Plant Physiol. (1988) 86, 117-1230032-0889/88/86/0117/07/$01.00/0

Internal CO2 Supply during Photosynthesis of Sun and ShadeGrown CAM Plants in Relation to Photoinhibition

Received for publication June 23, 1987 and in revised form September 7, 1987

WILLIAM W. ADAMS III*' AND C. BARRY OSMONDPlant Environmental Biology Group, Research School ofBiological Sciences, Australian NationalUniversity, P.O. Box 475, Canberra ACT 2601, Australia

ABSTRACr

Leaves of Kalancho#pinnata were exposed in the dark to air (allowingthe fixation of C02 into malic acid) or 2% 02,0% CO2 (preventing malicacid accumulation). They were then exposed to bright light in the presenceor absence of external CO2 and light dependent inhibition of photosyn-thetic properties assessed by changes in 77 K fluorescence from photo-system II (PSII), light response curves and quantum yields of 02 ex-change, rates of electron transport from H20 through QB (secondaryelectron acceptor from the PSI1 reaction center) in isolated thylakoids,and numbers of functional PSII centers in intact leaf discs. Sun leaves ofK. pinnata experienced greater photoinhibition when exposed to highlight in the absence ofCO2 if malic acid accumulation had been preventedduring the previous dark period. Shade leaves experienced a high degreeof photoinhibition when exposed to high light regardless ofwhether malicacid had been allowed to accumulate in the previous dark period or not.Quantum yields were depressed to a greater degree than was 77 Kfluorescence from PSII following photoinhibition.

Leaves of C3 plants grown in bright light are subject to pho-toinhibition following illumination at the light intensities expe-rienced during growth in the absence of internal or externalsources of CO2 (23, 24). Photoinhibition has been indicated byimpaired quantum yield, impaired light- and C02-saturated pho-tosynthesis, both measured in vivo, and by impaired PSII electrontransport activity in isolated thylakoids. Much the same photo-inhibitory responses are shown by shade plants after transfer tobright light in air, and the effects ofCO2 deprivation are additivein this case (25). It has been shown that photorespiration providesa significant internal source of CO2 which can mitigate photo-inhibition in sun grown C3 plants (10, 26, 27). That is, photores-piration in air makes it unlikely that CO2 deprivation due tostomatal closure could lead to photoinhibition (14, 22).Although stomatal closure is unlikely to lead to CO2 depriva-

tion in C3 plants, it is well established that stomata of CAMplants close so tightly as to preclude CO2 exchange in the lightduring deacidification (16, 19). During this period, high internalCO2 concentrations are generated by the decarboxylation ofmalic acid, and this internal CO2 source is utilized in photosyn-thesis (9). ManyCAM plants, especially stem succulents, respondto soil water deficits by maintaining tightly closed stomatathroughout the light period (29) and nocturnal recycling ofrespiratory CO2 through malic acid for subsequent photosyn-thesis. These studies led to the speculation (20, 22) that the

l Cufrent address: Lehrstuhl fiir Botanik II, Mittlerer Dallenbergweg64, 8700 Wurzburg, West Germany.

internal supply of CO2 derived from malic acid in CAM plantsserved to mitigate photoinhibition and maintain the integrity ofthe photosynthetic apparatus in these plants, in the absence ofCO2 exchange in the light. Although CAM plants grown in theshade are more susceptible to photoinhibition than those grownin bright light (3), the significance of the internal CO2 source inalleviating high light stress in these plants has not been examined.Preliminary experiments, based on manipulation of dark CO2fixation and malic acid pool size, were reported previously (21).This paper describes subsequent experiments in detail, and dem-onstrates the importance ofthe internal CO2 source in preventingphotoinhibition of sun-grown CAM plants.

MATERIAIS AND METHODSKalanchoe pinnata was grown from plantlets under either full

glasshouse light (sun-grown plants; peak of approximately 1500rmol quanta m 2 s-' in the winter and 2000 umol quanta m-2

s-5 in the summer, on a horizontal plane on a clear day) or undershade cloth (shade-grown plants; irradiance ranged from 15 to30% of full glasshouse irradiance depending on solar angle andcloud cover). All measurements of light were made with aquantum sensor (Li-Cor 190SB). Glasshouse air temperaturesranged from 14C at night to as high as 36C on some summerdays. Plants received water daily and nutrients (one-half strengthHewitt's) (30) three times a week. All plants were at least 3months old and only the terminal leaflet of tripinnate leaves ofthe third to fifth rank from the apex, in an east-west plane, wereused for the experiments described below.

