Tree Physiology 13, 107-l 17 0 1993 Heron Publishing-Victoria, Canada

Photosynthesis of oak leaves under water stress: maintenance of high photochemical efficiency of photosystem II and occurrence of non-uniform COz.assimilation

DANIEL EPRON’ and ERWIN DREYER’,*

1 INRA-Nancy, Laboratoire de Bioclimatologie et d’Ecophysiologie ForestitGres, Champenoux, F-54280 Seichamps, France

2 Author to whom correspondence should be addressed

Received October 22, 1992

Summary

Net CO2 assimilation rates (A), stomata1 conductance to water vapor (gW), photosynthetic 02 evolution in 5% CO2 (A,,,& and photochemical efficiency of photosystem II were monitored in leaves of young oak saplings (Quercuspetraea Matt. Liebl.) in response to increasing drought. Both A and gw declined rapidly as soon as predawn leaf water potential dropped below -1 .O MPa. The calculated intercellular concentration of CO2 first declined and then increased again as drought intensity increased, suggesting that both stomata1 closure and a decreased ability of mesophyll chloroplasts to fix available CO2 were involved in the drought-induced reductions in A and gw. However, this assumption was not supported by the observations that, with increasing drought, the decline in A max was limited, and the photochemistry of photosystem II and the quantum yield of light-driven electron transport remained stable. Autoradio- grams of 14C02-fed leaves revealed non-uniform assimilation rates during water stress. The conse- quences of a potential artifact induced by this patchiness on the calculation of intercellular CO2 concentration are discussed.

Keywords: assimilation rate, drought, gas exchange, leaf water potential, mesophyll conductance, stomata1 closure, stomata1 conductance.

Introduction

The effects of soil water depletion on leaf gas exchange of woody plants are well documented (for review, see Jarvis and Sandford 1986). Decreasing soil water content is thought to be responsible for drought-induced stomata1 closure. Drought- induced stomata1 closure is probably controlled both by metabolic signals that are transported from the roots in the drying zones of the soil to the shoots by means of xylem flux (Wartinger et al. 1990) and by leaf water status (Tardieu and Davies 1992). The resulting decline in stomata1 conductance will then limit net CO:! assim- ilation rates through a decrease in the intercellular CO2 concentration (c;).

However, in almost all studies of soil water depletion effects on leaf gas exchange, calculation of ci using models of leaf CO2 assimilation based on Fick’s law for gas diffusion (von Caemmerer and Farquhar 1981, Jones 1985) yielded a decline in Ci that was not as pronounced as predicted (cf. Teskey et al. 1986, Comic et al. 1987, Grieu et al. 1988, Epron and Dreyer 1990). This finding has led some authors to conclude that water deficits could impair both the diffusion of CO2 into the leaf and the ability of the mesophyll to fix available CO2. In contrast, measurements of

108 EPRON AND DREYER

chlorophyll a fluorescence or oxygen evolution in the presence of high CO:! concen- trations have shown that the photosynthetic apparatus is rather insensitive to leaf water deficits in herbaceous plants (Genty et al. 1987, Kaiser 1987). Similarly, we have observed that the photosynthetic apparatus of oak leaves is resistant to rapid leaf dehydration in darkness (Epron and Dreyer 1992). Kaiser (1987) and Comic et al. (1989) concluded that, in most cases, increased resistance to CO2 diffusion was entirely responsible for the drought-induced declines in photosynthesis.

Since Laisk et al. (1980) reported heterogeneity in stomata1 aperture over the leaf area, many authors have observed non-uniform gas exchange in water-stressed leaves (Downton et al. 1988b, Sharkey and Seemann 1989, Ni and Pallardy 1992) and suggested that this phenomenon may lead to overestimation of ci. However, because heterogeneity of stomata1 closure has only been reported in a restricted number of species, it may not be a widespread phenomenon (Guehl et al. 1991). Moreover, the real effect of this phenomenon on the calculation of ci is uncertain (Cheeseman 1991).

