Control Photosynthesis Stomatal Conductance in Ricinus ... · Control of Photosynthesis andStomatal Conductancein Ricinus communisL. (Castor Bean) byLeafto AirVapor Pressure Deficit1

Plant Physiol. (1992) 99, 1426-14340032-0889/92/99/1426/09/$01 .00/0

Received for publication December 3, 1991Accepted February 2, 1992

Control of Photosynthesis and Stomatal Conductance inRicinus communis L. (Castor Bean) by Leaf to Air Vapor

Pressure Deficit1

Ziyu Dai2, Gerald E. Edwards*, and Maurice S. B. KuBotany Department, Washington State University, Pullman, Washington 99164-4238

ABSTRACTCastor bean (Ricinus communis L.) has a high photosynthetic

capacity under high humidity and a pronounced sensitivity ofphotosynthesis to high water vapor pressure deficit (VPD). Thesensitivity of photosynthesis to varying VPD was analyzed by meas-uring CO2 assimilation, stomatal conductance (gj), quantum yieldof photosystem 11 ( and nonphotochemical quenching of chlo-rophyll fluorescence (qN) under different VPD. Under both medium(1000) and high (1800 micromoles quanta per square meter persecond) light intensities, CO2 assimilation decreased as the VPDbetween the leaf and the air around the leaf increased. The gsinitially dropped rapidly with increasing VPD and then showed aslower decrease above a VPD of 10 to 20 millibars. Over atemperature range from 20 to 40°C, CO2 assimilation and gs wereinhibited by high VPD (20 millibars). However, the rate of trans-piration increased with increasing temperature at either low orhigh VPD due to an increase in g5. The relative inhibition ofphotosynthesis under photorespiring (atmospheric levels of CO2and 02) versus nonphotorespiring (700 microbars CO2 and 2% 02)conditions was greater under high VPD (30 millibars) than underlow VPD (3 millibars). Also, with increasing light intensity therelative inhibition of photosynthesis by 02 increased under highVPD, but decreased under low VPD. The effect of high VPD onphotosynthesis under various conditions could not be totally ac-counted for by the decrease in the intercellular CO2 in the leaf (Ci)where Ci was estimated from gas exchange measurements. How-ever, estimates of C; from measurements of ,, and qN suggest thatthe decrease in photosynthesis and increase in photorespirationunder high VPD can be totally accounted for by stomatal closureand a decrease in Ci. The results also suggest that nonuniformclosure of stomata may occur in well-watered plants under highVPD, causing overestimates in the calculation of Ci from gasexchange measurements. Under low VPD, 30°C, high light, andsaturating C02, castor bean (C3 tropical shrub) has a rate ofphotosynthesis (61 micromoles CO2 per square meter per second)that is about 50% higher than that of tobacco (C3) or maize (C4)under the same conditions. The chlorophyll content, total solubleprotein, and ribulose- 1 ,5-bisphosphate carboxylase/oxygenaselevel on a leaf area basis were much higher in castor bean than inmaize or tobacco, which accounts for its high rates of photosyn-thesis under low VPD.

Castor bean (Ricinus communis L.) is a tropical and subtrop-ical shrub with high rates of photosynthesis. We have

' This work was funded in part by U.S. Department of Agriculturecompetitive Grant 90-37280-57606 and National Science Foundationgrants DMB-8512521 and DCB-8816322.

2Supported in part by Guangxi Agricultural College and Agricul-tural Ministry of the People's Republic of China.

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found that its photosynthesis is also very sensitive to humid-ity. Sensitivity of photosynthesis and stomata to changes inVPD3 between the leaf and the air in both C3 and C4 specieshas been reported in a number of studies, although the degreeof effect varies in both C3 and C4 species with decreasingatmospheric humidity (1, 8, 10-12, 17, 18, 22).The mechanisms of closure of stomata and decrease of

photosynthesis under low humidity have not been deter-mined. If low humidity directly affected the photosynthetictissue and decreased photochemical processes or carbon as-similation, a rise in Ci in the leaf could decrease gs. Thepossibility for inhibition of some component of the photo-synthetic process during photosynthesis under either lowhumidity or water stress was suggested from an earlier study(24) that indicated that, at a given Ci, the plants under lowhumidity or water stress had a lower A than control plants.However, in this study Ci was calculated by conventionalmethods from gas exchange data; recently it was found thatthis can result in an overestimation of the value of Ci if thereis nonuniform closure of stomata (5, 28). Heterogeneity instomatal closure has been reported in plants under waterstress, under high salinity, or after treatment with ABA (5-7,28), and it may occur under low humidity (17, 19).

Alternatively, low humidity might cause stomatal closuredirectly by causing a water stress in the epidermal tissue andguard cells due to excessive loss of water by transpiration.This effect of low humidity on stomata is consistent with adecrease in A under high VPD, which in some cases can beattributed totally to changes in g, based on the calculateddecrease in C1 (16). Closure of stomata under low humiditycould be the result of a decrease in bulk leaf water potentialas leaf-air VPD increases (12, 20, 23). However, there areother reports that indicate that stomata close at high VPDwithout changes in bulk leaf water potential (8, 30). Recently,Mott and Parkhurst (19) measured photosynthesis, transpir-ation, and stomatal conductance under different VPD be-tween the evaporating surface and the ambient gas made upof air or helox (a helium:oxygen mixture, 79:21) and sug-gested that the closure of stomata under low humidity was

3Abbreviations: VPD, vapor pressure deficit between leaf andatmosphere; A, CO2 assimilation rate; CABP, carboxyarabinitol 1,5-bisphosphate; C1, partial pressure of intercellular CO2 in the leaf; CO,partial pressure of external CO2 to the leaf; E, transpiration rate; g,,stomatal conductance to water; qN, nonphotochemical quenching; qE,component of nonphotochemical quenching due to membrane ener-gization; 4Il, photosystem II quantum yield.