All experiments were carried out with the gas exchange systemdescribed in von Caemmerer and Edmondson (7), with thefollowing changes. The leaflet was inserted into a chamber witha volume of 2.5 L and the flow of gases through the chamberwas maintained at 2 L min-', or at 5 L min-' when CO2 exchangewas measured. Light was provided by a metal halide lamp, anddifferent irradiance levels were obtained by changing the heightof the lamp and/or inserting neutral density glass filters. Leaftemperature (maintained at 15C during the dark and 27°Cduring the light) was monitored with a thermocouple insertedinto and parallel with the mesophyll. The vapor pressure deficitwas maintained at approximately 8 mbar during the dark and12 mbar during the light. The CO2 partial pressure of the airstream prior to entering the chamber was measured with aninfiared gas analyzer (model ZAR, Fuji Electric). Calculationsof the rates of CO2 exchange followed von Caemmerer andFarquhar (8).

Leaves were placed in the chamber in early afternoon andilluminated (sun leaves at 800 Mmol quanta m-2 s7' and shadeleaves at 325 ,umol quanta m-2 s-') for 5 to 6 h in air to ensuredecarboxylation of any remaining malic acid. This was followedby a 13 h night in either air (allowing fixation of C02 into malicacid, thereby providing an internal CO2 source for photosynthesis

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118 ADAMS AND OSMOND

Table I. Nocturnal Acid Accumulation and Fv/Fm (FluorescencefromPSII at 77 K) in K pinnata Leaves in Response to Different

Atmospheres in the DarkMean ± SE, (n).

Growth Dark AM Titratable PConditions Atmosphere Acidity

AEq g' fresh Fv/Fmwt

Sun Air 317 ± 8 (11) 0.804 ± 0.006 (12)2% O2 48 ± 3 (13) 0.781 ± 0.012 (16)

Shade Air 245 ± 12 (4) 0.829 ± 0.008 (8)2% 02 37 ± 0 (3) 0.786 ± 0.004 (4)

I0)

IE

cp

0E

0

S

IE

4

2

Time of day, hourFIG. 1. Diel patterns of CO2 exchange in air for 2 sun leaves ofK

pinnata (August), one exposed to 800 Amol quanta m-2 s-' for the first11 -h day followed by a day at 1200 Mmol quanta m-2 s-' (upper panel),the otierexposed to 800 Mmol quanta m-2 s' followed by a day at 1750Mmol quanta mi2 s-1. Net CO2 uptake for each leafwas identical for bothexposures.

during the subsequent light period) or in 2% 02, balance N2(preventing the fixation of CO2 from external sources and sup-pressing fixation of respiratory CC2). Following the dark treat-ment with either air or 2% 02, the leaves were exposed to lightin either air or 2% 02 and various parameters measured during

50

40

I0)CM

E

S

c

cJ0)x

_cw0

z

30

Plant Physiol. Vol. 86, 1988

0 200 400 600 800 1000 1200

Absorbed quanta, lmol mn"2 s 1

FIG. 2. Selected light response curves of photosynthetic 02 exchangeat 25C and 5% CO2 from sun-grown leaves ofK pinnata (August). Eachcurve was obtained at the end of a 4-h exposure to light in 2% 02, 0%CO2 at a leaf temperature of 27C. Closed circles = night of air (AMtitratable acidity = 307 ;Eq g7' fresh weight), exposure at 800 gmolquanta m-2 s-'; open circles = night of 2% 02, 0% CO2 (AM titatableacidity = 43 jEq g-' fresh weight), exposure at 1750 Mmol quanta m-2s

or at the end of this treatment. Levels of titratable acidity weredetermined on extracts of leaf punches which were weighed, cutinto small pieces, and boiled for 20 to 30 min in water. Theextracts were then titrated to an endpoint ofpH 7.0 with 0.01 NNaOH.