Because the oak trees that are widely distributed in the lowland forests of Europe suffer from periods of drought that are thought to be at least partly responsible for the occurrence of dieback (Becker and Levy 1982), it is of major importance to understand the physiological mechanisms involved in the reactions of oak trees to drought. To evaluate the nature of drought-induced disorders of the photosynthetic processes in oak leaves, we concurrently analyzed gas exchange under ambient conditions on the one hand, and oxygen evolution rates, photochemical efficiency of photosystem II (PS II) and quantum yield of light-driven electron transport in the presence of saturating light and CO2 concentrations on the other hand. Quantum yield of photosystem II was assessed by chlorophyll a fluorescence measurements (Genty et al. 1989). The occurrence of non-uniform gas exchange was monitored by means of autoradiograms of 14C02-fed leaves of oak seedlings subjected to various degrees of drought, to evaluate the effects of the phenomenon on the relationship between A and ci calculated from gas exchange data.

Material and methods

Plant material and experimental conditions

Seedlings of Quercus petraea (Matt.) Liebl. were grown in 5-liter pots filled with a l/l (v/v) mixture of sand and sphagnum peat fertilized with 2.0 g of Nutricote 100 (N/P/K, 13/13/13) supplemented with a mixture of trace elements and 4 g of magnesium chalk. The plants, which were irrigated daily, were grown in a naturally illuminated greenhouse from March to October 199 1.

One week before the onset of the experiments, twelve seedlings were transferred to a growth cabinet with a day/night air temperature (T,) of 24/16 “C, day/night relative humidity (RH) of 60/95%, and a 14-h photoperiod with a photon flux density (PFD) at the top of the plants of around 150 ymol rnp2 s-l. Drought was imposed by withholding water for up to 10 days. Predawn leaf water potential (‘I”,,) was used as

PHOTOSYNTHESIS OF OAK LEAVES UNDER WATER STRESS 109

an index of drought intensity. All measurements were made in a small climate chamber (PFD, 500 pmol m-* s-‘;

T,, 25 “C; RH, 40%; CO2 molar fraction in air (c,), 500 j,tmol mol-‘) after the seedlings had acclimated for one hour. Predawn leaf water potential (‘I’,& was monitored with a pressure chamber. Gas exchange was measured and exposure to 14C02madeon u y p f 11 ex anded leaves of the last growing flush. Chlorophyll fluores- cence and oxygen evolution capacity were recorded on leaf disks punched from narrow leaves.

Gas exchange measurements

Net CO2 assimilation rate (A, prnol m -* s-l) and stomata1 conductance for water vapor (gw, mm01 m -* s-l) were measured with a portable gas exchange system (LI 6200, Li-Cor, Lincoln, Nebraska, USA) inside the climate chamber. The intercellular CO:! concentration (ci, l.trnol mol-‘) was calculated according to von Caemmerer and Farquhar (198 1). Three relationships between A and ci, also called photosynthetic demand functions (Jones 1985), were established on well-watered plants by decreas- ing the ambient CO2 concentration (c,) from 600 to 100 pmol mol-’ in successive steps of about 50 pmol mall’.

Exposure to “COz and develop ment of autoradiograms

To determine potential heterogeneity in leaf photosynthesis, we used a 14C02 label- ing technique (Terashima et al. 1988, Sharkey and Seemann 1989, Ni and Pallardy 1992). Immediately after gas exchange measurements, the leaves were individually enclosed in a Plexiglas chamber and exposed to 14C02 for 2 min (specific activity, 40 mCi g-’ C; CO2 concentration, 500 pmol mol-‘). Each leaf was then immediately frozen in liquid nitrogen between two layers of aluminum foil. Autoradiograms of 28 labeled leaves were made by pressing the frozen leaves against X-ray films (X-OMAT AR, Kodak). Films were exposed for one week at -20 “C and then developed.

