remained constant during theinitial period of decline in RWC, but as leaf I,, reached -1.2 megapas-cals, stromal volume began to decline. The apparent maintenance ofstromal volume over the initial RWC decline during a stress cyclesuggested that chloroplasts are capable ofosmotic adjustment in responseto leaf water deficits. This hypothesis was confirmed by measuringchloroplast solute levels, which increased during stress. The results ofthese experiments suggest that stromal volume reduction in situ may beassociated with loss of photosynthetic capacity and that one mechanismof photosynthetic acclimation to low I,, may involve stromal volumemaintenance.

Chloroplast metabolism can be inhibited in plants subjectedto water deficits (11). These lesions can substantially contributeto the overall inhibition of photosynthesis in leaves of droughtedplants (8). Subcellular studies have indicated that low *"2 or I'may not directly inhibit photosynthetic capacity (6, 15, 20).Rather, these reports suggest that in water-stressed leaves, stromalvolume reduction may be the causal factor mediating photosyn-thetic inhibition.

Theoretical considerations (18) indicate that, with decliningleaf 'I' in situ, chloroplast volume reduction should essentiallyparallel relative water content decline (or more precisely, proto-plast volume reduction which occurs during cell dehydration).This assumption provided the basis for recent analyses of therelationship between protoplast volume and photosynthesis atlow 'I', (7, 14, 23). These studies have demonstrated that in leaftissue equilibrated to low '', a positive correlation exists betweeninhibition ofphotosynthesis and the degree of protoplast volumereduction. One mechanism facilitating cellular acclimation to

' New Jersey Agricultural Experiment Station, Publication No. 12149-14-87, supported by State and Hatch funds. This material is based uponwork supported by the National Science Foundation under grants DMB8414769 and 8706240.

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

low I' may be the maintenance of greater protoplast volume atlow I'. In a wheat cultivar that osmotically adjusted during anin situ drought cycle, subsequent exposure of leaf tissue to low*I' caused less reduction in both protoplast volume and nonsto-matally controlled photosynthesis (23). In a cultivar of wheatthat did not osmotically adjust, this acclimation was not dem-onstrated. These studies assumed that protoplast volume meas-urements reflected the degree of stromal volume reduction oc-curring at low 'I'.

In the study reported here, an attempt was made to directlyexamine the relationship between stromal volume reduction andphotosynthetic capacity at low I'.The photosynthetic capacity of chloroplasts isolated from

water-stressed plants is less inhibited in low I, medium thanthat of chloroplasts isolated from well-watered plants (3). Theshift in the optimum Is in vitro for chloroplast photosynthesiswas similar to the magnitude of leaf Is decline occurring in thedroughted plants. These results (3, 23) led to the hypothesis thatone aspect of both cell injury, and acclimation to low '', maybe mediated by the degree to which stromal volume is reducedduring leaf dehydration. As leaf Iw and *Is decline during waterstress, solutes could be accumulated and/or produced in thestroma. This could allow for maintenance of stromal volumeand photosynthetic capacity at low external Is. Alternatively,the chloroplast could dehydrate (to the same or a different extentas occurs in the rest of the cell) as stromal I,, equilibrates withexternal Is. Or, these two activities could be occurring simulta-neously, or at different phases of a drought episode.We attempted to examine the extent of "chloroplast osmotic

adjustment" during drought in two different ways. Intact chlo-roplasts were isolated from leaves of plants at different timesduring a drought cycle in media made isotonic to the decliningleaf I'. These preparations were used to measure stromal vol-umes and stromal osmoticum concentrations. These measure-ments allowed for calculation of stromal I' in droughted leaves.Studies were also undertaken to relate the degree of stromalvolume reduction to the level of inhibition in chloroplast pho-tosynthetic capacity during water stress.

MATERIALS AND METHODS

Plant Material. Spinach seeds (Spinacia oleracea, var 'WinterBloomsdale') were germinated in flats of vermiculite for 1 week.Seedlings (3/pot) were transplanted to pots containing 4.3 dmi3of 1: 1 peat/vermiculite in a growth chamber. Plants were irri-gated with standard commercial (Peters) fertilizer three times perweek and once with just water. The conditions in the growthchamber were: 22C and 50% RH constant, with an 11 h lightperiod (250 AE/m2/s). Plants were used after 6 to 8 weeks. Fullyexpanded, nonsenescing leaves were used for all measurements.Water stress cycles were initiated by withholding irrigation fromsome pots. These drought episodes generally lasted for 8 to 14 d.Water Relations. During the water stress cycles, leaf water

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WATER STRESS EFFECTS ON PHOTOSYNTHESIS

status was monitored by taking measurements 5 to 6 h into thelight period. Leaf I' was monitored with a pressure chamber(Soil Moisture Equipment Corp., Santa Barbara, CA). Leaveswere enclosed in plastic wrap, and the pressure chamber wallswere lined with wet paper towels during measurements. Leaf Iswas determined by measuring the 'I', of frozen and thawed leafdiscs (two/leaf) with a Wescor (Logan, Utah) HR33T microvolt-meter (operating in the hygrometric mode) and C-52 leaf cham-bers. Leaf turgor was calculated as the difference between meas-ured TI' and 'Is values. RWC was ascertained by measuring thefresh, rehydrated (minimum of 4 h floating on distilled water at4°C) and dry (80°C for a minimum of 2 d) weights of five 8-mmdiameter discs cut from a leaf. Control experiments indicatedthat between 4 and 24 h after spinach leaf discs (cut from bothwell-watered and stressed [50% RWC] plants) were placed ondistilled water, turgid weights were maximal and did not change.All measurements were taken on the same leaf, and a minimumof three leaves taken from plants growing in different pots wereused as treatment replicates for all water status measurements.Water stress-induced leaf osmotic adjustment was determinedby placing leaves (petioles were recut twice under water) ofstressed and well-watered plants in distilled water at 4°C for 24h. After rehydration, three discs were cut from each leaf fordetermination of 'Is at 100% RWC. Four separate leaves wereused for each treatment; 12 measurements of 's at 100% RWCwere therefore obtained for each treatment. All leaves sampledfor water status measurements were wiped with moist papertowels (to remove any fertilizer salts) prior to use.In Situ Photosynthesis. Photosynthetic rates of attached leaves

