Planta

Planta (1982)156:97-107

9 Springer-Verlag 1982

Photoinhibition of photosynthesis: effect on chlorophyll fluorescence at 77K in intact leaves and in chloroplast membranes of N e r i u m o l e a n d e r * Stephen B. Powles and Olle Bj6rkman Department of Plant Biology, Carnegie Institution of Washington, Stanford, CA 94305, USA

Abstract. The effect of exposing intact leaves and

isolated

chloroplast membranes of N e r i u m o l e a n d e r L. to excessive light levels under otherwise favorable conditions was followed by measuring photosynthetic CO2 uptake, electron transport and low-temperature (77K = - 196 ~ C) fluorescence kinetics. Photoinhibition, as manifested by a reduced rate and photon (quantum) yield of photosynthesis and a reduced electron transport rate, was accompanied by marked changes in fluorescence characteristics of the exposed upper leaf surface while there was little effect on the shaded lower surface. The most prominent effect of photoinhibitory treatment of leaves and chloroplasts was a strong quenching of the variable fluorescence emission at 6 9 2 nm (Fv,692) while the instantaneous fluorescence (Fo,692) was slightly increased. The maxim u m and the variable fluorescence at 734 nm were also reduced but not as much as FM,692 and Fv,692. The results support the view that photoinhibition involves an inactivation of the primary photochemistry of photosystem II by damaging the reaction-center complex. In intact leaves photoinhibition increased with increased light level, increased exposure time, and with decreased temperature. Increased CO 2 pressure or decreased 02 pressure provided no protection against photoinhibition. With isolated chloroplasts, inhibition of photosystem II occurred even under essentially anaerobic conditions. Measurements of fluorescence characteristics at 77K provides a simple, rapid, sensitive and reproducible method for assessing photoinhibitory injury to leaves. The method should prove especially useful in studies of the oc* C.I.W. - D.P.B. Publication No. 773

Abbreviations and symbols: P F D = p h o t o n flux area density; PSI, PSII = photosystem I, II; FM, Fo, Fv = maximum, instantaneous, variable fluorescence emission

currence of photoinhibition in nature and of interactive effects between high light levels and major environmental stress factors. Key words: Chlorophyll fluorescence - N e r i u m

-

Photoinhibition of photosynthesis - Photosynthesis (inhibition) - Photosystems I, II.

Introduction

Inhibition of photosynthesis can result when green plants are exposed to high light levels (for a review see Bj6rkman 1981). Sudden exposure of aquatic algae (Myers and Burr 1940; Kok 1956) or leaves of terrestrial plants (Bj6rkman and Holmgren 1963; Powles and Thorne 1981) to incident photon flux area densities (PFD) considerably higher than those at which the plants are grown, causes a gradual decline in photosynthetic rate in vivo. A similar photoinhibition phenomenon is observed when leaves are exposed to the same PFD at which the plants are grown but at low CO2 and Oz pressures which prevent or severely limit photosynthetic CO2 metabolism (Cornic 1976; Powles etal. 1979; Powles and Critchley 1980). Exposure of chillingsensitive plants to a combination of low temperature and high PFDs causes a similar type of inhibition (Powles et al. /982). In all of the above cases photoinhibition is manifest as a reduction in the rate of light-samrated photosynthetic COz uptake as well as a reduction in the photon (quantum) yield for CO2 uptake. These changes precede any detectable loss of bulk chlorophyll or visible lesions to the leaf. Photoinhibition is also evident as a reduction in the rate of photosynthetic electron transport of isolated chloroplasts (Powles and Critchley 1980; Critchley 1981 a, b; Powles and Thorne 198/).

0032-0935/82/0156/0097/$02.20

98

S.B. Powles and O. Bj6rkman: Photoinhibition and low-temperature fluorescence in Nerium

