Stomatal and Nonstomatal Limitations to Net ...Stomatal and nonstomatal limitations to Awere com-pared in three-month-old white oak (Quercus alba L.), post oak (Quercus stellata Wangenh.),

Plant Physiol. (1992) 99, 1502-15080032-0889/92/99/1 502/07/$01 .00/0

Received for publication December 9, 1991Accepted March 3, 1992

Stomatal and Nonstomatal Limitations to Net Photosynthesisin Seedlings of Woody Angiosperms'

Bing-Rui Ni2 and Stephen G. Pallardy*

School of Natural Resources, University of Missouri, Columbia, Missouri 6521 1

ABSTRACT

Comparative responses of net photosynthesis (A) to water stressin woody species from a variety of habitats were studied to assess

the relationship between photosynthetic attributes and droughttolerance. Stomatal and nonstomatal limitations to A were com-

pared in three-month-old white oak (Quercus alba L.), post oak(Quercus stellata Wangenh.), sugar maple (Acer saccharumMarsh.), and black walnut (Juglans nigra L.) seedlings during a

drying cycle. Relative stomatal limitation of photosynthesis (I) wasless than 50% in all species except for Q. stellata seedlings sub-jected to severe water stress. No significant changes in I were

observed in Q. alba and /. nigra before, during, and after drought.In A. saccharum, I was generally low and decreased significantlyunder water stress. Under well-watered conditions, A was highestin Q. stellata, intermediate in Q. alba, and lower in A. saccharumand /. nigra. High A in well-watered Q. stellata was associatedwith high stomatal conductance and carboxylation efficiency,whereas low A was associated with low stomatal conductance andcarboxylation efficiency in A. saccharum and low stomatal con-

ductance, low carboxylation efficiency, and high CO2 compensa-

tion point in 1. nigra. Under severe water stress, A, carboxylationefficiency, and stomatal conductance decreased substantially in allspecies; however, Q. stellata had the highest carboxylation effi-ciency and lowest CO2 compensation point under these conditions.After 5 days at high soil moisture after drought, stomatal andmesophyll components of A in A. saccharum and /. nigra had notrecovered to predrought levels, whereas they had completely re-

covered in Q. stellata and Q. alba. The photosynthetic apparatus,especially mesophyll components, of drought-tolerant Quercusspecies showed either less inhibition under water stress, superiorrecovery to predrought capacity, or both. Exposure of the leavesto "4CO2 indicated apparent asymmetric stomatal closure for mildlywater-stressed seedlings, but not for leaves of well-watered, se-

verely stressed, or rehydrated plants. These results suggest thatpatchy stomatal closure under mild water stress might be importantfor water stress-induced inhibition of photosynthesis, but not underthe more severe water stress imposed in this study.

As water stress develops, plants normally exhibit paralleldecreases in both CO2 fixation and &.3 This pattern of re-

1 Research funded by the U.S. Department of Agriculture, ForestBiology Competitive Grants Program. Missouri Agricultural Experi-ment Station Journal Series 11,342.

2 Present address: Department of Vegetable Crops, University ofCalifornia, Davis, CA 95616.

3Abbreviations: g stomatal conductance; A, net photosynthetic

sponse has led some to believe that stomatal closure isprimarily responsible for the inhibition of photosynthesisunder water stress. However, other studies have indicatedthat increased stomatal limitation under water stress is not ofsuch primary importance. Greater reductions in photosyn-thesis than leaf conductance have been observed for severalspecies (e.g. Gossypium hirsutum [10], Quercus rubra, Acerrubrum, Populus grandidentata [2], and Pinus taeda [22]), sug-gesting that nonstomatal factors significantly influence pho-tosynthesis in plants subjected to water stress. Some studiesemploying other species have not supported this contention(e.g. refs. 4 and 24), suggesting that there may be variationamong species in photosynthetic responses to water stress.

It is worth mentioning that both stomatal and nonstomatallimitations to photosynthesis exist regardless of plant waterstatus and may vary with species. Hence, interpretation ofwater stress effects on photosynthetic properties among spe-cies should ideally be based on how stomatal limitationchanges during water stress and after its relief, not on abso-lute values. Study of these patterns in various species maybe useful in revealing how differential limitation of stomataland nonstomatal components of photosynthesis under waterstress is related to drought tolerance.