were monitored using an A.D.C. infrared gas analyzer, massflowmeter, and broadleaf chamber (P.K. Morgan Inst., Andover,MA) set up as an open system. Air with varying [CO2] wassupplied to the system using an A.D.C. GM602 gas blender.Flow through the gas blender from compressed air cylinderscontaining either 950 or 100 ppm [C02] was maintained at 0.14MPa, and the gas mixture was supplied to the flowmeter at aflow rate (500 mL/min) in excess of that circulated through theanalyzer. Net CO2 uptake and leaf transpiration were monitoredat 1700 to 2000 ytE/m2/s, 20 to 25°C air temperature, and flowrates of 400 mL/min over 6.25 cm2 leaf area. Photosyntheticrates and internal leaf CO2 concentrations were calculated asdescribed previously (19). Four leaves taken from different potswere used as treatment replicates. Photosynthetic measurementsat a range of external [CO2] were made on the same leaves.Measurements were taken 4 to 5 h into the light period.

Chloroplast Isolation and Photosynthetic Measurements. Dur-ing water stress cycles, intact chloroplasts were isolated fromleaves as described previously (4) except that the isolation me-dium Is was matched to the leaf Ts. Leaf 'Is was measured onthese days 3 h into the light period, and chloroplasts were isolated5 h into the light period (i.e. as soon as Is was measured andmedia prepared). In addition to sorbitol, the isolation mediacontained 50 mM Hepes-NaOH (pH 6.8), 2 mM Na2EDTA, 1mM MgCl2, and 1 mm MnCl2. Photosynthesis was measuredusing 02 electrodes (Decagon, Pullman, WA) at 1500 ,AE/m2/sand 25°C in media containing varying sorbitol, 5 mM NaHCO3,0.25 mm KH2PO4, 1000 units/mL catalase, 50 mm Hepes-NaOH(pH 7.6), 2 mm Na2EDTA, 1 mM MnCl2, and 1 mM MgC92.Assay volume was 1 mL and contained chloroplasts equivalentto 35 ,ug Chl. Chloroplast intactness was monitored at 3000 ,uE/m2/s by measuring 1 mm K3FeCN-supported 02 evolution rates(in the presence of 5 mm NH4Cl used to uncouple photophos-phorylation) of chloroplasts (15 ,ug/mL). The media used forintactness assays were either made isotonic to leaf 'Is withsorbitol, or they lacked sorbitol. The difference between thesetwo rates was taken to be a measure of intactness.

Stromal Volume. The method of Heldt (12) was used to

ascertain the stromal volume of chloroplasts isolated in mediamade isotonic to the leaf I, during drought cycles. Chloroplastswere suspended in isolation medium with varying sorbitol afterisolation. Aliquots of this resuspension were mixed with 15 ,uCi/mL 3H20 and incubated for 5 min, and then 10 ,uCi/mL ["4C]sorbitol was added (the ['4C]sorbitol stock solution containedsufficient 3H20 to maintain a constant 3H concentration). Thesolution Is was maintained constant throughout these steps.After ['4C]sorbitol addition, 200 iL of the mixture (containing40 ,g Chl) was layered into 400 ,L microfuge tubes which had70 ,uL of 550 Dow Corning silicone oil layered on top of 20 ,Lof 14% HC104. The 550 silicone oil had a specific density of 1.07and viscosity of 125 cs. Tubes were then centrifuged in a Beck-man Microfuge B for 30 s. For each volume measurement, fourmicrofuge tubes were used as replicates. After centrifugation,aliquots ofthe incubation media above the oil layer were sampledfor radioactivity. The tubes were frozen, and then cut in the oillayer. The microfuge tube tips containing the HC104 with pel-leted chloroplasts were then placed in 1.5 mL microfuge tubeswith 380 ,uL H20, vortexed to resuspend the pellet and werecentrifuged to pellet any oil carryover. Aliquots of the aqueoussupernatant were sampled for radioactivity. Radioactivity meas-urements were made using a Beckman 3801 liquid scintillationspectrophotometer programmed for dual label, dpm counting.The silicone oil used in this study was not the standard oil

mixture used previously in this laboratory (19), which did notsupport the high specific density incubation media when centri-fuged in the microfuge. Therefore, precautions were taken toensure that volume measurements were not influenced by incom-plete chloroplast centrifugation through the oil layer. For eachvolume measurement, four additional microfuge tubes weremeasured with 20 gL of 40% Percoll (Pharmacia, Piscataway,NJ) replacing the HC104 layer. The tubes were used in a fashionsimilar to the tubes used for volume measurements, except thatno radioactivity was added to the chloroplast suspension. Thesetubes were carefully cut in the Percoll layer to avoid oil carryover.The chloroplasts were resuspended in 1 mL of 80% acetone and,after centrifugation, Chl was read using the method of Arnon(2).

Solute Measurements. Total stromal solutes were measuredusing chloroplasts isolated from leaves of plants exposed to insitu water stress cycles. For this study, the silicone oil microcen-trifugation technique was modified to allow for separation oflarge quantities of chloroplasts from the isolation medium. Sixbatches of 12 to 14 g deribbed leaves were cut into a 50 mL/batch of isolation medium on ice. After chloroplast isolation, thepellets were pooled and resuspended in 15 mL isolation medium.