Photoinhibition is also accompanied by effects on chlorophyll fluorescence. Measurements of fluorescence emission at room temperature from leaves previously exposed to excessive PFD show a reduced fluorescence yield (Critchley and Smillie 1981). Exposure of isolated chloroplast membranes to a high PFD caused similar reduction of fluorescence yield (Malkin and Jones 1968). Recent preliminary studies of fluorescence at 77K with chilling-sensitive plants exposed to high light levels at low temperature (Fork et al. 1981) and with plants subjected to water stress at high light levels (Bj6rkman et al. 1981), indicate that the resulting reduction in photosynthetic activity is accompanied by a reduction in the variable fluorescence component. Fluorescence measurements at 77K offer an important advantage in studies of photoinhibitory effects on chloroplast components involved in primary photochemistry. All photosynthetic reactions other than primary photochemistry are inhibited; therefore the fluorescence kinetics are much simpler than at room temperature, greatly facilitating interpretation. The objectives of the present study are 1) to gain further insight into the nature of the damage to the photosynthetic system that takes place when plants are exposed to excessive light levels by examining the effect on the fluorescence characteristics at 77K, and 2) to assess the usefulness of this technique as a tool for studies of the occurrence ofphotoinhibition in nature and for studies of possible interactions between light level and major stress factors such as drought. Materials and methods Plant materials. Nerium oleander L. plants were vegetatively propagated from stock material of a single clone maintained at Stanford. Vigna radiata (L.) R. Wilcz. cv. Berken Mung plants were grown from seed (Burpee Co., Riverside, Cal., USA). Both species were grown in a growth room at a temperature regime of 25 ~ C day-20 ~ C night. Illumination was from a bank of fluorescent tubes (VHO, cool-white, GTE; Sylvania, Winchester, Ky., USA) with a 14-h photoperiod. The incident photon flux area density (400-700 nm) at the height of the fully expanded leaves used in experiments was approx. 300 gmol m - z s- 1. Plants were grown in a mixture of perlite and vermiculite and fertilized four times daily with a nutrient solution containing 4 mM Ca z+, 7 mM K § 1 m M Mg z+, 2 m M N H +, 2 mM H z P O 4 , 1 4 m M NO~, i m M SO4z-, 0.1 mM Fe-ethylenediaminetetraacetic acid (EDTA), 0.5 gM Cu 2+, 2 gM Mn z+, 2 gM Zn z+, 25 gM BO~-, 50 ~tM C1- and 0.5 gM MoO4z- . The N. oleander plants used in the experiments shown in Fig. 1 and Fig. 2 were grown under 6% transmittance, black polypropylene shade cloth (Chicopee Lumite, Hummert Seed Co., St. Louis, Mo., USA) in a controlled-environment glasshouse during mid-summer. The mid-day PFD under the cloth was 140 gmol m -2 s -1. Other growth conditions were

similar to those described above. Oxalis oregana Nutt. was grown in a shade garden from vegetative propagules collected in the native redwood forest habitat in San Mateo Memorial Park, San Marco County, Cal., USA. Photoinhibition treatments. Photoinhibition was induced in intact attached leaves by exposing them to a P F D greater than that at which the plants were grown. The light source used for the treatments was a multivapor lamp (GTE Sylvania Metalarc, 1500 W), equipped with a diffusor and a water filter. In some experiments natural sunlight was used. A single intact attached leaf was sealed into the gas-exchange cuvette and exposed to a high PFD while other parameters that affect photosynthetic rate were held constant. Unless otherwise stated all experiments were conducted at a leaf temperature of 25 ~ C in a gas stream of normal air (35 Pa CO2, 21 kPa Oz, and a leaf-to-air water vapor pressure difference of 1-1.5 kPa). The leaf was held perpendicular to the light beam so that the upper leaf surface received direct radiation. The PFD reaching the lower surface from below was about 10% of that incident on the upper surface. Photoinhibitory treatments of chloroplast-membrane preparations from N. oleander leaves were made in the oxygenelectrode compartment described below. Chloroplast membranes (50 gg chlorophyll ml-1) were suspended in a medium containing 0.4 M sorbitol, 0.1 M Tricine chloride (pH 7.8) and 5 mM MgCI 2 . The suspension was then illuminated under constant stirring with the same light source that was used in measurements of electron transport (see below). Preliminary experiments showed that treatment of chloroplast-membrane suspensions for 15 min in the dark at 25~ and 20 kPa Oz resulted in substantial loss of chlorophyll and of electron-transport activity; these losses were greatly accelerated by light. Addition of catalase provided little or no protection. In contrast, there was no detectable loss of chlorophyll or electron-transport activity, and no changes in fluorescence characteristics when treatments were made at 5~ C in the dark, irrespective of whether the 02 pressure was 20 kPa or < 30 Pa. All subsequent photinhibition treatments with chloroplast membranes were therefore carried out at 5~ C. Gas-exchange measurements. Determinations of CO 2 and water vapor exchange were made with intact attached leaves using an open gas-analysis system as described by Ehleringer and Bj6rkman (1977). Calculations of intercellular CO2 pressure were as described by Bj6rkman et al. (1972). Chloroplast-membrane isolation. Chloroplast membranes were isolated from N. oleander leaves subjected to high PFD treatments and from untreated leaves (controls) from the same whorl. Midribs were removed and 2-4 g of leaf material cut into slices (approx. 1 mm x 5 ram) which were ground using a large pre-chilled mortar and pestle in 15 ml grinding medium, containing 0 . 4 M sorbitol, 0.1 M N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine (Tricine)-HCl (pH 7.8), 0.05 M sodium ascorbate, 5 mM MgC1 z and 2.5% (w/v) bovine serum albumin. The mixture was filtered through two layers of Miracloth (Calbiochem-Behringer Corp., La Jolla, Cal., USA) and the filtrate centrifuged for 5 min at 500 g. The pellet (unbroken ceils and starch) was discarded and the supernatant then centrifuged for 10 min at 2,200 g and the pellet (mostly broken chloroplasts) suspended in 1 ml of a solution identical to the grinding medium less sodium ascorbate and bovine serum albumin. Electron-transport determinations. Rates of photosynthetic electron transport were measured polarographically at 25 ~ C with a temperature-controlled, water-jacketed, Clark-type oxygenelectrode assembly (Rank Bros., Cambridge, U.K.), connected