Partitioning of the stomatal and mesophyll limitation ofphotosynthesis has usually been accomplished by indirectcalculation of C1 using gas exchange data. Such calculationsgenerally assume uniform stomatal aperture across a leaf.Asymmetric stomatal closure in some of the areoles of het-erobaric species under mild water stress (19, 20) and artificialABA treatment (5, 18, 21) has been reported. If such stomatalclosure occurs, the calculated Ci under water stress may nolonger be valid, thus rendering the calculated partitioningbetween stomatal and mesophyll components of photosyn-thesis an artifact (6, 13).

This study was conducted to evaluate the effects of waterstress on photosynthesis in seedlings of four species of woodyangiosperms with known differences in capacity to toleratedrought. The relative importance of stomatal and nonsto-matal limitations to photosynthesis was determined underwell-watered and severely water-stressed conditions, andafter rewatering. The possibility of asymmetric stomatal clo-sure also was examined by autoradiography under well-

rate; E, transpiration rate; Ca, ambient CO2 partial pressure; Ci,internal CO2 partial pressure; Cp, CO2 compensation point; 1, sto-matal limitation of net photosynthesis; CE, carboxylation efficiency;'', leaf water potential.

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STOMATAL AND NONSTOMATAL LIMITATIONS TO NET PHOTOSYNTHESIS

watered, mildly and severely water-stressed, and rewateredconditions.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Locally collected seeds of post oak (Quercus stellata Wan-genh.), white oak (Quereus alba L.), sugar maple (Acer sac-charum Marsh.), and black walnut (Juglans nigra L.) werestratified if necessary to break dormancy. These species wereselected because they differ substantially in natural distribu-tion across habitats varying in soil moisture availability (9).Q. stellata and Q. alba are distributed on xeric to very xericsites, whereas A. saccharum and I. nigra are generally re-stricted to mesic upland or riparian habitats (1, 8, 9). The oakspecies are considered more drought tolerant because theypossess higher capacity for osmotic adjustment (1), greaterdehydration tolerance (14), less leaf abscission under waterstress (7, unpublished observation), and the capacity to main-tain positive net photosynthesis to lower '' than A. saccharumorJ. nigra (17).

Seedlings were grown in a peat moss:sand:silt loam soilmixture (2:2:1) in 2.5-L pots in an evaporation-cooled green-house, with supplemental lighting from Na-vapor lamps tomaintain approximately 1000 ,mol -m2 *. s- PPFD at midday.All seedlings were kept well watered and fertilized withOsmocote (N:P:K = 14:14:14, Sierra Chemical Co., Milpitas,CA). Other macronutrients, Fe-EDTA, and micronutrientswere supplied by modified Hoagland solution (11). Whenplants were 3 months old, six seedlings of each species weremoved to a growth chamber containing gas exchange equip-ment (growth chamber conditions: 28/200C day/night; 400yumol-m-2 s-' PPFD at the plant tops for 14 h; and 60-70%RH) before gas exchange measurements. Although maximumlight levels were reduced, the growth chamber offered a farmore stable environment for gas exchange measurements.Five days of acclimation in the growth chamber were pro-vided to minimize the impact of physical disturbance imposedin moving plants from greenhouse to laboratory.

Gas Exchange Measurements

CO2 and H20 vapor exchange measurements were madeon fully expanded leaves of each species with an open IR gasanalysis system (see ref. 17 for a complete description of thesystem). Cuvette temperature was maintained at 25 ± 1°Cduring measurements. Leaf and air temperatures were mon-itored with thermocouples. Light intensity in the cuvette wasmaintained at 1000 ± 40 umol. m-2 -s-1 by means of a mul-tivapor lamp (General Electric, Inc.).Net photosynthesis (mol * m-2 * s-1) and E (mmol . m-2 . s-)

were measured at 7- to 8-Pa intervals over a range of Ca fromabout 60 Pa to the Cp of each species. Measurements weretaken when all the parameters were stable (usually 0.5-1 hafter sealing leaves in the cuvette). Ci was calculated by themethod of Farquhar and Sharkey (6). Stomatal limitation wasdetermined using the resistance analog method of Jones (12,13) because of the linear response of A to Ci for most curves.For a few samples with curvilinear A-Ci relationships, 1 wasestimated by the differential method of Jones (13). We pre-