Three mL of this resuspension was layered above 2 mL ofsilicone oil mixture (specific density of 1.0429) with a ratio (byweight) of 0.14167:0.6917:0.1667 of 200 (2 cs):550:710 DowCorning oils in each of five 16 mL, polyallomer, high-speedcentrifuge tubes. After centrifugation at 9000g in a Sorvall HB-4 swinging bucket rotor for 1 min, the tubes were frozen. Thefrozen tubes were then cut in the lower portion of the oil layerwith sharp knives (the blades were kept constantly hot by heatingover a flame and were changed often). Centrifuge tubes made ofpolyallomer were found to be more easily cut than the standardpolypropylene or polyethylene tubes. The chloroplast pellets wereresuspended in 1.5 mL of water and were centrifuged at 9500gfor 5 min. The supernatant was concentrated by drying in a ovenuntil it was reduced to 200 ,uL. Osmolality of the concentratedsupernatants was determined with an Osmette A osmometer(Precision Systems, Sudbury, MA). Total stromal solute concen-tration was calculated by assuming chloroplast stromal volumes,which were determined in separate experiments for plastidsisolated from well-watered and stressed plants. The ['4C]sorbitol(i.e. extrachloroplastic) volume measurements obtained in the

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SEN GUPTA AND BERKOWITZ

stromal volume studies were used to account for the contributionof isolation medium solutes in the chloroplast pellets. Threechloroplast pellets were used as replicates for solute measure-ments.The remaining chloroplast pellets were used to determine

stromal concentrations of specific solutes. For amino acids andreducing sugars, two pellets were used as replicates. Cations andanions were measured in only one pellet per treatment. Foramino acids and sugars, pellets were resuspended in 1.5 mLwater and centrifuged at 9500g for 5 min. One mL of thesupernatant was used for analysis. Amino acids were determinedusing the method of Moore and Stein (17). Proteins were precip-itated out with TCA. Amino acids were measured at 570 nm(Perkins-Elmer Lambda 3 spectrophotometer) after reaction withninhydrin. Isoleucine was used as a standard and for calculationofmolar concentrations. Reducing sugars were determined usingthe procedure of Somogyi (25), which involved sugar reactionwith copper and arsenomolybdate, and spectrophotometricmeasurement at 525 nm. Glucose was used as a standard, andglucose-6-phosphate was used for calculation of molar concen-trations.

Cations and anions were determined from 10 mL resuspen-sions of chloroplast pellets. Cations were determined usingatomic absorption spectroscopy (Perkin-Elmer 2280). For K+determination, the final supernatant dilution contained 0.2%(w/v) LaCl3. Anions were determined using ion exchange chro-matography (Dionex 2010i) with an AG4A prepacked columnand 2.2 mm Na2CO3/0.75 mm NaHCO3 eluent. ['4C]Sorbitol(i.e. extrachloroplastic) volume measurements were used to ac-count for the contribution of incubation medium K+, Mg2+, andCl- to stromal extracts. Specific solute concentrations were alsodetermined for water-stressed and well-watered leaves. LeafRWC was used to calculate molar concentrations. For reducingsugars, cations, and anions, leaves were ground in liquid nitrogen,dissolved in 10 mL of water, and centrifuged at 10,000g for 10min. The supernatant was used for analysis. For leaf reducingsugars, the mol wt of glucose was used to convert measurementsto molar concentrations. For amino acid determination, theground leaf material was further homogenized in 3 mL of 50mM Tris-HCl (pH 7.6), brought to 15 mL with more buffer, andcentrifuged at 10,000g for 30 min. For all experiments wherereplicates were taken, the data are reported as means ± the SE.All reagents were purchased from Sigma except for Hepes (Re-search Organics). Labeled compounds were obtained fromI.C.N., Inc., and silicone oils from William F. Nye, New Bedford,MA.

RESULTSStress Effects on Photosynthesis. Data presented in Figure 1

show the leaf water status of spinach plants during a typical insitu stress cycle. Leaf 'I', declined from -0.37 to -1.60 MPaafter 1 days without water. Leaf I, declined from -0.93 to-1.64 MPa over this period. Although leaf turgor declined, itremained positive over the entire stress period (Fig. Ib). Substan-tial dehydration (RWC reduction of 30%, Fig. IA) was associatedwith the 1.23 MPa decline in leaf *I',. Data presented in Table Iindicate that during in situ stress cycles, spinach plants undergoleaf osmotic adjustment. At the beginning of a stress cycle, leaf*I' was -0.38 MPa, and leaf Is at full turgor was -0.93 MPa.At the end of a stress cycle, when leaf 'K declined by 1.12 MPa,leaf osmotic adjustment was 0.31 MPa. Plants kept well wateredover this time period showed no change in 'K or leaf I,, at fullturgor (Table I).

Photosynthesis of attached leaves at a range of external [CO2](and calculated internal [CO2]) was measured on several daysduring the stress cycle shown in Figure 1. Calculation of leafphotosynthetic rates at similar internal leaf [Cr2] allows for

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1 3 5 7 9 11DAY OF STRESS

FIG. 1. Leaf RWC (A), turgor (B), and 'I', (closed symbols) and 's(open symbols) (C) during an in situ water stress cycle. Plants were lastwatered on d 1. All data (except leaf turgor, which is a calculated value)are means of at least three replications ± the SE.

