Zeaxanthin Heat Dissipation ofExcess Light Energy Nerium Exposed

Plant Physiol. (1988) 87, 17-240032-0889/88/87/0017/08/$0l .00/0

Zeaxanthin and the Heat Dissipation of Excess Light Energy inNerium oleander Exposed to a Combination of High Light andWater Stress'

Received for publication July 31, 1987 and in revised form November 30, 1987

BARBARA DEMMIG*, KLAUS WINTER, ALMUTH KRUGER, AND FRANZ-CHRISTIAN CZYGANLehrstuhl far Botanik II (B.D., K.W.) and Lehrstuhl far Pharmazeutische Biologie (A.K., F.-C.C.),Universitat Wurzburg, Mittlerer Dallenbergweg 64, 8700 Wurzburg, Federal Republic of Germany

ABSTRACT

Upon termination of watering of plants of Nerium oleander exposed tohigh light, photochemical efficiency became reduced as leaf water contentdecreased. Evidence is presented that this type of photoinhibition reflectsto a substantial degree radiationless dissipation of excitation energy, prob-ably mediated by the carotenoid zeaxanthin. During the imposition of waterstress, the zeaxanthin content of leaves increased at the expense of vio-laxanthin and a-carotene as a water deficit developed over a period ofseveral days. The increase in zeaxanthin content was linearly related toan increase in the rate of radiationless energy dissipation in the antennachlorophyll as calculated from the characteristics of chlorophyll a fluo-rescence measured with a pulse amplitude modulated fluorometer at roomtemperature. The increase in the rate of radiationless dissipation was alsolinearly related to a decrease in PSII photochemical efficiency as indicatedby the ratio of variable to maximum fluorescence. Leaves of well-wateredshade plants of N. oleander exposed to strong light showed a similarincrease in zeaxanthin content as sun leaves of the same species subjectedto drought in strong light. Shade leaves possessed the same capacity assun leaves to form zeaxanthin at the expense of both violaxanthin and j-carotene. The resistance of this species to the destructive effects of excesslight appears to be related to interconversions between 13-carotene andthe three carotenoids of the xanthophyll cycle.

Prolonged exposure of leaves to light levels where the exci-tation energy exceeds the capacity for orderly dissipation by thephotosynthetic system can lead to photoinhibition. Photoinhi-bition is characterized by a sustained decrease in the efficiencyof photon utilization by PSII photochemistry and is observed(a) when shade leaves are abruptly exposed to bright light whichgreatly exceeds the PFD2 experienced by the leaves during theirdevelopment and (b) when sun leaves acclimated to natural sun-light are exposed to additional environmental stresses such as

I Supported by the Deutsche Forschungsgemeinschaft and by the Fondsder Chemischen Industrie.

2 Abbreviations: PFD, photon flux density; A, antheraxanthin; Car,carotenoids; ,8-Car, 13-carotene; Cartota,, sum of all carotenoids; °Car,carotenoid in the ground state; 'Chl, chlorophyll in the singlet state;DW, dry weight; FW, fresh weight; FO, instantaneous fluorescence emis-sion; FM, maximum fluorescence emission; F, variable fluorescenceemission; KD, rate constant for radiationless energy dissipation; P, es-timated turgor pressure; ir, osmotic pressure; q,, water potential; qD,quenching coefficient for nonphotochemical fluorescence quenching; V,violaxanthin; Z, zeaxanthin.

water deficit or unfavorably high or low temperatures (for areview see Ref. 22). Under these conditions, a given light levelwhich previously was nonexcessive becomes excessive, becausethe utilization of energy through photosynthesis is decreased bythe additional stress treatment. The mechanism by which thephoton yield of photosynthesis is lowered in these two cases isprobably not the same (3, 8). Shade leaves exposed to brightlight show evidence of damage to PSII reaction centers, whereassun leaves exposed to a combination of high light levels andwater stress (Nerium oleander) or salinity stress (various speciesof mangroves) do not. Rather, in these sun leaves, the decreasein photochemical efficiency of PSII can to a large extent beaccounted for by a strong increase in thermal deactivation ofexcitation energy in the antenna Chl (3, 8). Excitation energy isthereby diverted away from the photosynthetic reaction centersand is no longer available for photochemistry. Although thisincrease in the rate of radiationless dissipation is associated witha reduction in the efficiency of photosynthesis at low light, thisdisadvantage is most likely outweighed by the benefit of pre-venting the accumulation of excess excitation energy at high light,whereby damage to the reaction centers is avoided. Regulationof the photon yield of PSII by varying rates of thermal deacti-vation has also been observed under less extreme conditions (27).