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PHOTOSYNTHESIS AND VAPOR PRESSURE DEFICIT

due to direct loss of water from epidermal and guard cellsunder a high rate of transpiration.

Besides questions about the mechanisms of inhibition ofphotosynthesis by low humidity, there are differences amongspecies in the degree of sensitivity of photosynthesis to lowhumidity. We have found that castor bean, a tropical andsubtropical C3 species, has a very high capacity for photosyn-thesis under low VPD, which is reduced dramatically underhigher VPD. The reasons for the high capacity for photosyn-thesis and the sensitivity of photosynthesis to low humidityin castor bean were investigated in the present study. Maize,a C4 species with high photosynthetic capacity, and tobacco,a C3 species with low photosynthetic capacity, were includedin the study for comparison.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Seeds of castor bean (Ricinus communis L.), tobacco, andmaize were germinated in a soil mixture containing peatmoss, vermiculite, and sand in a 2:1:1 ratio in pots 16 cm indiameter and 17.5 cm high. After 2 to 3 weeks, the seedlingswere selected for uniform size and one plant was maintainedper pot. Plants were watered every day and supplementedevery 2 or 3 d with a diluted solution of Peter's fertilizer. Allplants were cultivated in a growth room under a 16 h light(300C, VPD of 10-15 mbar water vapor)/8 h dark (180C,VPD of 5-8 mbar) cycle. The light intensity, provided by acombination of mercury vapor and high pressure sodiumlamps (P.L. Light Systems, Grimsby, Ontario, Canada), was550 to 650 ,umol quanta m-2 s-1 on the plant canopy. All ofthese species have amphistomatous leaves with homobaricminor veins (lacking a lateral bundle sheath extension be-tween the minor veins and the epidermis).

Gas Exchange Measurements

Simultaneous measurements of A and E were made on thethird or fourth youngest leaves from 5-week-old plants in anopen system using an Analytical Development Co. (ADC)IRGA (225-MK3) and a Bingham Interspace model BI-2 Con-troller System. Plants were well watered before experiments.The plant was placed in a controlled environment chamberand the selected leaf enclosed in a cuvette. The CO2 concen-trations in the leaf cuvette were maintained at the desiredlevels with the controller using a null balance system to keepa constant level of CO2. The leaf cuvette (Bingham InterspaceCo.) contained a dew point sensor for measurement of hu-midity. Leaf temperature was monitored using a copper-constantan thermocouple placed against the lower leaf sur-face. The maximum irradiance, provided by a 1000 W metalhalide lamp, was 1950 ,mol quanta m-2 s-1. A transparentplastic water tank containing 15 to 20 cm of water wasmounted above the growth chamber to absorb IR radiation.Varying light intensities were obtained during measurementof photosynthesis by placing cheesecloth layers between thelight source and the leaf cuvette.

Because water vapor concentration around the leaf wasdependent on the rate of transpiration and the flow rate ofdry air through the chamber, the VPD was controlled by

changing the flow rate of dry air through the cuvette. A fanin the chamber mixed the air. The boundary layer conduct-ance of castor bean leaves was measured using blotting paperrepresentative of the leaf area used. This conductance washigh (3.6 mol mr2 s-') and was independent of the flow ratesthrough the chamber (30-4500 mL L`). When measuringphotosynthesis under high CO2, or when varying the atmos-pheric levels of CO2, an ADC gas mixer was used to controlthe CO2 concentration in the cuvette (under either 2% or21% 02). Calculations of A, E, g0, and Ci from gas exchangemeasurements were according to von Caemmerer andFarquhar (32). Calculations of VPD were based on the dif-ference in the water vapor pressure in the leaf (assuming100% humidity at the measured leaf temperature) minus thewater vapor pressure in the air.

Measurement of $1 and qN4II and qN were determined from measurements with a

modulated fluorescence system (PAM fluorometer, Walzmodel 101). In this case (see Figs. 4 and 5), fluorescencemeasurements were made simultaneously with gas exchangemeasurements with a leaf enclosed in a cuvette as describedabove. The fiber optic probe was positioned above the cuvetteand slightly to the side so as not to interfere with the incidentillumination.

Before the beginning of each experiment (see 'Results"),the plant was kept in the dark for about 6 h, the modulatedlight turned on to obtain F,, and then a saturating pulse oflight was given to obtain maximum fluorescence (Fm). Afterthat, the actinic light (1500 ,smol quanta m-2 s-1) was turnedon. During the experiments, steady state fluorescence (F,)was monitored continuously and saturating pulses (10,000,umol quanta m-2 s-5, 800 ms duration) were applied auto-matically at 400-s intervals for periodic determination of Fm'(apparent Fm). II was calculated as (Fm' - Fs)/Fm' (10) andqN was determined as 1 - [(Fm' - Fo')/(Fm -Fo)] (31).