Table I. LeafOsmotic Adjustment in Water-Stressed Spinach LeavesLeaf '' at full turgor was measured on well-watered, control plants

prior to a stress cycle, the same plants after withholding water for 13 d,and age control plants that had been kept well-watered over the 13 dperiod. Leaf 'w was measured prior to hydration of leaves to full turgor.All data are means of four replicate leaves ± SE.

Lea*.at ull Degree ofTreatment Leaf LeafHyd at Full Osmotic

Adjustment-MPa -MPa MPa

Prior to stress 0.38 ± 0.02 0.93 ± 0.08Age control 0.41 ± 0.01 0.94 ± 0.06 0.01Stressed 1.50 ± 0.05 1.24 ± 0.03 0.31

evaluation of treatment effects on chloroplast photosyntheticcapacity in situ (8). As found previously with cotton plants (13),photosynthetic rates of the well-watered and stressed spinachplants were at or near maximum at internal [CO2] of between500 and 550 ppm (data not shown). The photosynthetic rateduring the stress cycle of attached leaves at approximately 300and 500 ppm internal leaf CO2 concentration is shown in TableII. Over the first six days of the stress cycle (when declinedby 0.63 MPa and RWC declined 12.5%), there was little to noinhibition of attached leaf photosynthesis (Table II) at either lowor high internal [CO2]. However, during the latter portion of thestress cycle, chloroplast photosynthetic capacity in situ was ap-parently affected. On d 9 and 11, photosynthesis at 300 ppminternal [CO2] was inhibited, and this inhibition was not reversedat substantially higher internal [CO2] (i.e. at approximately 500ppm). Gas exchange measurements were repeated on a secondset of plants subjected to in situ water deficits. Photosynthesis ata constant internal [CO2] again was found to decline (data notshown). These gas exchange measurements suggest that chloro-plast photosynthetic capacity declined in situ only during thelatter part of the stress cycle shown in Figure 1, when RWC

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202 Plant Physiol. Vol. 88, 1988

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Table II. Water Stress Effects on Chloroplast Photosynthetic Capacity in Situ and in VitroPlants were last watered on d 1, as shown in Figure 1. Chloroplast photosynthetic rates at saturating C02 (5 mM NaHC03) are shown for

preparations isolated and assayed in media exactly adjusted to leaf *,, except for d 1 and 3, when media I, was -1.04 MPa. Attached leafphotosynthetic rates at low (approximately 300 ppm) and high (approximately 500 ppm) internal [CO2] are shown. Photosynthetic rates ofchloroplasts are the means of three replications; leaf measurements were replicated four times. All presented data are means ± the SE.

Chloroplasts LeavesDay Low CO2 High C02of Photosynthetic

Stress rate Internal C02 rate Inhibition' IntemalC2 otosytet Inhibition

,umol/(mg Chl- h) % ppm Atmol/(m2. s) % ppm Imol/(m2.s) %1 129.8 ± 2.9 291.2 ± 5.2 12.68 ± 1.08 532.3 ± 19.9 22.69 ± 1.243 126.7± 1.3 2.4 319.1 ± 11.7 14.60± 1.28 511.2± 14.8 21.56± 1.20 5.06 124.7 ± 1.2 3.9 310.9 ± 7.6 12.17 ± 1.65 4.0 461.4 ± 18.0 21.72 ± 2.57 4.39 70.4 ± 2.9 45.8 289.1 ± 3.5 6.26 ± 0.37 50.6 487.2 ± 7.3 16.13 ± 2.41 28.9

11 36.9± 1.8 71.6 290.4± 5.8 5.51 ± 1.26 56.5 523.4± 12.2 11.40± 2.77 49.8a Photosynthetic rates are compared to the rates measured on d 1 of the stress cycle.

declined below approximately 75%. Interpretation of gas ex-change measurements with regard to photosynthetic capacity ata given internal [CO2] should be made with caution, however.Unpublished reports suggest that irregular, "patchy" stomatalclosure in water stressed leaves may cause internal leaf [CO2]calculations to be incorect (T Sharkey, personal communication).

Photosynthetic capacity of chloroplasts isolated from plantssubjected to the stress cycle shown in Figure 1 is also presentedin Table II. C02-saturated photosynthetic rates did not changein chloroplasts (isolated and assayed in solutions which closelymatched the declining leaf 'I,,) during the first 6 d of stress.During the latter part of the stress cycle, the photosynthetic rateof chloroplasts and leaves declined in parallel (Table II). Appar-ently, the photosynthetic capacity of chloroplasts isolated andassayed in isotonic media is a fairly good indicator of chloroplastphotosynthetic capacity under water stress in situ.

Further studies were undertaken to characterize water stresseffects on chloroplast photosynthesis. During the stress cycleshown in Figure 1, chloroplasts were isolated in media at varying'I,,, and photosynthesis was also assayed at varying reactionsolution T.s (Table III). Data shown in Table III indicate thatwhen isolation medium closely matched the leaf TI,, chloroplastintactness remained at or above 90% throughout the stress cycle(intactness for d 6 and 9 was 93.7 and 93.3%, respectively, datanot shown).When chloroplasts were isolated from well-watered plants (day

1) in standard 0.33 M sorbitol (-1.04 MPa) media, and thenexposed to low 'I, during photosynthetic measurements, photo-synthesis was greatly inhibited (Table III, lines a to d). This hasbeen shown in a number of studies previously (3, 5, 15). Reduc-ing isolation and reaction media I, slightly (down to -1.14 MPa)did not inhibit photosynthesis (lines a and e). Lowering isolationmedium slightly (down to -1.14 MPa) did not reduce the lowT, inhibition of photosynthesis (lines c and f) in these prepara-tions. Exposure of chloroplasts to low 'I, during photosynthesiscaused substantial inhibition, no matter what the isolation me-dium 'Is was (lines d, h, and i). These results differ substantiallywith recent work by Robinson (21), which suggested that whenchloroplasts are isolated in hypertonic (high sorbitol) medium,they become essentially insensitive to high sorbitol during pho-tosynthesis. Possibly, these plastid preparations may not havebeen as "leaky" to sorbitol as was found in Robinson's study.On d 3 of the stress, chloroplasts were again isolated in