S.B. Powles and O. Bj6rkman: Photoinhibition and low-temperature fluorescence in Nerium to a potentiometric recorder (Recordall 5000; Fischer Scientific CO., Pittsburgh, Pa., USA). A light beam from a tungstenhalogen lamp (GTE Sylvania Type DLS, 21.5 V, 150 W) was focussed with a lens system through an infrared reflecting filter (wide-band hot mirror; OCLI, Santa Rosa, Cal., USA) on the reaction mixture, contained in the electrode compartment. Whole-chain electron transport was determined by following the rate of oxygen uptake in a 4-ml reaction mixture containing 4 0 m M Sorensen's phosphate buffer (pH 7.8), 15 m M KC1, 5 mM MgC12, 1 m M methyl viologen, 1 m M NaN 3 and chloroplast-membrane preparation (containing 30-50 gg chlorophyll). Where desired, NHr (1 raM) was added as an uncoupler of photophosphorylation. Controls were run in the presence of 3,4-dichlorophenol 1,1-dimethylurea (DCMU, 4 ~tM). Photosystem I-driven electron transport was measured using the same system except that D C M U (4 gM) was added to block PSII activity and 2,6-dichlorophenol indophenol (100 gM) and sodium ascorbate (1 mM) were added as an electron donor. The total rates of PSI-driven electron transport (measured as oxygen uptake) have been divided by two since half of the electrons used in reducing the consumed oxygen are contributed directly by the hydrogen donor (Izawa et al. 1967). Fluorescence measurements. The apparatus for fluorescence measurements at liquid nitrogen temperature (77K) consists of a tungsten-halogen miniature lamp (Type FCR, 12 V, 100 W; GTE Sylvania), powered by a regulated DC power supply (Vista Model XRD, Clifford Industries, Camarillo, Cal., USA) which passes light to a bifurcated optical fiber bundle (No. 2030; Coming Glass Works, Science Products, Corning, N.Y., USA) via a lens system, a filter and a photographic shutter. The fiber bundle guides the light via a quartz rod (10 cm long, 10 mm diameter) to the sample. The fluorescence emitted from the sample is collected by the same quartz rod and then passes via a second fiber bundle and a filter to the photomultiplier (Type R928; Hamamatsu Corp., Middlesex, N.J., USA), driven by a high voltage power supply (Model TC 941; Tennelec, Oak Ridge, Tenn., USA), at 800 V. The current from the photomultiplier is passed through a 20 k ~ resistor and the voltage across this resistor is read, without further amplification, with a digital voltmeter (Digitec Model 277; United Systems Corp., Dayton, O., USA) and a potentiometric recorder (Recordall 5000; Fisher Scientific Co., Pittsburgh, Pa., USA). All components, except the light source, digital voltmeter and the recorder, are housed within a lighttight enclosure with the capability of permitting liquid nitrogen to be admitted while the sample is kept in total darkness. The entire apparatus is portable and has been found well suited for field use. Fluorescence measurements were made with leaf discs or with chloroplast-membrane preparations. Leaf discs (13 mm diameter) were punched from intact leaves immediately prior to measurement. In determinations with chloroplasts, these were isolated and treated as described above. A chloroplast-membrane suspension (0.5 ml; 50 gg chlorophyll ml-*) was applied to a 0.8 gm, 13-mm-diameter filter (Type A A W P 013 00; Millipore Corp., Bedford, Mass., USA) under reduced pressure, resulting in a deposit of a uniform thin layer of chloroplast membranes on the filter. The leaf disc, or the filter, was placed on a small, spring-loaded stainless-steel platform inside a 450-ml Dewar vessel and the sample lightly appressed against the lower, terminal end of the quartz rod. The sample then received a 5-min period at room temperature in darkness to ensure that photosynthetic reaction centers were in the oxidized state. Trial experiments confirmed that the fluorescence signal was constant when the dark period varied from 2-12 rain. Liquid nitrogen was then added to the Dewar vessel, in the dark,