Fluorescence from PSII (690 nm) from leaf tissues frozen to77 K for 4 min following a 7-min period of darkness wasmeasured with the system described in Adams (1). Light responsecurves and quantum yields (4) of 02 exchange were determinedfrom leaf discs at 25C and 5% CO2 as described previously (2,11).Uncoupled, light-saturated rates of electron transport through

PSII, from H20 through QB2 to DMQ, were determined polaro-graphically from suspensions of isolated thylakoids maintainedat 25C. Leaf tissues were sliced into small pieces and thenground for 6 s in a chilled medium (approximately 12 ml per 10cm2 oftissue) containing 350 mM sorbitol, 150 mm N-2-hydrox-yethylpiperazine-N'-3-propanesulfonic acid (EPPS), 20 mMNaCl, 5 mm MgCl2, and 0.5 mM NaEDTA, at a pH of 8.0(KOH), to which 1% PVP and 1% BSA were added just prior touse. This suspension was then filtered through 20 ,um nylon meshcloth and the filtrate spun at 2800g for 1 min. The supernatantwas discarded and the thylakoids resuspended in a small volumeofthe reaction medium (350 mM sorbitol, 50 mM Hepes, 20 mMNaCl, and 5 mM MgCl2, at pH 6.8, the experimentally deter-mined pH optimum). This suspension, which was kept on ice,

2 Abbreviations: Q13, the secondary electron acceptor from the PSIIreaction center-, DMQ, 2,5-dimethyl-p-quinone; Fo, initial, instantaneouslevel of fluorescence; Fm, maximum level of fluorescence; F,, variablefluorescence; PFD, photon flux density.

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119INTERNAL C02 AND PHOTOINHIBITION IN CAM

Table II. Evidencefor Different Degrees ofPhotoinhibition in Sun-Grown Leaves ofK pinnata underVarious Conditions in Which Internal and External Sources ofCO2 Have Been Controlled

Treatment Titratable . Electron Transport PSII Fluorescence

Dark Lighta Acidity H20-)DMQ at 77 Ki.Eq g-' mol 02 mol_' mmol 02 mol1' % change infresh wt quanta Chl s-' Fv/Fm

Air Air 318 0.085 137 -112%02 Air 49 0.090 105 -152%02 21%02 62 0.074 74 -172%02 2%02 49 0.076 50 -2721%02 2% 02 54 0.064 47 -25

'Treatment was 1750 umol quanta m-2 s-' for 4 h.

Ec00)

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o 100

00

0)

E

-80

0;

> 60

c 40

c

t- 20

a.

0 60 120 180 240 0 60 120 180 240

Exposure time, minutes

FIG. 3. Time course of changes in FI/Fm (77 K fluorescence fromPSII, closed circles) and electron transport through PSII from H20 toDMQ (open circles) in sun-grown leaves ofK pinnata (November) duringexposure to 1750 Mmol quanta m 2 s-1 in air left panel and in 2% 02(right panel). The leaf illuminated in air had been exposed to air duringthe previous night allowing accumulation of malic acid, while the leafilluminated in 2% 02, 0% C02 had been exposed to 2% 02, 0% CO2during the previous night preventing accumulation of malic acid.

was then immediately assayed. Each preparation was assayed aminimum of three times and the mean rate determined. Thereaction medium included 10 mM NH4Cl and 1 mm DMQ. TheChl concentration in the reaction suspension was determinedaccording to Arnon (6) following the electron transport assays.The number of functional PSII centers in intact leaf discs was

determined from the oxygen yield induced by single-turnoverflashes (approximately 3 ,us duration at half peak height). Theflashes were supplied by a xenon flash lamp (Stroboslave type1539-A) at a frequency of 4 Hz. The rate of oxygen evolution(minus dark drift), determined with a leaf disc 02 electrode, was

0.a

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0 0I120 240 360 480 6000 1 20 240 360 480 600

Time, minutesFIG. 4. Time course of changes in fluorescence from PSII at 77 K for

a sun-grown leaf of K. pinnata (November) during exposure to 1750zmol quanta m-2 s-' in 2% 02, 0% CO2 followed by recovery at 100Amol quanta m-2 s-' in air. The leaf had been prevented from accumu-lating malic acid during the previous 13-h night by exposure to 2% 02,

0% Co2.

compared with the flash frequency to give the oxygen yield perflash. The number of 02 molecules evolved per flash was multi-plied by 4 to give the number of functional PSII centers. Back-ground far-red light (approximately 17 gmol quanta m 2 s-,700-730 nm) was present during flash illumination in order tokeep PSI turning over and the plastoquinone pool in an oxidizedstate.