"standard" medium (0.33 M sorbitol, -1.04 MPa), or in mediumexactly adjusted to the leaf TI,, which was -0.80 MPa (lines j tom). When leaf 'I, is much greater than -1.04 MPa, matching

isolation and/or reaction solution I, to leaf *Is is injurious,although membrane integrity (i.e. intactness) is maintained.When chloroplasts were isolated on d 11 from water-stressed

plants, maximum photosynthetic rates (line n) were obtainedwhen isolation and reaction medium I, matched the ambientleaf I, of -1.64 MPa. Exposure of these plastids to hypertonicisolation and/or reaction media (lines o and q to s) still resultedin photosynthetic inhibition. Exposure of these plastids to hy-potonic isolation medium I, (-1.04 MPa) resulted in chloroplastrupture and inhibited photosynthesis (lines t and u). This wouldbe expected if the chloroplasts adjusted to a lowered ambient 'Isin situ. When chloroplasts were isolated from stressed plants thathad an I, of -1.64 MPa (d 11), photosynthesis was 45% higherthan the rates of chloroplasts isolated from well-watered plants(d 1) when reaction medium Is was -1.64 MPa (lines d and n).

Chloroplast Stromal Volume during in Situ Water Stress.Data presented in Figure 2 show chloroplast stromal volume atdecreasing RWC during a water stress cycle. During this droughtepisode, which lasted 14 d, leaf 'I', dropped from -0.29 MPadown to -1.9 MPa. As expected, initial leaf *I' depression frommaximum values was associated with a sharp decline in RWC.When leaf ',, dropped from -0.29 MPa (on d 1) to -0.83 MPa(on day 1), RWC declined by 22%. Despite this substantial lossof cell water during the initial phase ofthe stress, stromal volumeappeared to be maintained, averaging 20.9 ,uL/mg Chl over thisperiod (Fig. 2). As leaf RWC dropped below about 65% (andleaf 'I', approached -1.2 MPa), stromal volume was reduced,and declined further on the last day of measurement (d 14). In aseparate series of experiments (Fig. 1, Table II), chloroplastphotosynthetic potential was maintained during an initial phaseof a water stress cycle, despite substantial decline in RWC. Asleaf *I' dropped to -1.4 MPa (Fig. 1), chloroplast photosyntheticpotential dropped precipitously (Table II).

Further experiments were conducted to ensure that stromalvolume maintenance during the initial declines in leaf *'I andRWC, as shown in Figure 2, was not due to artifacts in the assayprocedure. The stromal volume measurement is based on meas-uring volume per unit Chl. Therefore, these measurements as-sume that Chl/chloroplast (or Chl/cell) does not change. As Chlbleaching is known to occur during water stress (1), studies wereundertaken to ensure that this effect did not influence the stromalvolume measurements in water-stressed tissue. Chl per unit freshand turgid (rehydrated) weight was monitored during the waterstress cycle (Fig. 2). Chl per unit fresh weight increased linearlywith decreasing RWC during the drought episode. However, Chlper unit turgid weight remained constant throughout the stresscycle until the last day, when some slight Chl destruction appar-

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SEN GUPTA AND BERKOWITZ Plant Physiol. Vol. 88, 1988

Table III. Photosynthesis at Varying Isolation and Reaction Media ',Chloroplasts were isolated from plants subjected to the stress cycle shown in Figure 1. When no standard

errors are shown for photosynthetic rates, the value is from a single assay. Otherwise, the value represents themean of at least three replications.

Days Leaf Isolation Reaction Chloroplast Photosynthesisof Medium Medium Intactness

Stress 'i'. Rate Inhibitiona

-MPa -MPa -MPa % Amol/mg Chl- h %a 1 0.93 1.04 1.04 92.0 129.8 ± 2.9b 1.04 1.14 121.1 6.7c 1.04 1.50 34.6 73.3d 1.04 1.64 25.4 80.4e 1.14 1.14 89.7 127.3 ± 3.0 1.9f 1.14 1.50 37.3 ± 1.8 71.3g 1.50 1.50 84.2 17.6 ± 0.6 86.4h 1.50 1.64 11.4 ± 0.0 91.2i 1.64 1.64 88.9 11.6 ± 0.6 91.1

j 3 0.80 1.04 1.04 92.5 126.7 ± 1.3k 1.04 0.80 55.7 ± 1.9 56.01 0.80 1.04 94.1 29.9 ± 4.3 76.4m 0.80 0.80 23.5 ± 2.1 81.5

n 11 1.64 1.64 1.64 92.5 36.9± 1.8o 1.64 1.94 30.5+ 1.2 17.3p 1.64 1.04 13.5 ± 0.6 63.4q 1.94 1.94 89.9 29.5 ± 1.3 20.1r 1.94 1.64 25.6 ± 0.0 30.6s 1.94 1.04 0 100t 1.04 1.04 40.5 12.8 ± 0.6 65.3u 1.04 1.64 6.0 83.7

a The calculated inhibition refers to the photosynthetic rate as compared to the maximum rate obtainedfrom chloroplasts isolated on that day of the stress cycle.