99

and 2 min allowed for equilibration of the sample to 77K. At this time the shutter was opened, admitting excitation light to the sample. Fluorescence kinetics were routinely monitored with the recorder and digital voltmeter. Experiments in which the fluorescence signal was simultaneously displayed on an oscilloscope showed that the potentiometric recorder provided a time resolution of the fluorescence kinetics adequate for the present purpose. The excitation beam was passed through a 470-nm, 10-nm half-bandwidth, interference filter (Ditric Optical Co., Hudson, Mass., USA), providing a PFD of 0.4 gmol m -2 s - t at the surface of the sample. The photosystem II (PSII) fluorescence emission from the sample was measured through a 692-nm, 12-nm half-bandwidth, interference filter (Ditric). The photosystem I (PSI) fluorescence was measured through a 734-nm, 13-nm half-bandwidth, interference filter (Ditric). The fluorescence kinetics were routinely recorded according to the protocol shown in Fig. 3. To ascertain that maximum fluorescence was reached, a neutral-density filter in the excitation beam was removed temporarily to increase the PFD to 4.5 gmol m -z s -1. When no further increase in fluorescence yield was obtained the filter was reinserted into the excitation beam, and the fluorescence immediately following was taken as the final value of maximum fluorescence.

Results

Effect of exposing intact leaves to a high PFD on the light-saturated rate and on the photon (quantum) yield of photosynthetic C02 uptake. When intact attached leaves of N. oleander, developed under a PFD of 140 gmol m - 2 S-1, were placed under a PFD of 2,000 gmol m -2 s -1 (25 ~ C, normal air), the rate of light-saturated CO 2 uptake gradually declined throughout the 4-h exposure (Fig. 1). This decline in light-saturated photosynthesis was not caused by a decrease in stomatal conductance. Rather, the decline occurred in spite of a rise in intercellular CO2 pressure (Fig. 1).

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Table 1. Effect on chloroplast electron transport rates at 25 ~ C of a 4-h exposure of an intact attached N e r i u m oleander leaf to a p h o t o n flux area density (PFD) of 2,000 pmol m - 2 s - 1 (at 2 5 ~ in air). Rates are expressed as ~mol 02 consumed (g- 1 Chl s - 1)

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Whole chain (H20 to methylviologen) Coupled rate 46 19 Uncoupled rate 59 34

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Hence, the reduction in photosynthetic rate is not attributable to factors which determine the diffusive transport of CO2 to the sites of COz fixation. Exposure of the same N. oleander leaves to high PFD resulted in a pronounced reduction of the light-limited CO2 uptake. As shown in Fig. 2, the photon yield before the exposure to a PFD of 2,000 gmol m -2 s -1 was 0.057 CO 2 per photon and it fell to 0.026 CO2 per photon after 4 h exposure. As with light-saturated photosynthesis, the reduction in photon yield occurred in spite of the fact that the intercellular CO2 pressure was higher after than it was before the exposure to a high PFD.