ferred this method to that of Farquhar and Sharkey (6)because: (a) we often were unable to bring Ci to 34 Pa dueto stomatal closure at high extemal CO2 partial pressure, and(b) it reduces to the resistance analog model when the A-Cirelationship is linear, thus providing more comparable esti-mates of 1.Gas exchange measurements were first made on well-

watered seedlings. Subsequently, seedlings were removedfrom the cuvette, water was withheld, and plants were al-lowed to slowly deplete soil water until WI was -2 to -3MPa, at which point gas exchange was measured again. Thisusually took 9 to 13 d, depending on the species. Somewhatdifferent I, levels were employed because of differences indrought tolerance capacity among the four species: J. nigraand A. saccharum showed complete stomatal closure at sub-stantially higher '' than did the two Quercus species. Addi-tionally, these levels of stress represented realistic levels ofwater stress encountered during severe droughts in the field(1, 8). After measurements of A and E under water stress,seedlings were rewatered and maintained under high soilmoisture for 5 d. Gas exchange measurements were thenconducted for a third time on rewatered seedlings.

'I and Leaf Area Measurement

', were determined with a pressure chamber immediatelyafter each gas exchange measurement (23). To minimize theeffect of any changes in source-sink relations over the courseof the experiment as a result of leaf harvesting, I' was notmeasured for well-watered seedlings. Thus, the same leavescould be used to measure photosynthesis of well-wateredand water-stressed seedlings. This procedure limited removalto only one leaf during the three measurements of photosyn-thesis. It was assumed that any initial difference in ', ofwell-watered seedlings had a negligible influence on photo-synthesis. Leaf area was measured using a leaf area meter(LI-3000, Li-Cor, Inc.).

Determination of Cp and CE

Cp was determined by extrapolating the linear portion ofthe A-Ci curve to the abscissa. CE was calculated as the slopeof the A-Ci curve within 2 Pa of Ci above the compensationpoint.

Data Analysis

Data were analyzed by analysis of variance using thegeneral linear models procedure of SAS (SAS Institute, Inc.,1982). Mean comparisons were conducted using protectedLSD statistics.

4CO2 Autoradiography

"4CO2 exposure was conducted in a fume hood at roomtemperature on fully expanded leaves of the four species ina 4-L gas exchange cuvette (made for the LI-6200 PortablePhotosynthesis System, LI-COR, Inc.). Light intensity ofabout 750 Ismol. m-2*s- was provided by a 500-W quartzlamp (QL-500-WL, Regent Lighting Corp., Burlington, NC).The temperature of the cuvette was maintained between 26

1 503



Plant Physiol. Vol. 99, 1992

and 28.50C by placing a layer of water between the light andthe cuvette and by passing the laboratory air through icewater before it entered the cuvette. The partial pressure ofCO2 of the laboratory air varied between 35 and 37 Pa. Oneattached leaf was sealed in the cuvette for about 20 min toallow the leaf to acclimate to the cuvette conditions. ', of anadjacent, illuminated leaf was then measured and the leaf inthe cuvette was exposed to either 15 ,Ci of 14CO2 for 60 sfor well-watered and rewatered plants or to 50 ACi for 75 sfor water-stressed plants. After exposure and purging of thecuvette for 60 s, the leaf was immediately clamped betweenaluminum plates that had been prechilled in liquid N2. Afterfreezing, the leaf was placed in contact with x-ray film (KodakX-Omat RP, Eastman Kodak) and exposures were carried outat -60 to -800C for 1 to 2 weeks. This experiment was firstperformed on well-watered seedlings. Water was then with-held and seedlings were allowed to deplete soil water at arate similar to that in the gas exchange experiment. Exposureswere conducted on leaves of both mildly and severely water-stressed plants. The severely stressed seedlings were thenrewatered and kept at high soil moisture for 5 d, after which"4CO2 exposure was again performed. Exposures were repli-cated two to three times for each species across water stresstreatments; autoradiographs of adequate quality were ob-tained except in a few instances (see 'Results').