RESULTS AND DISCUSSION

Leaves of K. pinnata, grown in sun or shade, exhibited greatlyreduced levels of nocturnal acid accumulation when kept in 2%02 in the dark for 13 h (Table I). This result, similar to thatobtained by Moyse (17) with N2 atmospheres, provided a readymeans of controlling internal CO2 supply in the subsequent lightphase. However, we found that N2 atmospheres led to flaccidleaves, for unknown reasons, and this complication could beavoided in 2% 02. Several other complications also arose fromthe protocol employed in these experiments. One is that Fv/Fm

air 2% 02

360 peq acid g1FW 63 peq acid g FW

0~~~~~~

0~~~~~

0 C | Fe/Fm I"0%

'01.. .1. sol~~~PSI... .~~~~~~~~~"

e.fSun

K. pinnata

1750 100 pmol quanta m2 s

2% 02 air

0*

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ADAMS AND OSMOND

Table III. Photoinhibition in Leaves ofSun-Grown K pinnatafollowing CO2 Deprivation in Bright Light

Leaves were kept in 2% 02 in dark, then 3 h at 1750 1imol quantam-2 s-1 in 2% 02 followed by recovery at 100 ;mol quanta m-2 s-' inair (November).

PSII ElectronExperiment Fluorescence Transport Functional

at 77 K H20-DMQ P511Centersmmol 02 mmol mol-

Fv/Fm mol' Chi Chl

No. 1 control 0.83 132 2.34Photoinhibited 0.58 97 0.52Recovery 3 h 0.74 141 2.67Recovery 7 h 0.76 153 2.34

No. 2 control 0.79 170Photoinhibited 0.53 118Recovery 3 h 0.76 172

was usually slightly reduced in leaves exposed to 2% 02 overnightrelative to those exposed to air (Table I). Another is that 2% 02in the light substantially delayed deacidification in tissues whichhad been kept in air in the dark. For instance, in one typicalexperiment the level of titratable acidity dropped from 360 to 81jsEq acid g7l fresh weight during the 4-h exposure to 1750 ltmolquanta m 2 s-' in air, whereas in 2% 02 it only decreased from350 to 309 j&Eq acid g7l fresh weight. Therefore, controls wereusually plants exposed to air in the dark and light and treatmentswere usually plants kept in 2% 02 in the dark and light.Another aspect ofthe controls used in these experiments which

required evaluation at the outset was the intrinsic sensitivity ofK. pinnata to photoinhibition in bright light. Some CAM plantsshow light dependent reduction in dark CO2 fixation capacitywhen exposed to high PFD for several days (18). Figure 1 showsthat, in the relatively short-term experiments employed here,leaves of sun-grown K pinnata did not show any evidence oflight dependent reduction in maximum rates of CO2 uptake inair in the late afternoon. Moreover, increasing the PFD from800 to 1200 or 1750 Mmol quanta m 2 s-' did not alter theamount ofCO2 fixed in the light or the dark. That is photosyn-thetic CO2 fixation in these plants was light saturated at 800Amol quanta m-2 s'-, but higher PFD did not impair CO2 uptakewhen air levels ofCO2 were available.Comparison of Figures 1 and 2 shows that the C02-saturated

rate of02 exchange is much greater (5-8 times) than the rates ofphotosynthetic CO2 uptake achieved in air in the late afternoon.This feature ofCAM plants, noted earlier by Spalding et al. (28)in comparisons of the rate of deacidification and photosynthetic

CO2 exchange in Sedum praealtum and more recently for ratesof deacidification and photosynthetic 02 exchange versus CO2exchange in Opuntia stricta (1), suggests that the photosyntheticapparatus is optimized for rapid and efficient photosynthesisunder the high CO2 concentrations prevailing during deacidifi-cation, rather than for photosynthesis in air.