DAY OF STRESS

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FIG. 2. Chloroplast stromal volume (closed symbols), leaf Chl perunit fresh weight (C), and per unit turgid (i.e. rehydrated to 100% RWC)weight (0), plotted as a function of declining RWC during an in situstress cycle. Paired measurements are shown for each Chl determination.RWC and stormal volume measurements are the means of at least threereplications ± the SE. The leaf Iw on d 1, 9, 11, 12, and 14 was -0.29 ±0.01, -0.53 ± 0.03, -0.83 ± 0.03, -1.24 ± 0.07, and -1.90 ± 0.09 MPa,respectively. The [14C]sorbitol volumes for chloroplasts isolated duringthe stress cycle were 25.5, 38.1, 26.6, 39.7, and 38.1 gL/mg Chl, respec-tively, on d 1, 9, 11, 12, and 14. When this experiment was repeated a

second time (results described in text), the ['4C]sorbitol volumes ofchloroplasts isolated at the beginning and end of a stress cycle were 38.0and 34.1 uL/mg Chl, respectively.

ently occurred. The constant Chl/turgid weight value during thestress indicates that stromal volume per cell was maintainedduring most of the stress cycle. The constant increase in Chl/fresh weight during the stress was due to cell water loss ratherthan changes in Chl/cell. The stromal volume measurementswere repeated in a separate experiment with a second set ofplants. Over seven days of stress, leaf Iw dropped from -0.29 ±0.02 MPa down to -1.03 ± 0.06 MPa. Stromal volume at thebeginning and end of this second stress cycle was 17.46 ± 1.08uL/mg Chl and 21.31 ± 1.62 ,uL/mg Chl, respectively. The initialvolume measurement was somewhat lower than the 20.9 uL/mgChl volume maintained at the beginning of the first stress cycle(Fig. 2). However, no stromal volume reduction occurred duringa 0.74 MPa drop in leaf Iw over this second stress period.

Chloroplast Solute Accumulation. If stromal volume is main-tained during at least part of a drought episode, as is suggestedin this report, then solute accumulation must be occurring in theplastid. If stromal volume does not decrease at all while RWCdeclines during the initial phase of a drought cycle, then it canbe hypothesized that osmotic adjustment in the chloroplastoccurs in excess of that occurring in the vacuole and cytoplasm.The only other explanation for this effect (as shown in Fig. 2) isthat the symplast volume and water content of the leaf cells(which was not measured) remained constant during the initialRWC decline; with leaf dehydration being associated only withapoplastic water loss. This cell response to drought is unlikely(24). Therefore, stromal volume maintenance during a droughtepisode should be associated with substantial solute accumula-tion in the chloroplast.An attempt was made to document this effect (Table IV).

During this drought cycle, which lasted 9 d, leaf 'w dropped by1.33 MPa. At the end of the stress, RWC declined to 59%. Large

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quantities of chloroplasts were isolated from these plants, andused to measure stromal solute levels. Assuming stromal volumesof 20.9 gL/mg Chl and 16 ,L/mg Chl for well-watered andstressed leaves, respectively, (as suggested by the experimentshown in Fig. 2), stromal solute concentrations were calculated(Table IV). With control (well-watered) plants, leaf I, was meas-ured at -1.18 MPa; leaf solute concentration was calculated tobe 462 mm. Stromal solute concentration measurements weresimilar, although slightly lower: 388 mM. With stressed plants,leaf solute concentration was calculated to be 657 mm. Assuminga stromal volume of 16 ,uL/mg Chl, the stromal solute concen-tration of stressed plants was measured at 648 mm. These results,then, support the validity of the stromal volume measurementsreported here (Fig. 2). If stromal volume did drop substantiallyin conjunction with RWC decline and was not picked up in ourvolume assay, then the calculated stromal solute concentrationof 648 mm would have been far lower than the value (657 mM)predicted from leaf measurements.The level of some solutes that have been previously shown to

be major osmotic constituents of spinach chloroplasts (16) weremeasured individually (Table IV). Amino acid, reducing sugar,and K+ concentrations were all found to substantially increasein the stroma of stressed chloroplasts. The increase in the meas-ured levels of these specific solutes accounted for 83% of thetotal increase in measured chloroplast solutes.

Chloroplast solute measurements were also made in a secondexperiment. Here, total chloroplast solute levels were not meas-ured directly with osmometry. This allowed use ofthe chloroplastextracts for the measurement of a broader range of individualsolutes. Leafsolute levels were also monitored in this experiment.These results are shown in Table V. During this stress cycle,which lasted 9 d, leaf *I' dropped by 1.19 MPa. The reductionin RWC (25%) was not as great as occurred during the experi-ment shown in Table IV. Therefore, 20.9 ,uL/mg Chl was usedas a stromal volume for both control (well-watered) and stressedplants. This value is suggested by the results shown in Figure 2.The chloroplast solute profiles shown in Table V again reveal

the increase in concentration of several solutes in the stressedchloroplast. Amino acids, reducing sugars, and K+, along withCl-, account for a substantial portion of the total solutes meas-ured in both control and stressed chloroplasts. The increase inthe concentration of these major stromal solutes also accountedfor a substantial portion of the solute accumulation occurring in

Table IV. Stromal Solute Accumulation in Response to in Situ WaterStress

Control and stress values were determined on plants prior to and aftera 9 d stress cycle. Leaf *, was converted to solute concentration by usinga KCI standard curve and thermocouple psychrometer measurements.Total chloroplast solute measurements were calculated from osmometricmeasurement of stromal extracts separated from incubating media andstromal volume measurements made during the experiment shown inFigure 2. All presented data (except for the calculated leaf solute concen-

trations) are the means of at least three replications ± the SE.