RESULTS

Gas Exchange Patterns

With decreasing C. and Ci, net photosynthesis decreasedin all species (Fig. 1, A-D). In moist soil, Q. stellata had thehighest A at ambient C., with progressively lower A in Q.alba, A. saccharum, and J. nigra (Table I). The last two speciesshowed similar photosynthetic rates when well watered and,based on CE and Cp estimates, were the least photosynthet-ically efficient species at Ca of 34 Pa.Water stress greatly reduced A in all species (Table I). No

significant difference in A among species was detected underwater stress, although the level of water stress was not thesame for all species. Upon rewatering, photosynthetic capac-ity recovered fully within 5 d in both Q. alba and Q. stellata,the two drought-tolerant species (Fig. 1, A and B; Table I),but was only about half its predrought value for the moredrought-sensitive A. saccharum and J. nigra (Fig. 1, C and D;Table I).

Similar to the response of A to water stress, gs decreasedsubstantially in all species under water stress. Although g5recovered completely within 5 d at high soil moisture in thetwo Quercus species, it only partially recovered in A. sac-charum and J. nigra (Table I). As was the case for photosyn-thetic rates, the highest gs under well-watered conditions wasfound in Q. stellata, which had gs values about twice thoseof other species.

Stomatal limitation of photosynthesis was less than 50%under all conditions and for all species, except for Q. stellataseedlings subjected to severe water stress (Table I). Stomatallimitation varied between 40 and 50% in Q. alba and J. nigra,and no significant differences (P c 0.05) were detected amongwell-watered, water-stressed, and rewatered plants of these

species. The lowest 1 under well-watered conditions wasobserved in A. saccharum (34%), and this value decreased to19% when I' fell to -2.36 MPa. On the other hand, Q.stellata showed the opposite response, with increased 1 undersevere water stress (Table I).At all soil moisture levels, changes in Ci were apparently

attributable to both changes in stomatal conductance andchloroplast activity. Although a large decrease in g& underwater-stressed conditions was observed in Q. alba (Table I),Ci remained the same as that observed in well-watered andrewatered seedlings, indicating that the stomatal and meso-phyll components of photosynthesis were equally inhibited.After 5 d in moist soil after drought, both mesophyll andstomata of Q. alba had completely recovered from waterstress (Table I). In J. nigra, Ci was unchanged after rewatering,but gs did not recover completely from inhibition induced bywater stress. In Q. stellata seedlings, low Ci observed in water-stressed seedlings indicated that the mesophyll componentswere less responsive to dehydration than were stomata.Among the four species, A. saccharum alone exhibited in-creased Ci under water stress. Ci remained high in A. sac-charum even after 5 d of rehydration, indicating persistentmesophyll inhibition by water stress.The decrease of A under water stress generally was accom-

panied by decreasing CE and increasing Cp (except for Q.stellata, which showed no change in Cp) (Table I). CE re-mained significantly reduced and Cp significantly higherafter rewatering in A. saccharum and J. nigra. In moist soil, Q.stellata had the highest and A. saccharum the lowest CEvalues. The highest Cp was found in J. nigra when wellwatered.

4C02 Autoradiography

No asymmetry in stomatal closure was found for leaves ofwell-watered or rehydrated plants of any species (with Q.stellata shown as an example in Fig. 2, A and D). In mildlywater-stressed plants (-1.8 to -2.1 MPa), uptake of labelwas clearly asymmetric across the leaf in Q. stellata, Q. alba,and A. saccharum (Fig. 2, B, E, and F). When plants of themore drought-sensitive J. nigra were subjected to slightly lessnegative I' (-1.62 MPa), uptake of 14CO2 could not bedetected (not shown). Plants subjected to more severe levelsof water stress imposed during gas exchange measurementsexhibited either no detectable level of label in the leaves (Q.alba, A. saccharum, J. nigra, not shown) or only a uniformpattern of fixation (Q. stellata, Fig. 2C) under the exposuretimes allowed. We were unable to obtain autoradiographs ofadequate quality for A. saccharum seedlings under severelystressed and rewatered conditions.

DISCUSSION

Asymmetric stomatal closure was not observed for well-watered plants and plants rehydrated for 5 d after beingseverely water stressed. However, in agreement with Sharkeyand Seemann (20), exposure of mildly water-stressed leavesto 14CO2 showed that for three of four species, the uptake ofthe label was not uniform over the leaf, strongly suggestingpatchy stomatal closure. No asymmetric distribution of label

1504 Nl AND PALLARDY




0 1 0 20 30 40 50Co2 partial pressure (Pa)

.-

U,

E0E:1.4:-

15

10

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0

10

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060

(C) Acer saccharumAmbient

I n scl O, 13,

Intercellular

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0 1 0 20 30 40 50Co2 partial pressure (Pa)