Figure 2 shows the light response curves for photosynthetic 02

evolution in K pinnata leaves exposed to 800 ,umol quanta m-2s' for 4 h in 2% 02 after a night in air (titratable acidity followingdarkness = 307 ,uEq g-' fresh weight) and to 1750 ,umol quantam-2 s-' for 4 h in 2% 02 after a night in 2% 02 (titratable acidityfollowing darkness = 43 ,Eq g-I fresh weight). Illumination inthe absence of an internal CO2 supply (malic acid), and in theabsence of external C02, led to a reduction in quantum yield, a

reduction in photosynthesis at high PFD, and a reduction in Fl/Fm of PSII fluorescence. These changes suggest that photoinhi-bition of these sun-grown plants depends on low malic acidcontent and high PFD.These indications were confirmed in other experiments with

leaves of sun-grown plants (Table II). As an additional measure

of the occurrence of photoinhibition, the rate of PSII mediatedelectron transport from H20 to DMQ was measured. Comparedwith leaves which had access to an internal and/or externalsource of C02, leaves exposed to high light in the absence ofeither experienced greater reductions in quantum yield, light-saturated electron transport, and Fv/Fm fluorescence from PSII.Treatment in air following 2% 02 in the dark prevented photo-inhibition, although the most sensitive parameter, electron trans-port activity, decreased to some extent. The ability of photores-piration to prevent photoinhibition through the maintenance ofC3 plants at the CO2 compensation point in 21% 02 at high lightwas previously demonstrated (24, 26). However, treatment of K.pinnata in 21% 02 in the light, which would permit photorespi-ration to proceed, was not very effective, possibly because theflowing gas stream flushed photorespiratory CO2 from the cham-ber. Perhaps for the same reason, 21% 02 in the dark, whichwould permit respiration to proceed, did not result in a significantincrease in malic acid synthesis, although this may also indicatethat 2% 02 did permit respiration to continue. These experimentsestablish that in the absence of an internal CO2 supply (malicacid) and external C02, this CAM plant shows evidence ofphotoinhibition in bright light. The results are comparable tothose obtained with C3 plants (24, 26) and demonstrate that inCAM plants, internal refixation of CO2 from malic acid decar-boxylation, like recycling of photorespiratory CO2 in C3 plants,mitigates photoinhibition.

In most of these experiments with leaves of sun-grown plantsin which photoinhibition was brought about by CO2 deprivationin bright light, changes in PSII electron transport were greater

Table IV. Evidencefor Photoinhibition ofShade-Grown Leaves ofK pinnata in High Light Regardless ofWhether an Internal Source ofCO2 (Malic Acid) Is Present or Not

TitratbleQantum Electron P5I1Treatment Titratable Quantum Trnsport FluorescenceAcidity Yield H20--DMQ at 77 K

AEq9- mol 02 mmol 02fresh wt molt' mol-' Chl % change in

quanta sF/Air (dark/light')

800 umol quanta m 2 s-' 225 0.094 84 -91750 pmol quanta m 2 s-' 237 0.047 44 -38

2% 02 (dark) air (light")800 pmol quanta m-2 sg' 36 0.090 79 -51750 jsmol quanta m'2 s1 38 0.054 41 -27

"Leaves exposed to light for 4 h.

120 Plant Physiol. Vol. 86, 1988

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INTERNAL CO2 AND PHOTOINHIBITION IN CAM

0.83i_ air

0.6

Shade0.4 -K pinnata

0.2 :100 pimol m 2 s

e ^

80 [

60

40

20

i aqn

100 pmol quanta m 2s

0 120 240 360 480 600 720 0 120 240 360 480 600

121

FIG. 5. Time course of changes in flu-orescence from PSII at 77 K for a shade-grown leafofK pinnata (November) dur-ing exposure to 1750 ismol quanta m-2s-1 in air followed by recovery at 100,umol quanta m-2 s' in air. The leaf hadbeen allowed to accumulate malic acidduring the previous night by exposure toair.

Time, minutes

Table V. Photoinhibition ofLeaves ofShade-Grown K pinnataExposed to Bright Light in Air

Leaves were kept in air in the dark, then 4 h at 1750 ,mol quanta m-2s-' in air, followed by recovery at 100 umol quanta m-2 s-' in air(November).