Effect of exposing leaves to high PFD on the activity of photosynthetic electron transport by isolated chloroplast membranes. Table 1 summarizes the results of experiments in which the rates of light-saturated photosynthetic electron transport were determined with chloroplast membrane preparations, isolated from N. oleander leaves before and after exposure of the leaves to a high PFD. A 4-h exposure to a PFD of 2,000 gmol m - 2 s- 1 caused substantial inhibition of both coupled and uncoupled wholechain electron-transport activity while PSI-driven electron-transport activity was little affected. Thus, the inhibition is mainly attributable to an inactivation of reactions associated with PSII. Experiments in which isolated chloroplast membranes were exposed to high PFD gave similar results (see below). Rates of light-limited electron transport were not measured; however, previous studies indicate that photoinhibition affects light-saturated and lightlimited electron transport activity to a similar extent both when isolated chloroplasts (Jones and

Chloroplast membranes isolated from the two untreated leaves on the same whorl served as the control. Chloroplast membranes from b o t h the control and treated leaves were isolated 1 h after end of the treatment and electron transport rates assayed immediately. Each value shown in the mean of three determinations b Ascorbate-dichlorophenol indophenol c The total rate of O2 uptake has been divided by two (see Methods)

Kok 1966; Bj6rkman et al. 1972) and when intact leaves (Powles and Critchley 1980) are exposed to excess light.

Effect of exposing leaves to a high PFD on fluorescence emission at 77K. All experiments presented below on the effect of exposing intact leaves to high PFDs were made on leaves of plants grown at a PFD of 300 lamol m-2 s-1 (incident on the upper leaf surface). Figure 3 shows the PSII fluorescence-induction kinetics (measured at 692 nm) for the upper leaf surfaces before and after a 3-h exposure to a PFD of 1,650 ~tmol m -z s -1. These kinetics shown that upon excitation, fluorescence instantaneously reaches the F o level, followed by a gradual increase to a maximum level (FM). This rise from Fo to FM at 77K reflects the gradual accumulation of Q-, the reduced form of the primary electron acceptor of PSII (Butler and Kitajima 1975). (At 77K reoxidation of Q cannot take place and reduced Q therefore accumulates.) A 3-h exposure to a high PFD resulted in a substantial reduction in FM,692 and a slight increase in Fo,692, measured on the upper leaf surface. The fluorescence-induction kinetics at 77K, illustrated in Fig. 3, showed little variation among different samples from the same leaf, or from different leaves, subjected to identical treatments. Table 2 summarizes the results of experiments i n which intact attached N. oleander leaves were treated for 3 h at a PFD of 1,750 gmol m-2 s-1. The FM,692 was inhibited by about 50% after a 3-h exposure. The variable fluorescence (Fv,692 = FM,692--Fo,692 ) was inhibited by about 60%.

S.B. Powles and O. Bj6rkman: Photoinhibition and low-temperature fluorescence in Nerium 1 O0

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Table 3. Effect on fluorescence emission characteristics at 77K (lower leaf surface) of exposing the upper surface of Nerium oleander leaves a for 3 h to a PFD of 1,650 pmol m - 2 s - 1

Characteristic

Before exposure (mean + SD) b

After exposure (mean 4- SD) b

Change

Characteristic

Before exposure (mean_ SD) b

After exposure (mean +_SD) b

Change (%)

F o,692 FM,692 Fv,692 Fo,734 FM,734 Fv,734 Fv,734~ Fv,692 j

9.7 _+0.2 54.3_+0.1 44.7___0.2 185.1_+6,2 260.9_+10.5 76.6-+4.7

11.1 4- 0.3 29.1 _+1.5 18.0--+1.3 163.2_+6.5 201.9+7.3 38.7_+0.9

+ 14.4 --46.4 --59.7 -11.8 --22.6 --49.5

11.8+_0.3 78.9_+1.7 67.14-1.6 296.6 416.3_+9.8 119.7

12.1-1-0.2 73.5+_4.6 61.4_+4.6 267.8 386.0+_14.4 118.2

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Table

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" Leaf temperature during exposure was 25 ~ C b Each value is the mean of duplicate determinations for three different leaves (n = 6)

As shown in Table 2, exposure to a high PFD also affected the fluorescence characteristics at 734 nm. The Fo,734 was slightly reduced while FM,734 and Fv,734 were inhibited by about 23% and 50%, respectively. Since Fv,734 was less affected than Fv,692, the ratio Fv,734/Fv,692 increased by about 36%. The effect on fluorescence characteristics of the lower leaf surface was much smaller (Table 3). The reduction in Fv,692 and Fv,734 was less than 10%. These results are expected since during the treatment the light beam was directed toward the upper surface on the leaf and the light reaching the lower surface was restricted to reflected light from below (10% of the PFD incident on the upper surface) plus the light transmitted by the leaf ( < 2 % at 470nm and 680nm and < 9 % at 550 nm). It should be noted that the lower leaf