Dissipation of excitation energy via pathways other than PSIIphotochemistry leads to fluorescence quenching. One compo-nent of fluorescence quenching which relaxes within 30 s upondarkening (16, 17) has been termed high energy state quenching(14, 15). A second component of fluorescence quenching showsslower relaxation kinetics (typically between 30 to 60 min) upondarkening (8, 9). Although radiationless dissipation does reducethe photon yield of PSII photochemistry, it is generally not con-sidered to be photoinhibition as long as it is reversible within 30to 60 min. After long-term exposure of N. oleander to a com-bination of high light and water stress, PSII photochemical ef-ficiency took 1 to 2 weeks to recover (3, 5). In this case, theprotective response, increased radiationless dissipation, contrib-utes to a lasting limitation to photosynthesis after the stress isalleviated.We have recently begun to explore the mechanism of fluo-

rescence quenching which reflects increased radiationless dissi-pation in the antenna Chl under photoinhibitory conditions. Whena pronounced, yet relatively rapidly reversible (within 30-60min), increase in the rate of radiationless dissipation was inducedwithout damage to PSII (exposure of intact sun leaves to 2%02, zero CO2 in low light), a strong and reversible increase inthe content of the carotenoid zeaxanthin was observed (10). Thisincrease was linearly related to the increase in radiationless dis-sipation as indicated by the decline in fluorescence. Thus, zeax-anthin may act as a fluorescence quencher competing with both

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Plant Physiol. Vol. 87, 1988

fluorescence emission and photochemistry for excitation energy.Since protection occurs at the expense of lowered photochemicalefficiency, one would expect that the formation of such an 'al-ternative quencher' is tightly controlled. Indeed, zeaxanthin isformed in a light-dependent reaction in the xanthophyll cycle(12, 30) which is present in all photosynthetic systems examinedthus far. This cycle is fine-tuned to sense the point at which lightbecomes excessive (12, 30), and formation of zeaxanthin occurs

only under such conditions (10, 24).The present paper examines the changes in fluorescence char-

acteristics and pigment composition of N. oleander leaves sub-jected to high light and water stress. In plants which were no

longer irrigated, the fluorescence yield, determined by meas-

urements at 77K, was previously found to decrease during thefirst few h of each consecutive light period, to remain at thisdecreased level, and to be incapable of recovery overnight (5).Consequently, a progressive and sustained quenching of fluo-rescence over a series of days was observed as the water deficitdeveloped. We have investigated the quantitative relationshipbetween the lowering of fluorescence (i.e. the increase in radia-tionless dissipation) and the zeaxanthin content of N. oleanderleaves subjected to such a treatment.

MATERIALS AND METHODS

Plant Material. Plants of Nerium oleander L. (Apocynaceae),an extremely drought-tolerant evergreen C-3 shrub, were estab-lished in 5-L plastic pots filled with garden soil, watered daily,and received Johnson's nutrient solution once per week (28).They were kept in a glasshouse at about 30/15°C (day/night) and30/40% RH. Natural daylight was supplemented by Osram PowerStar metal halide lamps (HQI-T 2000W/D), which provided con-

tinuous illumination of 1000 ,umol photons m -2 - for 12 h (from7 AM-7 PM). After about 3 months, when plants had a height of50 to 70 cm, experimental leaves were oriented in a horizontalposition to minimize differences and changes in light intercep-tion. One group of plants (experiment 1) which had been watereddaily was transferred to a growth cabinet (12 h light, 800 ,molphotons m-2 s- , 25°C, 50% RH/12 h dark, 15°C, 80% RH).After 2 more weeks with optimal water supply, irrigation was