PSH Electron FunctionalExperiment Fluorescence Transport PSII Centers

at 77 K H20-.)DMQmmol 02 1

Fv/Fm mol' Chl mmol mol

No. 1 control 0.84 128 1.84Photoinhibited 0.48 51 0.87Recovery 2 h 0.71 98 2.02Recovery4h 0.73 118 2.45Recovery 6 h 0.72 123 1.81No. 2 control 0.80 143Photoinhibited 0.45 62Recovery 1.5 h 0.58 96Recovery 3 h 0.63 124Recovery 6 h 0.75 134

than changes in fluorescence. Figure 3 compares the time courseof the former two parameters in control and photoinhibitorytreatments, and shows that inhibition of PSII electron transportexceeded the changes in fluorescence in the absence of CO2.Further time courses of changes in 77 K fluorescence and elec-tron transport during photoinhibition in leaves of sun-grownplants deprived ofCO2 are shown in Figure 4 and Table III. Thedecrease in FvIFm during the exposure to high light in thepresence of 2% 02 was due to a massive quenching of Fm and asmaller, but significant, quenching of Fo. This quenching wasrapidly reversible upon transfer of the leaf to low light and air,and Fv/Fm recovered within 4 to 5 h (Fig. 4).These changes in fluorescence are consistent with those be-

lieved to indicate the operation ofa mechanism whereby energyin excess of that which can be utilized for photosynthesis andphotorespiration is diverted from the reaction centers through

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0

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r2= 0.627

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0.00.00 0.025 0.050 0.075 0.100 0.125

1

Quantum yield, mol 02 mol quanta

FIG. 6. Relationship between the quantum yields of 02 exchange andFvI/Fm (fluorescence from PSII at 77 K) in sun-grown (open circles) andshade-grown (closed circles) leaves of K pinnata subjected to variousdegrees of photoinhibition.

nonradiative dissipation, thus protecting them from photoin-hibitory damage (12, 13, 15). According to this model an increasein Fo is indicative ofdamage to PSII. However, these fluorescencechanges were regularly accompanied by a decrease in PSII elec-tron transport activity, and a decrease in functional PSII reactioncenters as measured by flash yield, both ofwhich were reversiblewithin 3 h (Table III). It is possible that an increase in Fo did

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ADAMS AND OSMOND

occur in these experiments but that it was masked by concurrentquenching. Alternatively, it is possible that in spite of the pho-toprotective dissipation of excitation indicated by the fluores-cence changes, lesions occurred elsewhere in the photosyntheticapparatus which were responsible for impaired PSII electrontransport and PSII reaction center function.Some of these experiments were repeated with leaves of shade-

grown K pinnata. Table IV shows that neither external norinternal sources of CO2 were effective in protecting againstphotoinhibition when leaves were exposed to 1750 ,mol quantam-2 s-'. These results are similar to those obtained previouslywith shade-grown K. daigremontiana (3). In the experimentsdescribed in Table IV, unlike those in Table II, there was goodproportional agreement between the changes in quantum yield,electron transport, and fluorescence. However, the time courseof changes in fluorescence exhibit quite a different pattern ofresponse to that shown in Figure 4. In the leaves of shade-grownK. pinnata photoinhibited in air, the decrease in Fv/Fm was dueto an increase in Fo as well as a decrease in Fm (Fig. 5). Recoverywas slower, incomplete, and primarily due to an increase in Fm.Even after 24 h, Fo remained higher than the initial control value.This pattern of photoinhibition was associated with much thesame reversible reductions in PSII electron transport activity andfunctional PSII reaction centers as observed previously (cf TableV and Table III). In these experiments it is presumed that, inspite of the potentially photoprotective processes indicated bythe decrease in Fm (due to an increase in nonradiative dissipationof excess energy), the increase in Fo indicates photoinhibitorydamage, and it was not surprising to find this associated withimpaired PSII electron transport and flash yield.The possibility of photoinhibitory damage at sites in addition

to the PSII reaction center is indicated by plotting the relationshipbetween Fv/Fm and quantum yield measured in leaves of sun-and shade-grown plants which had been subjected to differenttreatments resulting in various degrees of photoinhibition. Theregresssion line (Fig. 6), which was very similar to that observedpreviously in K. daigremontiana photoinhibited in air (3), doesnot pass through the origin. This implies that the greater changein quantum yield than in fluorescence could involve factors otherthan the balance of excitation dissipation in the reaction centers,as interpreted by current models of in vivo fluorescence. Themore perfect correlations obtained by Demmig and Bjorkman(12) with C3 plants were based on quantum yield and fluores-cence measurements after periods of recovery in air at low PFD.Their period of recovery may have allowed repair of additionalsites of damage other than the PSII reaction center, perhapsbetween the water-splitting apparatus and QB (see Fig. 3, andcompare rates of recovery in Tables III and V), or differencesbetween C3 and CAM plants may explain the difference betweenthese correlations. However, the relationships between the twodifferent responses behind the change in Fv/Fm and the similarityof changes in PSII function indicated in vivo and in vitro in thedata sets for sun-grown and shade-grown leaves cannot, at pres-ent, be explained.