Estimates of Ci at High VPD by a Combination of GasExchange and Fluorescence Measurements

To estimate Ci from fluorescence and gas exchange meas-urements at high VPD where there may be nonuniformclosure of stomata, the relationship between (bi/A and Ciwas first established under normal conditions at low VPD.This relationship was then used to estimate Ci from measure-ments of the change in qbjI/A with increasing VPD. Alterna-tively, the relationship between qN/A and Ci was establishedunder low VPD, and this was used to estimate C, frommeasurements of the change in qN/A with increasing VPD(see 'Results'). This is similar to the method used by Downtonet al. (6) to estimate Ci under water stress. They also measuredtotal qN, although at the time it was called qE.

Total Leaf Water Potential Measurement

Four leaf discs for each experiment, 8 mm in diameter,were harvested following treatment of leaves under differentVPD during gas exchange measurements, placed in thermo-couple psychrometer chambers, and allowed to equilibrate

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Plant Physiol. Vol. 99, 1992

for 50 to 70 min. The chambers were connected to a psy-chrometric microvoltmeter (model MJ 55, Wescor Company).After the thermocouple was cooled for 15 s, readings weretaken from the psychrometer output. The actual water poten-tial values for the leaves were determined according to thestandard curve generated using different concentrations ofNaCl with the chambers.

Rubisco, Chl, and Soluble Protein MeasurementsBefore gas exchange measurements, the area of the leaf to

be enclosed in the cuvette was marked and traced on paperand the area measured with a portable area meter (LI-3000,Li-Cor, Inc.). Following the gas exchange measurements, theleaf was removed from the cuvette and the area enclosedwas cut out and divided into two halves. One half of the leafsection was immediately immersed and stored in liquid nitro-gen until assays of Rubisco, total soluble protein, and Chlcontent were made, and the other half was cut into smallstrips and put into a test tube containing 10 mL of 95%ethanol for Chl extraction. The Chl samples were kept in thedark and incubated at room temperature (usually 2 d) untilall of the Chl was extracted. Then 20 to 50 ,uL of the clearsupematant was removed and diluted to 1 mL and the Chldetermined spectrophotometrically (34).

Quantification of Rubisco was achieved by binding of theenzyme to its substrate analog CABP (3, 33). CABP binds toboth carbamylated and decarbamylated Rubisco, though thedissociation constant of binding with the noncarbamylatedenzyme is higher. 14C-Labeled CABP was synthesized ac-cording to Collatz et al. (3). Frozen leaf tissue was groundcompletely with 10 volumes (w/v) of grinding buffer (100mM Bicine, pH 8.0, 25 mM MgCl2, 10 tsM leupeptin, 1 mmPMSF, 10 mm NaHCO3, and 12.5% glycerol), and 5% (w/w)insoluble PVP. Following centrifugation of the homogenatefor 5 min at 15,000g, 25 ,L of supematant were added to100,uL of 0.04 mM 14C-CABP and allowed to incubate for 45min at room temperature. Protein (including 14C-CABP-labeled Rubisco) was precipitated by addition of 125 gL of40% PEG 4000 in 100 mm Bicine (pH 8.0) and 25 mM MgC12,followed by incubation at 250C for 10 min. After centrifu-gation for 5 min at 15,000g, the pellet was washed with 250,uL of 20% PEG 4000 containing 20 mM MgCl2 and centri-fuged again at 15,000g for 5 min. The resulting pellet, con-taining 14C-CABP-labeled Rubisco, was resuspended in 100,gL of 100 mm Bicine (pH 8.0) and 10 mn-m MgCl2. Scintillationfluid was added to the sample and 14C was counted with ascintillation counter. The amount of Rubisco protein in thesample was calculated based on binding of 8 mol CABP permol of enzyme and assuming a molecular mass of Rubiscoof 550 kD. Total soluble protein in the 15,000g supematantwas determined by the method of Bradford (2), and theamount of Rubisco as a percentage of total protein wascalculated.

RESULTS

Effects of VPD on Photosynthesis Rate of Castor Beanversus Tobacco and MaizeThe influence of VPD on A, E, and gs was examined with

castor bean (C3) versus tobacco (C3) and maize (C4), with

increasing VPD under medium (1000 ,umol quanta m-2 s-)and high (1800 Amol quanta m-2 s-1) light intensity, at 30°Cand defined atmospheric conditions (21% 02, 345 gbar C02)(Fig. 1). In all three species, A decreased with increasingVPD. The degree of decrease in A with increasing VPD wasgreatest in castor bean and tobacco, whereas A in maize wasrelatively insensitive to increase in VPD up to 30 to 35 mbar.When comparisons were made of changes in A on a relativebasis (taking the rate at the lowest VPD as 100%), castorbean and tobacco showed a similar decline in A with increas-ing VPD, whereas maize, again, showed much less sensitivityto increasing VPD. For tobacco and maize the sensitivity ofA, on a relative basis, to increasing VPD was higher underhigh light intensity than under moderate light intensity.Under high light intensity, the CO2 assimilation rate in

castor bean was much higher than that of tobacco. Also,under low VPD and high light, A in castor bean was close tothat in maize. However, under high VPD, A in castor beanwas much lower than in maize due to its greater sensitivityto high VPD.Under low VPD, g, was higher in castor bean than in

tobacco or maize. In maize, the degree of decrease in gs withincreasing VPD was less than that in castor bean or tobacco.Castor bean always maintained a higher gs than eithertobacco or maize at any given VPD, which results in castorbean having the highest E at any given VPD. Castor bean

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Figure 1. The responses of A, percentage of maximum A, g,, and Eof castor bean, tobacco, and maize to different VPD under twolight intensities (1000 ± 30, 1800 ± 50 itmol quanta m-2 s-').