Chlorophyll fluorescence and oxygen evolution Fluorescence measurements were made with a modulated fluorometer (PAM 101, Walz, Germany) on leaf disks (10 cm*) enclosed in an oxygen electrode chamber (LD2/2, Hansatech, U.K.) that was flushed with humidified air containing 5% CO*. Both the leaf chamber and the incoming air were maintained at 25 “C. Only the central part of the leaf disk (2 cm*) was illuminated through the fluorometer fiber optics. Before the fluorescence measurements, the leaf disks were dark adapted for 10 min. Initial fluorescence (F,) was determined by applying a modulated measuring light (0.1 pmol m-* s-‘) at a frequency of 1.6 kHz. A 700-ms pulse of high intensity

* white light (7000 pmol m- s -’ . KL1500, Schott, Germany) was used to determine , maximal fluorescence (F,). Then, the central part of the leaf disks was continuously illuminated with actinic white light (1200 pmol rn-* s-l ; KL1500, Schott, Germany) for 20 min. The steady-state value of fluorescence (F’) was then recorded and a second pulse of high intensity white light was imposed to determine maximal

110 EPRON AND DREYER

fluorescence in the light-adapted state (F,‘). The actinic light was removed and F, recorded. This sequence of operations is summarized in Figure 1. Photochemical efficiencies of PS II in the dark (Fv/Fm), of open PS II reaction centers (F,‘/F,‘), and of PS II in the light (AF’IF,‘) were calculated according to Genty et al. (1989):

FJF, = (F, - FdIFm

AF’IF,’ = (F,’ - F’)/F,’

F,‘/F,’ = (F,’ - Fd)IFm’.

Before switching off the actinic light, oxygen evolution rates (A,,& were determined by closing the electrode chamber and following the increase in 02 partial pressure for 3 min. Calibration and calculation of A,,, were made according to Delieu and Walker (198 1).

Results

The response of leaf gas exchange of Quercus petraea seedlings to increasing drought is shown in Figure 2. Both net CO2 assimilation rates (A) and stomata1 conductance to water vapor (gw) decreased in response to the drought-induced decline in predawn leaf water potential (Yw,). Stomata1 conductance exhibited high variability at optimal water supply (‘I”,, > -0.5 MPa). Below -0.5 MPa, the drought-induced decline in g, was gradual, leading to low g, values at -3.0 MPa.

Figure 1. Fluorescence signals and measured parameters. Dark-adapted leaf disks (10 min) were enclosed in a chamber flushed with air containing 5% CO2. They were first illuminated with a modulated light (L,,) and initial fluorescence (F,) was recorded. A 700-ms pulse of high intensity white light (L,) was used to determine maximal fluorescence (F,,,). Then, the leaf disks were continuously illuminated with an actinic white light (La, 1200 pmol m -2 ss’) for 20 min. The steady-state value of fluorescence (F’) was recorded and a second pulse of high intensity white light was imposed to determine maximal fluorescence in this light-adapted state (Fm’). The actinic light was removed and F,’ recorded. A measurement of 02 evolution (A,,& was performed during the last three minutes before switching off the actinic light.

--

PHOTOSYNTHESIS OF OAK LEAVES UNDER WATER STRESS 111

300

67

2 200

'E S

.E % 100 M

0

12 47

'WI N

'E 8 ';j

3 : 4

0

0.7

0.5 2 ;m

0.3

0.1

a. :

.

I I

b. 8

2

C. : l *

-4 -3 -2 -1 0

Ywp WW

Figure 2. Relationships between predawn leaf water potential (Yw,) and (a) stomata1 conductance to water vapor (,gw), (b) net CO2 assimilation rate (A), and (c) ratio of intercellular CO2 to atmospheric CO2 (ci/c,) during the progression of drought. Each value represents an individual measurement, n = 12. Curves were fitted by eye.