stopped (d 0) and measurements begun. Another group of plants(experiment 2), which remained in the glasshouse, was exposedto a series of drying cycles. Each drying cycle lasted for 5 to 7d during which irrigation was completely stopped. At the end ofeach drying cycle, when the experimental leaves showed signsof wilting, the soil was rewatered to full pot capacity. Measure-ments were taken during the seventh drying cycle and beyond.Water Relations. Determinations of leaf water potential and

of the osmotic pressure of leaf sap were made psychrometricallyon leaf discs at 30.0°C with a series of thermocouple psychro-meters (model C-52) equipped with appropriate electronic cir-cuitry. The time required for equilibration between the watervapor pressure of the leaf sample and that of the psychrometerchamber varied from approximately 2 h at 4' = -1 MPa to 5 hat 41 = - 4 to - 5 MPa. Determinations of 7r were made on thesame discs used for the water potential measurements followingbreakage of the cell walls by freezing and thawing. Turgor pres-sure P was estimated from 4' - ir. For dry weight determinationssamples were dried at 80°C to constant weight.

Fluorescence Measurements and Pigment Analysis. From eachleaf, one disc was removed for fluorescence measurements andfour subsamples, which were pooled into two sets, were takenfor pigment analysis (Fig. 1). Chl a fluorescence was measuredat room temperature using a pulse amplitude modulation fluo-rometer (model PAM 101; H. Walz, Effeltrich, F.R.G.) (23) as

described previously (10). All fluorescence measurements were

made on the upper leaf surface and were preceded by a 5-minperiod of complete darkness. Fluorescence was excited with a

aY I

FIG. 1. Schematic diagram of how samples for fluorescence meas-

urements (F) and pigment analysis (samples 1 and 2) were taken from

a given leaf.

measuring beam of weak light from a pulsed light-emitting diodeto obtain FO, which designates the fluorescence level when allreaction centers of PSII are open. Maximum fluorescence yield,FM, was determined by application of a 1 s pulse of saturatinglight to transiently close all reaction centers. The variable fluo-rescence, FV, was obtained from the expression FV = FM - F,The units for fluorescence yield given in this paper are arbitrary.The leaf sample and fiber optic probe of the fluorometer were

maintained at a constant distance. Furthermore, the PFD of theweak and the saturating beam were the same in all experimentsand the gain of the measuring system remained constant through-out the investigation. Therefore, differences in the fluorescencevalues between different samples reflect differences in the truefluorescence yields. The ratio FVIFM corresponds to PSII pho-tochemical efficiency (see also Refs. 8, 10). The quenching coef-ficient qD (=1 - Fv'IFv) was calculated, according to the cal-culation of high energy state quenching, qE (23, 29), from theratio of the variable fluorescence, FV', of treated samples andvariable fluorescence, FV, of a control leaf at the end of the 12-h dark period. The rate constant of radiationless energy dissi-pation, KD, was calculated according to Kitajima and Butler (13)from FM = KFIKF + KD where FM is the fluorescence at closedPSII traps, in a saturating light pulse, at different times duringthe drying cycle, and KF is the rate constant of fluorescence. Forall leaves KF was arbitrarily set to be 1 and constant throughoutthe treatment (3). To obtain fluorescence characteristics typicalof a nonphotoinhibited sun leaf, a N. oleander plant which hadbeen supplied with ample water at all times was kept shaded (at200 instead of 800 ,umol photon m-2 s-1) for two 12-h lightperiods. The zeaxanthin content at the end of the subsequent12-h dark period, when fluorescence measurements were taken,was zero, and FV/FM had increased to 0.847, a value typicallyfound in nonphotoinhibited leaves (4). The corresponding valuesof initial, maximum, and variable fluorescence were FO = 1.96,FM = 12.71, and Fv = 10.76. These fluorescence values were

used as reference values for the calculation of qD and KD (Figs.5 and 6). KD in a nonphotoinhibited control leaf was taken as

12.5 (cf. 3). Pigments were analyzed quantitatively after sepa-ration by TLC (7, 26). Further procedures and calculations wereas described previously (10).