1428 DAI ET AL.




Table I. Photosynthesis Rate, Chl Content, Total Soluble Protein, and Rubisco Activity and Content inLeaves of Various Plants

Measurement Unit Castor Bean Tobacco Corn

PhotosynthesisNonphotorespiring conditiona gmol m-2 s51 60.9 40.3 46.6

Amol mg-' Chi h-1 449 446 460Photorespiring conditionb Omol m-2 s-, 42.1 20.5 43.8

Amol mg- Chl h-' 310 227 434Chl content mgm2 488 325 365Total soluble protein content mg m-2 8540 4720 4980Protein/Chi mgmg-' Chl 13.0 10.8 9.5Rubisco content mg mg-' soluble protein 0.43 0.49 0.37

mg m-2 3660 2300 1830a Measured at 30°C, 700 Mbar C02, 2% 02, 1800 ± 30 Mmol m-2 s-1 light intensity and 3 ± 0.7

mbar VPD. b Measured at 30°C, 345 gbar CO2, 21% 02, 1800 ± 30 Mmol m-2 S-1 light intensityand 3 ± 0.7 mbar VPD.

was analyzed in more detail because of its high capacity forA and high sensitivity of A to increasing VPD.

Photosynthetic Rate, Rubisco, Chi, and Soluble ProteinContent in Castor Bean, Tobacco, and Maize

To analyze further the basis for the high capacity forphotosynthesis in castor bean, measurements were made ofA, Rubisco, Chl, and soluble protein content on a leaf areabasis in comparison to tobacco and maize (Table I). Undernonphotorespiring (low 02 and saturating C02), low VPD,300C, and high light conditions, castor bean had the highestA, whereas tobacco and maize had lower A on a leaf areabasis. Under normal atmosphere (photorespiring in C3plants), and with other conditions the same as above, A incastor bean was similar to that in maize, while that in tobaccowas much lower. Under nonphotorespiring conditions, A incastor bean was about 50% higher than that in tobacco ormaize on a leaf area basis; the rates, on a Chl basis, weresimilar in all three species because castor bean has more Chlper leaf area. Tobacco and maize had a similar amount ofChl, soluble protein, and Rubisco per unit leaf area, whereasthe levels of these components in castor bean were about1.5- to 2-fold higher.

Effect of VPD on Photosynthetic Rate in Castor Beanunder Different Light Intensities and Temperatures

The high sensitivity of photosynthesis to humidity in castorbean was further evaluated under different environmentalconditions. The effects of VPD on A, g5, and Ci under varyingleaf temperature and light intensity are shown in Figure 2.At both 1000 and 1800 ,mol quanta m-2 s-1, A, and g,decreased more rapidly with increasing VPD at the lowertemperature (200C) than at the higher temperatures (30 and350C) (Fig. 2). Also, at both light intensities g5 decreasedrapidly with increasing VPD from 2 to 3 mbar up to approx-imately 15 mbar, but at higher VPD g5 decreased more slowlyand reached a minimum value. Ci, calculated from gas ex-change data, decreased up to a point with increasing VPD;however at higher VPD C1 increased. Thus, A continued todecrease at high VPD without a decrease in the value of Ci

.%40

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Figure 2. The responses of A, percentage of maximum A, gs, andC, to varying VPD under different leaf temperatures (20, 30, 35°C)and light intensities (1000 ± 30, 1800 ± 50 Amol quanta m-2 S-1) incastor bean.

I

1429




calculated from gas exchange data. As VPD increased thepoint of rise in the calculated Ci occurred earliest at 200C,next at 300C, and last at 350C. This correlates with gs decreas-ing more rapidly with increasing VPD at the lower tempe-rature (200C) followed by 300C and 350C.

In another experiment the temperature was increased from20 to 400C, at 1000 or 1800 ,umol quanta m-2 S-1 andmeasurements made on A, gs, and E at either 3 or 20 mbarVPD (Fig. 3). At the low VPD A was high and g, increasedwith increasing temperature up to 400C. At the high VPD, Awas inhibited by more than 50% under both light intensitiesand g, was low over the entire temperature range, but gsgradually increased with increasing temperature. At both lowand high VPD the rate of transpiration, E, increased withincreasing temperature due to the increase in g5, whereas thetemperature optimum for A was 300C. Also, water loss asindicated by E was as great at high VPD as at low VPD,despite the much lower g& at high VPD.