The variability in A at optimal water supply was much lower than that of g, (mean values around 13 pmol m-’ SK’). The decline in A with decreasing ‘I’,, was gradual and rates were still positive (around 2 pmol mm2 SK’) at -3.0 MPa.

In response to increasing drought intensity, the ratio of intercellular CO2 concen- tration to atmospheric CO2 concentration (ci/ca) displayed a strong decline to low values of about 0.4 at around -2 MPa, but the ratio increased again as ‘PW, declined to -3 MPa (Figure 2~).

112 EPRON AND DREYER

Figure 3 shows the mean A versus ci relationship (demand function) obtained by decreasing c, in three well-watered plants. Most of the data points fell along the regression line, but seven to eight values deviated significantly, and among them there were two data points with ci values around 300 pmol mol-’ and A values below 3 pm01 mm2 SK’.

In response to increasing drought intensity, rates of 02 evolution under saturating conditions (A,,,=, 5% CO2 and 1200 pmol mm2 s-’ PFD) were maintained at much higher rates than A (Figure 4a). Only a very limited decline in A,,, was observed in response to increasing drought (mean A,,, values declined from 29.9 ltmolO2 m-* s-’ in well-watered plants to 25.7 pmol 02 m-* s-’ in drought-stressed plants). In severely drought-stressed plants that exhibited A values close to 0, A,, was reduced by only 30% compared to that of well-watered plants. At ‘PWp values above -0.5 MPa, a linear regression between A,, and YWp yielded a significant correlation (A,,, = 29.8 + 3.19 YWp, ? = 0.32, P = O.OS), but the correlation was not significant at Y,, values below -0.5 MPa (Amax = 21.8 + 0.20 YWp, r* = 0.001).

In the presence of high CO2 concentrations and saturating PFD, neither the photochemical efficiency of PS II in the dark (FJF,) or in the light (M’IF,,,‘), nor the photochemical efficiency of open PS II reaction centers (F”‘/F,,,‘) were signifi- cantly reduced in response to decreasing YWp (Figure 4b). Linear regressions on ‘I’,, were not significant for F,/F, or AF’/F,’ (mean values 0.795 5 0.013 and 0.356 + 0.041, respectively). They were significant, although with a very low slope, for F,‘/F,’ (F,‘/F,’ = 0.594 + 0.0213 YWp, r2 = 0.265, P = 0.05, mean, 0.566 * 0.045). These observations indicate that the photochemistry of PS II, light-driven electron transport, and enzymatic reactions requiring ATP and NADPH from chloroplasts were not significantly affected by the leaf water deficits induced by soil water depletion.

16 , I

0 100 200 300 400

Ci (pm01 mol-l)

Figure 3. Relationships between calculated intercellular CO2 concentration (c,) and net CO2 assimilation rate (A) measured at an ambient CO2 concentration of 500 pmol mol-’ during progression of drought. Each value represents an individual measurement, IZ = 12.The regression curve was fitted to data points (not shown) from A versus c, relationships established on three well-watered plants by incrementally decreasing the ambient CO;? concentration from 600 to 100 pm01 mol-’ (r = 0.987).

PHOTOSYNTHESIS OF OAK LEAVES UNDER WATER STRESS 113

a.

1 -4 -3 -2 -1 0

Ywp NW

Figure 4. Relationships between predawn leaf water potential (‘I’,,) and (a) oxygen evolution rate (A,,,& or (b) photochemical efficiency of PS II in the dark (FJF,, 0) of open PS II reaction center (F”‘/Fd, 0) and of PS II in the light (AF’IF,‘, A) during progression of drought. Each value represents an individual measurement made in air containing 5% CO2, II = 12. Solid lines represent linear regressions fitted to all data points; dashed line represents linear regression fitted to data recorded on water-stressed seedlings (Y’,, < -0.5 MPa).