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" Leaf temperature during exposure was 25 ~ C b Each value, except F o,734, is the mean of duplicate determinations for three difference leaves (n=6). The value for F o,734 is the mean of duplicate determinations for a single leaf only

surface is at least as susceptible to direct exposure to high PFD as the upper surface (data not shown). Results similar to those reported for N. oleander were also obtained when intact leaves of Vigna radiata and Oxalis oregana were exposed to a high PFD although the inhibition in the latter plant (an extreme shade species) was more severe than in N. oleander (data not shown). Time course of and effect of PFD level on fluorescence characteristics of intact N. oleander leaves. Figure 4 shows that inhibition of FM,692 and Fv,692 could be detected within 10 min of exposure to a PFD of 1,650 gmol m -2 s-1 and that this inhibition increased with increased exposure time. No significant inhibition was observed at a

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P F D level of 300 gmol m - 2 s - 1 (the P F D level under which the leaves had developed) over the 250-rain exposure but substantial inhibition occurred at a P F D of 825 pmol m - 2 S-t and the inhibition became more severe at 1,650 gmol m-2 s-t. Exposure to natural sunlight at 1,650 gmol m -2 s -1 (400-710 nm) gave results (not shown) quantitatively very similar to those obtained with the Metalarc lamp in spite of the different spectral photon distribution. These high P F D treatments did not cause any detectable loss of bulk chlorophyll or visible symptoms of damage to the leaves even in those cases where Fv,692 was inhibited by as much as 90%.

Effect of exposure temperature on the fluorescence characteristics of intact leaves. Figure 5 shows the results of experiments in which intact, attached N. oleander leaves were exposed to a P F D of 1,000 gmol m - 2 s - 1 (normal air) for 100 min over a range of temperatures. Inhibition o f F M,692 occurred at all treatment temperatures. However, the inhibition was most severe at the lowest treatment temperature (5.6 ~ C) and the degree of inhibition decreased with increased treatment temperature (Fig. 5 A). The Fo,692 increased slightly following high P F D treatment at lower temperatures. The inhibition of FM,734 was unaffected by temperature and the ratio of FM,734/FM,692 therefore increased with decreased treatment temperature (Fig. 5 B). Other experiments show that treatment at temperatures above 40 ~ C causes severe inhibition of Fv,692. However, contrary to the effects below

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40 ~ C, this inhibition was largely independent of whether the P F D during the treatment was high or low, and the inhibition was characterized by a very marked rise in Fo,692. There is little doubt that this inhibition is caused by heat damage to the photosynthetic system. Investigation of such heat damage is, however, beyond the scope of the present study (for a review of heat damage, see Berry and Bj6rkman 1980).

Effect on fluorescence characteristics of the 02 and C02 pressure during exposure of intact leaves to a high PFD. As shown in Fig. 6A, the inhibition of F v,692, induced by exposing N. oleander leaves to a high P F D at normal air pressures of oxygen (21 kPa), was the same irrespective of whether the treatment was made at a normal (35 Pa) or at an elevated (95 Pa) CO2 pressure. Similar results were also obtained at a low 02 pressure of only 1 kPa (Fig. 6 B). These results show that neither CO2 enrichment nor low 02 pressure significantly influences the inhibition of Fv,692 caused by exposure of leaves to a high PFD.

S.B. Powles and O. Bj6rkman: Photoinhibition and low-temperature fluorescence in Nerium

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I 150

Table 4. Effect on electron transport rates at 25 ~ C of a 15-rain exposure of chloroplast membranes from Nerium oleander leaves at 5~ C to a P F D of 2,000 gmol m -2 s -1, or in darkness. Rates are expressed as gmol 02 consumed (g-1 Chl s-1) Characteristic

Time, minutes Fig. 6A, B. Time course of Fo,692 and F M,692 (upper leaf surface) of N. oleander leaves exposed to a P F D of 1,650 gmol m 2 s-X at 25 ~ C and a COz partial pressure of either 35 Pa (solid symbols) or 95 Pa (open symbols). The oxygen partial pressure in A and B was 21 and 1 kPa, respectively

(A) Treatment at 20 kPa 0 2 Whole chain 135+_23 PSI a 147_+13 (B)

Treatment at