Estimates of Changes in Ci and Photosynthetic Rate inCastor Bean from Measurements of 4,, and qN

As noted in the introduction, nonuniform stomatal closurecan cause errors (overestimates) in the calculation of Ci fromgas exchange measurements. If nonuniform closure of sto-mata occurs under high VPD, this would make gas exchange

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Figure 3. The responses of A, g,, and E to varying temperatureunder two light intensities (1000 ± 30, 1800 ± 50 gmol quanta m2s-') and two VPDs (3 ± 0.7, 20 + 0.4 mbar) in castor bean. Openand filled circles represent the temperature responses under 1800± 50,umol quanta m-2 s-1. Open and filled triangles indicated thetemperature response under 1000 ± 30 Mmol quanta m-2 s-'. At agiven light intensity the temperature was changed from low to highduring the experiment.

estimates of Ci unreliable for analysis of limits on photosyn-thesis due to stomatal or nonstomatal causes. Thus, fluores-cence measurements were made as a means of evaluating thebasis for inhibition of photosynthesis by high VPD.

First, as a control, the external CO2 was varied under lowVPD (without stress) and A and II were determined as afunction of Ci at 300C and 1500 Amol quanta m-2 s-' (Fig.4a). As Ci decreased from 700 to 70 ,bar, 4b1 was maintainedat a higher level than A due to an increased partitioning ofelectrons from PSII to photorespiration (see 'Discussion').Thus, the ratio of 4NI/A increased with decreasing Ci. Thiswas especially marked at C, below 150 ,ubar. In a secondexperiment, the influence of increasing VPD on A and sIIwas determined (Fig. 4b). As VPD increased, A decreasedmore rapidly than (4n, resulting in an increase in the 411/Aratio. This suggests that the decreased Ci at high VPD causeda decrease in A and an increase in the partitioning of electronsto photorespiration.From measurements of the relationship between bi/A and

Ci under low VPD (Fig. 4a), Ci was estimated from themeasured 4',,/A ratios under varying VPD. As shown inFigure 4c, with increasing VPD there was a decrease in Ci,estimated from measurements of the 411/A ratio (Ci(2)). How-ever, when calculated from gas exchange measurements, Cifirst decreased, and then increased, with increasing VPD at32 mbar and above (Cj(l), Fig. 4c). This indicates an overesti-mation of Ci, based on gas exchange measurements, at highVPD. Figure 4d shows the decrease in measured A withincreasing VPD (from Fig. 4b) versus the predicted decreasein A based on the change in Ci(2) from fluorescence analysis(Fig. 4c). The results show a near identical decline in boththe measured A and that predicted from estimates of C1derived from measurements of tI.A similar approach was also used to estimate Ci under high

VPD, based on changes in qN (Fig. 5) (5, 6). As Ci increaseddue to changes in the [CO2] around the leaf under low VPD,A increased and qN decreased (Fig. 5). Changes in qN/A undervarying VPD were then used to estimate Ci(3) from the rela-tionship between Ci and qN/A under low VPD. Ci(3) decreasedwith increasing VPD, but as shown in Figure 4c, Ci(l) calcu-lated from gas exchange measurements first decreased andthen increased with increasing VPD above 32 mbar. There isa similar decline in both the measured A with increasingVPD and that predicted from estimates of C1 from measure-ments of qN (Fig. 5d). These results again suggest that, athigher VPD, estimating C1 by analyzing changes in fluores-cence is more accurate than estimating Ci from gas exchange,because of nonuniform stomatal closure under higher VPD.An equation developed by Downton et al. (5, 6) to determinemodeled Ci strictly from gas exchange measurement of Aunder stressed versus nonstressed conditions was also tested,and the results were used to predict A at varying VPD. Thisequation depends on the assumption that part of the stomataare completely closed as others open with normal conduct-ance under patchy conditions. However, this equation didnot give quite as close a fit to the measured A as didpredictions from fluorescence analysis (data not shown).

Photosynthetic Light Responses in Castor Bean underPhotorespiring and Nonphotorespiring Conditions atDifferent VPDThe effects of light intensity on photosynthesis in castor

bean were examined at 300C under photorespiring (21% 02

O A 3 mbar'VPD* * 20 mbor VPD

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1430 DAI ET AL.




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and 354 ,bar CO2) versus nonphotorespiring (2% 02 and 700,ubar CO2) conditions (Fig. 6). Under normal ambient atmos-phere and low VPD, there was an increase in A with increas-ing light intensity up to 1800 ,umol quanta m-2 s-', whereasunder high VPD, A saturated at about 800 jtmol quanta m-2s-' and then declined thereafter. Under high VPD, A wasless than half that measured under low VPD, particularly athigh light intensities. Under nonphotorespiring conditionsand low VPD, A increased with increasing light intensity,reaching very high rates (60 /mol CO2 m-2 s-') at 2000 ,molquanta m-2 s-, the equivalent of full sunlight. Under non-photorespiring conditions and high VPD, A remained highand was near saturating at approximately 800 ,umol quanta

-2 -1m sUnder photorespiring conditions at low VPD, g, increased

drastically as light intensity was increased from 100 up to1200 ,umol quanta m-2 s-' (Fig. 6). However, under photo-respiring conditions at high VPD, g, was very low at all lightintensities. Under nonphotorespiring conditions (high CO2and low 02) and low VPD, g, was lower than under photo-respiring conditions. Again, under nonphotorespiring condi-tions, gs was low under both low and high VPD. The results

Figure 4. a, Changes in A, 4X,,, and ratio of (Dn/C A with varying C, under low VPD (3 ± 0.7 mbar)

at 1500 /smol quanta m-2 s-' and 30°C in castorbean. b, Changes in A, 411, and 4ki,/A with vary-ing VPD. c, Changes in C, calculated from gasexchange (Cj(l)), and C, calculated from thechange in ratio of 411/A (Ci(2)) at varying VPD. d,Changes in measured A and in predicted Awith varying VPD. The measured A was from

d panel b. For A predicted from Ci(2), the value ofCi(2) at each VPD (panel c) was used to calculatethe rate of A that would be expected based onthe relationship between A and C, at low VPDin panel a.