Autoradiograms of water-stressed leaves (Figures 5B-D) displayed a non-uniform distribution of t4C, whereas autoradiograms of well-watered leaves showed a uni- form labelling pattern (Figure 5A). Non-uniform photosynthesis was observed in leaves of seedlings in which Y,, values were below - 1.2 MPa.

Discussion

Both net CO2 assimilation rates (A) and stomata1 conductance to water vapor (sW) in ambient CO2 and limiting irradiance declined in response to decreasing predawn leaf water potential (YWp). The declines in A and gW were gradual, and significant rates of A and g, were recorded at Y’,, values near -3.0 MPa, indicating that Quercus petruea is rather tolerant to drought, as has already been described for this species by Epron and Dreyer (1990), and by Bahari et al. (1985) for Q. alba and Q. rubra, and Ni and Pallardy (1991, 1992) for Q. alba and Q. stellatu.

Figure 5. Representative autoradiograms of leaves of Q. petruea seedlings frozen immediately after a 2-min exposure to “C02. The 14C02 labeling was done in a Plexiglas chamber immediately after gas exchange measurements. White areas have fixed “COz. Predawn leaf water potentials were -0.30 (A), -1.95 (B),-2.15 (C), and-2.90 MPa(D).

The gradual decline in A with decreasing ‘I”,, occurred at higher calculated intercellular CO2 concentrations (CJ than expected if stomata1 closure alone limited A, indicating that both stomata1 and non-stomata1 factors could contribute to the reduction in A during drought (cf. Teskey et al. 1986, Comic et al. 1987, Grieu et al. 1988, Epron and Dreyer 1990).

However, the measurements of 02 evolution (A,J and chlorophyll a fluorescence at 5% CO* and saturating h-radiance did not support the hypothesis of a non-stomata1 limitation of net CO2 assimilation rate during drought. Despite an apparent decrease in the ability of mesophyll to fix available CO2 in drought-stressed seedlings, neither the photochemical efficiency of PS II in the dark (F,/F,) or in the light (M’IF,‘), nor the photochemical efficiency of open PS II reaction centers (F,‘/F,‘) were reduced in response to decreasing ‘I’,,. These findings confirm previous observa- tions on Quercus petruea leaves subjected to rapid dehydration in the dark (Epron and Dreyer 1992) and on drought-stressed Helianthus annuus, Gossypium hirsutum, Phaseolus vulgaris and Elatostema repens (Ben et al. 1987, Genty et al. 1987, Comic et al. 1989). The small decrease in A,,, in drought-stressed leaves, which was not correlated with the intensity of drought, was observed as soon as stomata1 conduc- tance was reduced to about 100 mmol m- s ’ -’ Many factors may explain this slight . reduction in A,,,. For instance, Chaves (1991) reported that misleading results can be obtained with the leaf disk oxygen electrode if CO2 saturation is not fully achieved, because of high boundary layer resistance in the electrode chamber. A CO2 concentration as high as 17% was required to determine the maximal rate of 02 evolution in dehydrated leaves of Phaseolus vulgaris (Comic et al. 1989). On the other hand, Graan and Boyer (1990) reported that photosynthesis of ABA-treated leaves of Helianthus alznuus was restored at 3% CO;? in a gas exchange chamber with a much higher boundary layer conductance than that in our leaf disk electrode. In contrast to our results, the decline in COz-saturated photosynthesis observed by Graan and Boyer (1990) was closely related to the decrease in leaf water potential and leaf dehydration that occurred under high irradiance conditions.

PHOTOSYNTHESIS OF OAK LEAVES UNDER WATER STRESS 115

The absence of any effect of drought on PS II photochemistry and quantum yield of light-driven electron transport in the drought-stressed leaves indicates that the photosynthetic apparatus of Quercus petruea leaves is rather resistant to leaf water deficits imposed by soil water depletion, and is able to perform at high rates provided there is a sufficient supply of CO2 to the chloroplasts. These data do not support the hypothesis, deduced from calculations of Ci, of a non-stomata1 limitation of net CO2 assimilation rate during drought.