Measured Parameter Control Stress

Leaf 'I', (-MPa) 0.28 ± 0.05 1.61 ± 0.16RWC (%) 95.8 ± 4.9 58.9 ± 0.3Leaf *I (-MPa) 1.18 ± 0.15 1.80 ± 0.21Leaf solutes (mM) 462 657Chloroplast solutes:

Total (mM) 388.1 ± 15.7 647.7 ± 10.6Amino acids(mM) 62.8 ± 0.3 125.5 ± 4.4

Reducing sugars

(mM) 64.0 ± 0.8 159.8 ± 0.4K+ (mM) 75.3 133.3

Table V. Solute Profile ofLeaves and Chloroplasts PreparedfromWell- Watered (Control) and Water-Stressed Plants

Control values were determined prior to an 8 d stress cycle. Leaf 'swas converted to solute concentration by using a KCI standard curveand thermocouple psychrometer measurements. All data with standarderrors are means of at least three replications.

Leaf ChloroplastSolute

Control Stress Control Stress

mM mm M mM

K+ 108.2 ± 7.4 160.2 ± 7.1 53.6 95.7Ca2+ 32.1 ± 4.0 33.0 ± 0.8 25.0 21.3Mg2+ 29.9 ± 1.6 32.2 ± 2.9 21.1 24.6C1- 58.2 ± 5.8 112.4 ± 3.2 58.0 152.6NO3- 35.8 ± 3.6 59.0 ± 2.3 0.7 2.9So42- 40.3 ± 4.1 53.5 ± 4.6 5.1 5.2HP042- 25.4 ± 0.6 46.4 ± 1.0 6.9 12.4Reducing sugars 97.0 ± 9.3 192.5 ± 6.3 35.6 83.9Amino acids 109.4 ± 6.6 161.8 ± 6.7 94.8 163.3

Sum of Solutes 536.0 851.0 300.8 561.9

Leaf solutes (mM) 386 579 386 579Leaf Is (-MPa) 0.96 ± 0.11 1.56 ± 0.09Leaf *w (-MPa) 0.36 ± 0.00 1.55 ± 0.05RWC (%) 93.5 ± 0.5 68.5 ± 0.7

the stressed chloroplast. It should be noted that betaine has beenrecently shown to accumulate in the chloroplast of salt-stressedspinach (22). Studies documenting increases in leaf proline dur-ing water stress are widespread in the literature. However, totalquaternary ammonium compounds (an unspecific assay of be-taine) were undetectable, and proline was found in only traceamounts in both control and stressed chloroplasts in the experi-ment shown in Table V (data not shown).

Extrapolating from leaf T, measurements (Table V), chloro-plast stromal solute levels should have been 386 and 579 mm inthe control and stressed chloroplast, respectively. The sum of themeasured solutes was 78% (for control) and 97% (for stress) ofthese values.The specific solute analyses as presented in Tables IV and V

further support the validity ofthe stromal volume measurements(Fig. 2), as presented in this report. Especially with the stressedchloroplast, the sum of the individually measured solutes is veryclose to the value predicted from leaf I, measurements. Thiscorrelation suggests that the stromal volumes used to calculatethe solute concentrations in these two experiments were fairlyaccurate. In the first case (Table IV), RWC declined to 59%.Stromal volume reduction (from 20.9 ,uL/mg Chl down to 16,uL/mg Chl) was assumed to be only 23%. In the second case(Table V), stromal volume was hypothesized not to change withstress, while RWC declined to 69%.

Solute profiles of well-watered and stressed leaves are alsoshown in Table V. It appears that K+, Cl-, amino acid, andreducing sugar concentrations increased in the stressed leaves, aswas shown to occur in the chloroplast.

It should be noted that the sum of the measured leaf solutesexceeds the values predicted from the leaf TI, measurements, forboth stress and control plants. This suggests that a substantialproportion of the measured solutes was osmotically inactive, inboth control and stressed leaves. Hence, leaf solute profiles aspresented in Table V should be interpreted with caution. Ionsbound to membranes, cell walls, and proteins, while not contrib-uting to the osmotically active pool of solutes in the cell, wouldnot be discriminated from free solutes in these assays. Anotherexplanation for the discrepancy between the values for leaf soluteconcentrations obtained by summing the individually measuredsolutes, and calculating the values by conversion from leaf Is

205



SEN GUPTA AND BERKOWITZ

measurements, is that the individual osmotic coefficients of thesolutes listed in Table V may be substantially different from thevalue used to convert leaf Is to solute concentration.

DISCUSSION

The results presented in this report indicate that cell water lossand stromal volume reduction may not be occurring at the sametime or at the same rate with decreasing leaf"I' during a droughtepisode. Apparently, stromal volume can be maintained duringthe initial phase of a drought episode, even though total cell

water (measured as RWC) declines. Although not quantitative,electron micrograph studies (9) have shown chloroplast shrinkageat pronounced tissue dehydration (RWC of 47%). The swellingof chloroplasts prepared from water-stressed tissue that is shownin some electron micrograph studies (10) can be attributed toartifacts due to improper aqueous fixative 'I,.

Evidence has been presented in this report that strongly sug-

gests that the chloroplast has the capability to undergo substantialosmotic adjustment during initial leaf 'I', decline. Stromal vol-ume maintenance can be attributed to solute accumulation abovethat occurring in the cell as a whole. Data presented in Figure 2suggest that the capability for stromal volume maintenanceseems to break down rather suddenly during a drought as RWCapproaches 60% and leaf 'I', drops down to about -1.2 to -1.5MPa.