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24 32 40bar)

on E show, again, a high rate of water loss at high VPDdespite the low g5. The relative inhibition of A under photo-respiring conditions versus nonphotorespiring conditions wasmuch higher under high VPD (31 mbar) than under low VPD(3 mbar) (Fig. 7). With increasing light intensity, the relativeinhibition of A by photorespiring conditions increased underhigh VPD, but decreased under low VPD. Those differencesmay be related to changes in Ci under low versus high VPD,and under different light intensities.

Effects of CO2 on Photosynthesis in Castor Bean underDifferent VPD

The effects of varying levels of atmospheric CO2 on A incastor bean were determined under three different VPDs (Fig.8). Increasing VPD not only lowered the maximum rate ofphotosynthesis, it also caused an apparent saturation of pho-tosynthesis at a lower C.. At low VPD (3 mbar), gs increasedgradually with increasing CO2, reaching a high, maximumvalue around 900 ,ubar CO2 (Fig. 8). However, at 20 and 30mbar VPD, g5 decreased steadily with increasing C., indicat-ing stomatal closure by high CO2. At VPD of 20 and 30 mbar,

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Figure 5. a, Changes in A, qN, and ratio of qN/A with varying C, under low VPD (3 ± 0.7 mbar)at 1500,umol quanta m-2 s-1 and 30°C in castorbean. b, Changes in A and qN with varying VPDin castor bean. c, Changes in C, calculated fromgas exchange (C(l)) and in C, calculated basedon the change in qN (Ci(3)) with varying VPD incastor bean. d, Changes in measured A and inpredicted A with varying VPD. The measuredA is the same as in Figure 4b. For A predictedfrom Ci(3), the value of Ci(3) at each VPD (panelc) was used to calculate the rate of A that wouldbe expected based on the relationship betweenA and C, at low VPD in panel a.

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n




345 ,Abor CO2. 21% 02 700 ubar CO2, 2% 02

60

45

30

15

0

2400

1800

1200

600

0

6

4

2

0

0 400 800 1200 1600 2000 0 400 800 1200 1600 2000

Light ( Aumol m s )

Figure 6. The responses of A, g,, and E to varying light intensitiesat 30°C under photorespiring (345 Mbar CO2 and 21% 02) versusnonphotorespiring (700 j.bar CO2 and 2% 02) conditions at twoVPD (3 ± 0.7, 31 + 2 mbar) in castor bean.

E decreased with increasing CO2. However, under low VPD,E was low and increased gradually with increasing CO2.

DISCUSSION

Several notable observations about photosynthesis in cas-tor bean can be made from this study. Under low VPD, castorbean maintains a high capacity for photosynthesis and highstomatal conductance with increasing CO2 concentration (Fig.8) and has a lower sensitivity to photorespiring conditions(Figs. 6 and 7); increasing VPD causes a marked loss incapacity for photosynthesis (Figs. 1-3, 6, and 8). The highcapacity of photosynthesis (leaf area basis) in this C3 species

v 70

C 600

-50

.0

C4C 40

o 3 mbar VPD *31 mbar VPD /

I.

400 800 1200 1600 2000

Light (,umol m s )

Figure 7. Percent inhibition of photosynthesis in castor bean byphotorespiring conditions (APR measured at 345 ,Lbar CO2 and 21%02) versus nonphotorespiring conditions (ANPR measured at 700,ubar CO2 and 2% 02) with increasing light intensity at low (3 ± 0.7mbar) versus high (31 ± 2 mbar) VPD. Percent inhibition wascalculated from the data in Figure 6 as [(ANPR - APR) X 100]/ANPR.

n

N1

75

60

E 45

E 30

%- 15

0

1000to

N750

E-6 500EE 250o!T 0

a8n

N 6E

4

E 2

0

0 300 600 900 1200 1500

CO (Abar)

Figure 8. The responses of A, g,, and E to varying C. under 300Cand 1800 ± 50 Amol quanta m-2 s at three different VPD (3 ± 0.7,19.4 ± 1 and 30.2 ± 1 mbar) in castor bean.

is apparently due to a high level of photosynthetic compo-nents: a higher Chl, Rubisco protein, and total soluble proteinper leaf area compared with the C3 species tobacco and theC4 species maize (Table I). However, at 300C castor bean wasonly able to photosynthesize at rates comparable to maize ifthe RH was quite high; at low RH maize has a distinctadvantage due to its lower sensitivity to high VPD (Fig. 1).C3 plants are generally considered to have a lower capacityfor photosynthesis than C4 plants under atmospheric condi-tions and relatively high temperature. However, this is notalways the case. For example, under optimum levels of Nnutrition the C3 species Chenopodium album has a high ca-pacity for photosynthesis per unit leaf area, equivalent tothat of the C4 species Amaranthus retroflexus (21).