10

-%

U)

E

E:4:

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0

10

5

n60 0 1 0 20 30 40 50

CO2 partial pressure (Pa)

Figure 1. Response of photosynthesis to Ca and Ci for representative seedlings of Q. stellata (A), Q. alba (B), A. saccharum (C), and /. nigra(D) during well-watered (0), water-stressed (O), and rewatered (A) phases of a drying cycle. Data fit by cubic spline (Ca) and linear regression(C).

was detected in mildly stressed J. nigra leaves; however,considering the sensitivity of stomata to water stress in thisspecies (17), nearly complete stomatal closure might not, inthis case, be unexpected.As discussed by Terashima et al. (21), patchy stomatal

closure will result in an overestimation of Ci. The degree ofoverestimation and consequent impact on A-Ci relationshipsdepend very much on the actual pattern of closure (i.e.bimodal versus a truncated normal distribution of apertures),variability of average gs in leaf areoles, and the biochemistryof fixation (3). Using a modeling approach based on theseattributes, Cheeseman (3) concluded that the impact of asym-metric stomatal closure on A-Ci curves would be minor under

many conditions. However, larger deviations from correct A-C, relationships were suggested if A was not CO2 saturated(i.e. the most common situation in the present study). Thus,interpretation of A-Ca relationships in these species undermild water stress might be problematic.

In contrast to the situation under mild water stress, no

patchy stomatal closure was detected in seedlings subjectedto severe water stress, although the photosynthetic rate ap-

peared to be very low. This severe water stress cannot beconsidered unnatural, because similar conditions may de-velop during drought in natural forests (1, 8). Under severe

stress, apparently all stomata were nearly closed, and therewere no differences in label uptake between presumably

15

10

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En

0E1-

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(B) Quercus albaAmbient

Intercellular

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(D) Juglans nigraAmbient

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Table I. Means of ',, Photosynthesis (A), C, at 34 Pa of Cuvette C02 Partial Pressure, I, g,, CE, and Cp in Seedlings of Q. stellata, Q. alba,A. saccharum, and 1. nigra under Well-Watered (W), Water-Stressed (S), and Rewatered (R) Conditionsa

Q. stellate Q. alba A. saccharum 1. nigraParameterb

W S R W S R W S R W S R

ll - 2.64*" 1.75' - 2.55*t 1.63t - 2.36*t 1.21 _ 1.84** 1.45tA 10.95t 1.97*t 11.36t 5.97* 1.01*t 4.74* 4.121 0.80**l 2.03*§ 4.73§ 0.52**t 2.70*'Ci 19.6' 15.1* 22.111 180ot 18.5* 18.415 22.5's 27.3*' 25.2 I1.9t 20.8* 19.6'(%) 421 54*1 33t 47* 49* 46* 34*f 19*§ 27t 42t 44* 45$

g5 1 26E 1 7*f 149' 62* 1 0*1 49* 76* 1 7** 39*' 56* 6**t 31 *1CE 0.691 0.23*' 0.70' 0.49* 0.09** 0.38* 0.26*§ 0.04* 0.12§ 0.41* 0.03*$ 0.31*Cp 5.5t 5.7t 5.3t 5.8t 7.0* 6.2t 5.6t 8.4*§ 6.1t 9.1*$ 10.3"1 10.8*

a Within a species, means of different stages of the drying cycle that are superscripted with * (P c 0.05) or ** (P c 0.01) are significantlydifferent. Among the four species, means at a given stage of the drying cycle are significantly different (P c 0.05) if superscripted withdifferent symbols (t, * 5, 11). hUnits for '' =-MPa, A = ,Umol.m-2.-s, C, = Pa, g, = mmol-m-2-s-1, Cp = Pa, CE =,4molm-2.-- Pa'.

Figure 2. Representative autoradiographs of leaves exposed to 14CO2. A, Leaf of well-watered Q. stellate (T, = -1.03 MPa). B, Leaf of mildlystressed Q. stellata ('I = -2.14 MPa). C, Leaf of severely stressed Q. stellata (I, = -2.55 MPa). D, Leaves of rewatered Q. stellata (', = -1.10MPa). E, Leaf of mildly stressed Q. alba (T4 = -1.88 MPa). F, Leaf of mildly stressed A. saccharum (T' = -1.79 MPa). Autoradiographs wereused as negatives so that light areas in the panels indicate 14C02 uptake.