Chloroplast photosynthetic potential during in situ waterstress, as measured i-n situ and in vitro, was monitored in thisstudy (Table II). The results of this work show a very sharpdecline in chloroplast photosynthesis during the latter portion ofa drought cycle. The sudden loss in stromal volume maintenancecapacity as demonstrated in another experiment (Fig. 2) may beassociated with this water stress inhibition of chloroplast metab-olism. The two experiments, however, should be compared withcaution, as they represent studies using different sets of plantssubjected to different drought episodes. Nonetheless, the resultspresented in this report are consistent with a correlative associa-tion between chloroplast photosynthetic potential and stromalvolume during water stress. The inability to maintain stromalvolume may cause photosynthetic inhibition. Conversely, theinhibition ofphotosynthesis (and the resultant reduction in soluteand energy generation) may facilitate loss in chloroplast osmoticadjustment capability, and hence, volume maintenance.The studies of low Is effects on isolated chloroplasts in vitro

(Table III) extend the conclusions of previous work in this area(5, 6, 15, 23). The results presented in Table III indicate thatwhen chloroplasts are isolated from stressed leaves which havelowIs, subjecting the plastids to a further decrease in I' in vitrocauses inhibition of photosynthesis. This conclusion is consistentwith the hypothesis developed from in vitro studies (6) that onemechanism of low 'I', inhibition of photosynthesis may beosmotic dehydration of the stroma. The results presented inTable III also support our assertion (23) that chloroplast osmoticadjustment facilitates acclimation of the photosynthetic mecha-nism to water stress. When assayed at low I,, chloroplastsisolated from stressed plants had higher photosynthetic rates thanplastids isolated from control plants.The solute analysis experiments (Tables IV and V) show that

chloroplast osmotic adjustment can occur during drought, and

possibly to an extent above that occurring in the cell at large.However, specific solute profiles did not reveal the productionof a single solute, or metabolic pathway that was "turned on"dunng water stress. We interpret the results of these experimentsas indicating that during stress, there is a general increase in thelevel of chloroplast solutes that are already present in the stromaas major osmotica.

LITERATURE CITED

1. ALBERTE RS, JP THORNBER, EL Fwscus 1977 Water stress effects on the contentand organization of chlorophyll in mesophyll and bundle sheath chloroplastsof maize. Plant Physiol 59: 351-353

2. ARNON DI 1949 Copper enzymes in chloroplasts. Polyphenoloxidases in Betavulgaris. Plant Physiol 24: 1-14

3. BERKOWITZ GA 1987 Chloroplast acclimation to low osmotic potentials. PlantCell Rep 6: 208-211

4. BERKowITz GA, M GIBBS 1982 Effect of osmotic stress on photosynthesisstudies with the isolated spinach chloroplast. Generation and use of reducingpower. Plant Physiol 70: 1143-1148

5. BERKOWITZ GA, M GIBBS 1982 Effect of osmotic stress on photosynthesisstudies with the isolated spinach chloroplast. Site specific inhibition of thephotosynthetic carbon reduction cycle. Plant Physiol 70: 1535-1540

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

7. BERKOWITZ GA, KS KROLL 1988 Acclimation of photosynthesis in Zea maysto low water potentials involves alterations in protoplast volume reduction.Planta (in press)

8. FARQUHAR GD, TD SHARKEY 1982 Stromatal conductance and photosyn-thesis. Annu Rev Plant Physiol 33: 317-345

9. FELLOwS RJ, JS BOYER 1978 Altered ultrastructure of cells of sunflower leaveshaving low water potentials. Protoplasma 93: 381-395

10. GILEs KL, MF BEARDSELL, D COHEN 1974 Cellular and ultrastructural changesin mesophyll and bundle sheath cells of maize in response to water stress.Plant Physiol 54: 208-218

11. HANSON AD, WD HITz 1982 Metabolic responses of mesophytes to plantwater deficits. Annu Rev Plant Physiol 33: 163-203

12. HELDT HW 1980 Measurement of metabolite movement across the envelopeand of the pH in the stroma and the thylakoid space in chloroplasts. MethodsEnzymol 69: 604-613

13. HUTMACHER RB, DR KRIEG 1983 Photosynthetic rate control in cotton. PlantPhysiol 73: 658-661

14. 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

15. 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. Planta153: 416-422

16. KAISER WM, H WEBER, M SAUER 1983 Photosynthetic capacity, osmoticresponse and solute content of leaves and chloroplast from Spinacia oleraceaunder salt stress. Z Pflanzenphysiol113: 15-27

17. MOORES, WH STEIN 1948 Photometric ninhydrin method for use in thechromatography of amino acids. J Biol Chem 176: 367-388

18. NOBEL PS 1983 Biophysical Plant Physiology and Ecology. W. H. Freeman,San Francisco, pp 83-86

19. PIER PA, GA BERKOWITZ 1987 Modulation of water stress effects on photo-synthesis by altered leafK+. Plant Physiol 85: 655-661

20. RAO IM, RE SHARP, JSBOYER 1987 Leaf magnesium alters photosyntheticresponse to low water potentials in sunflower. Plant Physiol 84: 1214-1219

21. ROBINSON SP 1985 Osmotic adjustment by intact isolated chloroplast inresponse to osmotic stress and its effects on photosynthesis and chloroplastvolume. Plant Physiol 79: 996-1002

22. ROBINSON SP, GP JONES 1986 Accumulation of glycinebetaine in chloroplastsprovides osmotic adjustment during salt stress. Aust J Plant Physiol 13: 659-668

23. SEN GUPTA A, GA BERKOWITZ 1987 Osmotic adjustment, symplast volume,and nonstomatally mediated water stress inhibition of photosynthesis inwheat. Plant Physiol 85: 1040-1047

24. SINCLAIR TR, MM LUDLOW 1985 Who taught plants thermodynamics? Theunfulfilled potential of plant water potential. Aust J Plant Physiol 12: 213-217

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206 Plant Physiol. Vol. 88, 1988



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