Photosynthesis in castor bean was very sensitive to changein humidity; A decreased in a near linear or curvilinearfashion with increasing VPD (Figs. 1 and 2). Despite thedecrease in gs with increasing VPD, the rate of water loss bytranspiration was usually high at high VPD. At a given VPD,however, castor bean has a higher E than maize or tobacco,due to its higher gs (Fig. 1).With increasing VPD there was a decline in A, while Ci,

calculated from gas exchange, decreased at first but thenincreased at high VPD (Fig. 2). An unexplainable increase inCi, calculated from gas exchange measurement at high VPD,was previously observed (26). Such an increase in Ci was alsopredicted in a model when there is nonuniform stomatalclosure (13). These results indicate that patchy stomatal clo-sure may occur at high VPD. There is also evidence from

1-1

7

_E0

EU)

N

E

-3E-rIE

0)

EhJEEw

I

1 432 DAI ET AL.




several recent studies for nonuniform stomatal closure duringstress caused by withholding water (5), by salinity (7), or bythe application of ABA, which simulates these stresses (6,28).To use changes in PSII Chl fluorescence in conjunction

with gas exchange techniques to obtain an accurate estimateof C1, we measured two components that are related to theway energy absorbed by PSII is utilized (4)II) or dissipated(qN). In C3 plants, at a given light intensity and leaf tempe-rature and under low VPD, decreasing C1 by changing thelevel of CO2 in the atmosphere causes a decrease in photo-chemistry by PSII (reflected in a decrease in 4)lj) and anincrease in nonphotochemical quenching of absorbed energy(qN). 41i is indicative of PSII activity, because bl x lightintensity x the fraction of incident light absorbed by PSIIequals the PSII activity (in these experiments light intensityis constant). Because more of the energy utilized in photo-chemistry is partitioned into photorespiration with decreasingCi, the ratio of 1ii/A also increases with decreasing Ci. Theserelationships, when measured under low VPD where thereis high stomatal conductance and accurate calculations of Cifrom gas exchange data, can be used to estimate Ci underhigher VPD. First, C, was calculated by gas exchange methodswhile varying atmospheric CO2 under low VPD (with simul-taneous measurements of A, E, (DI, and qN). The relationshipbetween the change in C, and the ratio of 4ki,/A or qN/A,established with varying CO2 under low VPD, was then usedto calculate Ci from the change in 4II/A or qN/A with varyingVPD.A comparison of the decrease in A versus the change in 4VI

with increasing VPD (Fig. 4) provides some initial insight asto whether high VPD inhibits photosynthesis by stomatalclosure or by direct inhibition of photochemistry/carbon me-tabolism. If high VPD inhibits photosynthesis due to stomatalclosure, this should result in a decrease in Ci, which, in turn,would decrease photosynthesis. However, in this case bII willnot decrease as rapidly as A because a decrease in Ci (e.g. bydecreasing [CO2] around the leaf) will result in an increase ofenergy partitioning from PSII into photorespiration (4, 14)and, thus, will decrease A more rapidly than 'bI.The results of estimating Ci from fluorescence analysis ((I,

and qN) with castor bean (Figs. 4 and 5) indicate that there isa continual decrease in Ci with increasing VPD. This is incontrast to the results based on gas exchange data, where C1decreased and then increased with increasing VPD (Figs. 2,4c, and 5c). An earlier study with cotton (26) also reportedan increased Ci at high VPD while A decreased. These resultsindicate that stomatal closure, and the resultant decrease inCi, account for the decrease in photosynthesis in castor beanunder high VPD. The deviation between the two methods ofcalculating Ci, under the conditions used (1500 ,mol quantam-2 s-', 30°C; Figs. 4 and 5), becomes apparent at and above32 mbar VPD and is greatest at the highest VPD (38 mbar),which suggests that, at high VPD, there is nonuniform closureof stomata.We found that Ci and A values predicted from measure-

ments of qN at varying VPD gave a closer fit to the measuredvalues of A than those predicted from measurements of qE, acomponent of nonphotochemical quenching due tomembrane energization (data not shown). Although the rea-

sons for this are not certain, it may be that during the courseof the measurements under both low and high VPD thereare changes in the partitioning of nonphotochemical quench-ing between qE-type quenching and other forms of nonpho-tochemical quenching.

In castor bean, photosynthesis was more sensitive to pho-torespiring conditions under high VPD than under low VPD,regardless of the light intensity (Figs. 6 and 7). This is,presumably, due to a lower Ci under high VPD, because it iswell known that decreasing C1 causes an increase in 02inhibition of photosynthesis (25). Values of Ci calculated fromgas exchange measurements under photorespiring conditionswere lower under higher VPD than under lower VPD (resultsnot shown, but the difference may be even greater if the C1value at high VPD is overestimated). With increasing lightintensity the relative inhibition of photosynthesis due tophotorespiring conditions increased under high VPD (Fig. 7).This may be due to a decrease in Ci under high light intensitywhere there is low stomatal conductance. A complete analysisof these phenomena would require estimates of Ci fromfluorescence measurements. In contrast, there was a decreasein relative inhibition of photosynthesis by photorespiringconditions under low VPD with increasing light intensity.This likely occurs because O2-insensitive photosynthesis canoccur under conditions where the capacity for photosynthesisis high, but is limited by triose-P utilization (27). With in-creasing irradiance, the relative inhibition of photosynthesisby 02 also decreased in potatoes under a VPD of 11 to 15mbar (15).High VPD caused an inhibition of A in castor bean over a