In conclusion it seems clear that the internal source of CO2provided by the nocturnal fixation of CO2 into malic acid can

provide photosynthetic tissues of CAM plants adequate protec-tion against photoinhibition during deacidification when stomataare closed. If no internal source of CO2 is available then photo-inhibition ensues, just as it does in CO2 deprived C3 plants.Despite the apparent protection against photoinhibition afforded

by the internal supply of CO2 in these short-term experimentswith sun-grown leaves ofK. pinnata, it has recently become clearthat CAM plants growing in full sunlight under natural condi-tions may experience photoinhibition, often to a severe degree(1, 4, 5). Some degree of CAM-idling (29) probably occurs in

many CAM plants under natural conditions, and it is likely that

the CO2 supplied in this manner is insufficient to prevent pho-toinhibition in conditions of full sunlight. Afterall, deacidifica-tion is usually complete in about 4 h and if stomata remainclosed, photorespiratory CO2 cycling is likely to be the only othersource of CO2 available. This may mitigate photoinhibition inC3 plants for several hours (26) but its long-term efficacy has notbeen demonstrated. It should be noted that the photoinhibitedCAM species 0. basilaris, growing in Death Valley, exhibitedonly 10% of the nocturnal acidification observed in K. pinnata(4). In 0. basilaris the internal CO2 pool is clearly of limitedsignificance, and processes involved in the nonradiative dissipa-tion of excitation may be more important. Our experiments alsoindicate that, in shade-grown CAM plants, the internal supplyof CO2 probably does not provide significant protection againstphotoinhibition, should these plants experience bright light. Likeshade-grown C3 plants, they will be photoinhibited when exposedto high light even in the presence of adequate internal or externalCO2 sources.

Acknowledgments-We would like to thank S. C. Wong, W. Coupland, and P.Groeneveld for construction of the 77 K fluorescence system, W. S. Chow for theflash yield determinations of functional PSII centers, and I. Terashima for helpfuladvice and comments on the manuscript.

LITERATURE CITED

1. ADAMS WW III 1988 Photosynthetic acclimiation and photoinhibition ofterrestrial and epiphyticCAM tissues growing in full sunlight and deep shade.Aust J Plant Physiol. In press

2. ADAMSWW III, K NISHIDA, CB OSMOND 1986 Quantum yields ofCAM plantsmeasured by photosynthetic 02 exchange. Plant Physiol 81: 297-300

3. ADAMS WW III, CB OSMOND, TD SHARKEY 1987 Responses of two CAMspecies to different irradiances during growth and susceptibility to photo-inhibition by high light. Plant Physiol 83: 213-218

4. ADAMS WW III, SD SMITH, CB OSMOND 1987 Photoinhibition of the CAMsucculent Opuntia basilaris growing in Death Valley: evidence from 77Kfluorescence and quantum yield. Oecologia 71: 221-228

5. ADAMS WW III, I TERASHIMA, E BRUGNOLI, B DEMMIG 1988 Comparisons ofphotosynthesis and photoinhibition in the CAM vine Hoya australis andseveral C3 vines growing on the coast of eastern Australia. Plant Cell Environ.In press

6. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenol oxidasesin Beta vulgaris. Plant Physiol 24: 1-15

7. CAEMMERER S VON, DL EDMONDSON 1986 Relationship between steady-stategas exchange, in vivo ribulose bisphosphate carboxylase activity and somecarbon reduction cycle intermediates in Raphanus sativus. Aust J PlantPhysiol 13: 669-688

8. CAEMMERER S VON, GD FARQUHAR 1981 Some relationships between thebiochemistry of photosynthesis and the gas exchange of leaves. Planta 153:376-387

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