Stomata exhibit a wide variation in their aperture over the leaf surface (Laisk et al. 1980), and non-uniform gas exchange may occur if patches of stomata remain open and other patches close completely. Autoradiograms of 14C02-fed leaves revealed that non-uniform gas exchange occurred in the drought-stressed leaves of Quercus petruea. Non-uniform gas exchange has also been observed in water-stressed leaves of Vitis viniferu, Nerium oleander, Eucalyptus filicifoliu, Phuseolus vulgaris, Quer- cus stelluta, Quercus albu, Acer succhurum (Downton et al. 1988b, Sharkey and Seemann 1989, Ni and Pallardy 1992), in leaves of Heliunthus UY~JZUUS and Vitis viniferu fed with abscisic acid (Downton et al. 198&z, Terashima et al. 1988) and in leaves of Oleu europeu and Arbutus unedo in response to low atmospheric humidity (Loreto and Sharkey 1990, Beyschlag et al. 1990). It is noteworthy, that the patches we observed were not strictly delimited by leaf ribs as expected in these strictly heterobaric leaves, in which lateral diffusion of CO* within the leaf is thought to be strongly restricted (Terashima et al. 1988).

Laisk (1983) demonstrated that calculations of mesophyll resistance and Ci from models based on Fick’s law for gas diffusion are erroneous if net CO2 assimilation and transpiration are not uniformly distributed over the leaf area. Terashima et al. (1988) concluded that the apparent non-stomata1 inhibition of photosynthesis by abscisic acid, which was based on the finding that the calculated values of Ci were higher than the values predicted by models, was an artifact that could be ascribed to patchy stomata1 closure.

We conclude that the discordance between the maintenance of optimal PS II photochemistry and quantum yield of light-driven electron transport in drought- stressed leaves and the apparent decrease in the ability of the mesophyll to fix available CO2 supports the idea that the ci calculation is misleading in drought- stressed leaves of oak seedlings. The occurrence of patchy stomata1 closure may be one cause of this discrepancy, but our data do not support the hypothesis of a generalized patchiness, and the real consequences of patchiness on the ci calculation may only be limited. The existence of a significant resistance to the liquid diffusion of CO2 from intercellular spaces to the chloroplastic stroma may be another cause for the discrepancy. Potential decreases in mesophyll conductance during drought are poorly documented, but may result from decreases in intercellular volume as a result of turgor loss in mesophyll cells (Levitt 1980, Comic et al. 1989), apoplast acidification, or changes in the solubility of CO2. Loreto et al. (1992) have shown that oak species have lower mesophyll conductances (higher resistances) than many herbaceous species. A drought-induced decrease in chloroplastic CO2 concentration, calculated from the ratio of carboxylation to oxygenation rates, coupled with the

116 EPRON AND DREYER

maintenance of a constant ci has been reported for both Triticum aestivum (Renou et al. 1990) and Phase&s vulgaris (Comic and Briantais 1991).

We conclude that, under low light conditions, water deficits imposed rapidly (during a few days) limit photosynthesis by reducing the supply of CO2 to chloro- plasts as a result of decreases in stomata1 conductance in response to soil drying. The use of calculated ci to support the hypothesis of non-stomata1 limitation to net CO2 assimilation may be misleading because of stomata1 patchiness or changes in meso- phyll conductance to CO*, or both.

Acknowledgments

These experiments were partly performed at the Ecole Nationale d’Agronomie et des Industries Al- imentaires (ENSAIA), Laboratory of Plant Sciences, Nancy-Vandoeuvre. The authors are grateful to C. Robin and P. Grieu for their help and for having provided 14COz and climate chamber facilities. The help of J.M. Gioria in growing the plants and performing measurements is acknowledged.

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