1506 N AND PALLARDY




4closed' areoles and the rest of the leaf. These changes in thepattern of label uptake suggest that asymmetric stomatalclosure may be a transient response of stomata to the pro-gressive development of water stress typical of naturaldroughts. Furthermore, the lack of observable asymmetricclosure under severe stress supports the validity of our esti-mates of Ci and 1.Our gas exchange measurements were conducted under

environmental conditions that were similar, but not identical,to those imposed during '4CO2 exposures. Hence, it is appro-priate to assess the impact of these differences on the validityof stomatal limitation estimates derived from A-Ci relation-ships in the gas exchange experiment. During 14CO2 expo-sures, there was lower PPFD (750 versus 1000 ,mol m-2 s-')and higher Ca (1-3 Pa) and cuvette temperature (1-3.50C)].TI in severely water-stressed plants used for "4CO2 exposuresalso varied somewhat from that of plants on which gasexchange measurements were made because of our inabilityto control precisely rates of soil moisture depletion in eitherexperiment. However, on average, these ', differences wereno greater than 0.2 MPa, except for Q. alba, where '' was0.5 MPa higher in seedlings used for gas exchange measure-ments (not shown). Given these small differences, it is un-likely that plants exposed to severe water stress in either ofthe two experiments had qualitatively different patterns ofstomatal closure.

In agreement with others (e.g. refs. 2, 4, 10, 15, 16), ourresults indicated that nonstomatal limitation to photosyn-thesis was generally more important than stomatal limitationunder all conditions. The only exception to this pattern wasin severely stressed seedlings of Q. stellata, which showedslightly greater stomatal limitation. In well-watered plants,Q. stellata exhibited the highest photosynthetic rate, coupledwith the highest & and CE. The lower photosynthetic ratesof A. saccharum and J. nigra may partially be explained bylow CE in A. saccharum and high Cp in J. nigra.As has been reported in other studies (e.g. refs. 2, 3, 15,

22), gs was closely coupled with A in this experiment. How-ever, in the present study, CE also was closely correlatedwith A. Furthermore, decreased A under water stress wasgenerally accompanied by an increase in Cp. The relation-ships among A and ga, CE, and Cp indicate that both stomataland nonstomatal components of photosynthesis were influ-enced by water stress.Apart from these general relationships, the relative changes

of stomatal and nonstomatal factors under well-watered,water-stressed, and rewatered conditions differed amongspecies in a fashion that can be related to their distributionacross habitats varying in soil moisture availability. Undersevere water stress, increased stomatal limitation in xeric Q.stellata suggests that the mesophyll components of photosyn-thesis were relatively resistant to dehydration injury. On theother hand, the mesophyll of drought-sensitive A. saccharumleaves apparently was more susceptible to dehydration be-cause stomatal limitation decreased under water stress. In Q.alba and J. nigra, stomatal limitation did not change withplant water status. However, although A and gs recoveredfully in Q. alba within 5 d, A and gs were still depressed inseedlings of 1. nigra after rewatering. This pattern of responsein J. nigra was coupled with constant Ci, indicating that

mesophyll components of photosynthesis in this species stillhad not completely recovered after rewatering even thoughthis species was never exposed to the low AY, imposed onother species. In contrast, patterns of A, gs, and constant Ciin Q. alba indicated full recovery of the entire photosyntheticapparatus in this species. The results of this study supportprevious assertions of the pronounced sensitivity of J. nigrato leaf dehydration (14, 17), and emphasize the importanceto drought-tolerant plants of a robust photosyntheticapparatus.

Because we were unable to obtain good autoradiographsof leaves of severely stressed and rewatered A. saccharum,our conclusions for the photosynthetic responses to waterstress in this species must be considered cautiously. However,because the other species showed patterns of response thatwe believe permitted valid assessment of Ci and stomatal andnonstomatal limitation, there is less reason to believe thatleaves of A. saccharum should behave differently.

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1508 NI AND PALLARDY

Larrea divaricata. Carnegie Inst Washington Year Book 76:328-335

17. Ni BR, Pallardy SG (1991) Response of gas exchange to waterstress in woody angiosperm seedlings: net photosynthesis, leafconductance, and water use efficiency. Tree Physiol 8: 1-9

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