wide range of temperatures from 200C to 400C (Fig. 3). Theseresults are in contrast to those of Thompson et al. (29), whofound a depression of photosynthesis in Citrus jambhiri athigh temperature when vapor pressure in the air around theleaf was low, but not when it was high. Although high VPDcaused low stomatal conductance at both high and low VPD,g5 increased with increasing temperature. There are differ-ences among species in the reported responses of stomata totemperature (1).With varying atmospheric levels of CO2, the most striking

effects were the steady decrease of gs with increasing CO2under high VPD and the lack of closure of stomata withincreasing CO2 under low VPD (Fig. 8). Although high C1 iswell known to cause a decrease in g5 (17), under low VPDthe stomatal conductance continued to increase up to 900,ubar and was maintained up to 1500 ,ubar CO2. This suggeststhat sensitivity of stomata to high CO2 in castor bean may belinked to VPD. With increasing atmospheric CO2 under lowVPD, the ratio of Ci/Co was constant (not shown). This iscommonly observed, although the mechanism for maintain-ing this ratio is not known (18).To determine whether VPD affected leaf water potential,

we measured the total leaf water potential initially and aftertreatment of leaves at different RHs. At 1800 timol quantam-2 s-', 300C, and low VPD, the total water potential of theleaf was -13.3 bars; after gradually increasing the VPD overabout 3 to 4 h to 36 mbar, the total water potential of theleaf was -13.6 bars. Thus, the leaf water potential was onlyslightly more negative after exposure of the leaf to a highVPD. The results indicate that, in a short-term treatment, leaf

1433




water status is not affected by varying RHs. However, inresponse to high VPD, water deficits may be localized in theguard cells and epidermis, such that little difference may beobserved in the total leaf water potential (19).

In summary, the tropical C3 plant castor bean has a highphotosynthetic capacity under high humidity conditions,comparable to that of the C4 plant maize. This high capacityis sustained by high Chl, soluble protein, and Rubisco contentin the leaf. Under high humidity, photosynthesis respondsfavorably to high temperature, high light, and high CO2concentrations. The inhibitory effect of low humidity onphotosynthesis in castor bean is mainly, if not entirely, dueto a lowered CO2 concentration in the leaf, which is causedby stomatal closure.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Keith Mott for his interest andcomments on the subject, Dr. Alan Black for suggestions and use ofhis equipment for leaf water potential measurement, Dr. Stewart S.Higgins for help in data analysis, and Dr. C. B. Osmond for providingCABP. The authors also wish to thank Sandy Edwards and Dr.Walter Oberhuber for helpful comments on the manuscript.

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8. El-Sharkawy MA, Cock JH (1986) The humidity factor instomatal control and its effect on crop productivity. In RMarcelle, H Clijsters, MV Poucke, eds, Biological Control ofPhotosynthesis. Martinus Nijhoff, Boston, pp 187-198

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15. Ku MSB, Edwards GE, Tanner CB (1977) Effects of light, carbondioxide, and temperature on photosynthesis, oxygen inhibitionof photosynthesis, and transpiration in Solanum tuberosum.Plant Physiol 59: 868-872

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18. Mott KA (1990) Sensing of atmospheric CO2 by plants. PlantCell Environ 13: 73 L-737

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25. Sharkey TD (1988) Estimating the rate of photorespiration inleaves. Physiol Plant 73: 147-152

26. Sharkey TD, Imai K, Farquhar GD, Cowan IR (1982) A directconfirmation of the standard method of estimating intercellu-lar partial pressure of CO2. Plant Physiol 69: 657-659

27. Sharkey TD, Stitt M, Heineke D, Gerhardt R, Raschke K,Heldt HW (1986) Limitation of photosynthesis by carbonmetabolism. II. O2-insensitive CO2 uptake results from limi-tation of triose phosphate utilization. Plant Physiol 81:1123-1129

28. Terashima I, Wong SC, Osmond CB, Farquhar GD (1988)Characterization of non-uniform photosynthesis induced byabscisic acid in leaves having different mesophyll anatomies.Plant Cell Physiol 29: 385-394

29. Thompson CR, Stolzy LH, Taylor OC (1965) Effect of soilsuction, relative humidity and temperature on apparent pho-tosynthesis and transpiration of rough lemon (Citrus jambhiri).Proc Am Soc Hortic Sci 87: 168-175

30. Turner NC, Schulze E-D, Gollan T (1985) The responses ofstomata and leaf gas exchange to vapor pressure deficits andsoil and leaf water content. II. In the mesophytic herbaceousspecies Helianthus annus. Oecologia 65: 348-355

31. Van Kooten 0, Snell JFH (1990) The use of chlorophyll fluo-rescence nomenclature in plant stress physiology. PhotosynthRes 25: 147-150

32. von Caemmerer SV, Farquhar GD (1981) Some relationshipsbetween the biochemistry of photosynthesis and the gas ex-change of leaves. Planta 153: 376-387

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1 434 DAI ET AL.


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