RESULTS

Water Relations of N. okander Leaves during Drought. Upontermination of watering, leaves of those plants which had been

kept well-watered until then (Table I; Fig. 2) lost turgor as soon

as their water content dropped below 250% of DW. There was

no evidence for osmotic adjustment, i.e. ir fell merely as a con-

sequence of the water loss (Table I). Plants which had experi-enced several drying cycles (experiment 2), showed a lower leafwater content in percent of DW upon watering (d 0). They ex-

hibited a positive turgor pressure at a water content (205% of

DW) at which the leaves of plants not pretreated by water stress

showed zero turgor pressure (Fig. 2). This cannot be explained

0*1' ~0*

18 DEMMIG ETAL.

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ZEAXANTHIN, HEAT DISSIPATION, AND WATER STRESS

Table I. Time Course of Changes in Water Relations of Leaves of N. oleander following Termination of WateringWater potential (4,), osmotic pressure (ir), estimated turgor pressure (P), fresh (FW), and dry (DW) weight were determined in leaf samples

harvested at the end of the 12-h dark period.Time

Exp. after FW DW Water,g, pExp. End of Area 1 Area l Content

Watering

d gm-2 % DW MPa1 0 472 134 252 -0.40 -1.35 0.95

1 500 134 273 -0.58 -1.45 0.873 460 130 253 -0.50 -1.42 0.926 478 136 250 -0.43 -1.32 0.8910 482 139 246 -0.73 -1.13 0.4012 413 136 204 -1.75 -1.69 014 356 136 162 -2.32 -1.96 (0)17 346 136 155 -3.63 - 3.08 (0)19a 277 139 99 -4.76 -4.00 (0)19b 279 142 97 -4.60 - 3.73 (0)

2 0 405 133 205 -0.83 -1.42 0.594 418 137 205 -1.22 -1.81 0.596 391 155 152 -2.61 -2.62 0.018 340 152 123 -3.18 -3.12 010 291 152 92 -4.18 NDa ND

a Not determined.

v}0)

L.

in

L.

a

0

-1.0

-2.0

-3.0

-4.0

1.6 X20 ,~0

-1.0 2 10

p ~~~~~0.8 w

-3.0

0

3' -5.0 0

250 200 150 100

Water content, 0/0 of DWFIG. 2. Water potential (4,), osmotic pressure (ir), and estimated tur-

gor pressure (P) as a function of leaf water content. (O, A), Samples ofplants from experiment 1 (plants well irrigated until onset of the stresstreatment); (0, A), samples of plants from experiment 2 which had beensubjected to repetitive drying cycles before measurements were begun.

by osmotic adjustment in the prestressed plants, since values of7r of previously well-watered and of prestressed plants were thesame at a given leaf water content (Fig. 2). The differences inturgor pressure and water potential at a given water contentbetween the two groups of plants may be due to a higher exten-sibility of the cell walls in the prestressed plants.

Effect of Drought on Fluorescence Characteristics. Figure 3shows changes in fluorescence yield at open, FO, and closed, FM,PSII reaction centers, as well as changes in photochemical effi-ciency of PSII as indicated by the ratio of variable to maximumfluorescence, FVIFM (4, 8, 10). Measurements were taken at theend of the respective 12-h dark periods as the water deficit de-veloped. As leaves progressively lost water, FM fell and Fo rose

slightly such that F, (not shown) had dropped to about 25% of

End of dark period

4)_

c

U)L-

LL

10

8

6

4

2

0

0.8

0.6

0.4U-

tLO

0.2

0

250 200 150 100

Water content, °/ of DWFIG. 3. Fluorescence characteristics, FO, FM, and FV/FM, of N. olean-

der as a function of leaf water content. Fluorescence measurements were

made at the end of the 12-h dark period on each consecutive day as thewater deficit developed. (A, E, 0), Data obtained with plants of ex-

periment 2 where data points are the mean of two subsamples takenfrom the same leaf; (A, U, 0), plants of experiment 1 where the datapoints at the high leaf water content represent the mean of five differentleaves with two subsamples each and where the data points at the lowleaf water content represent the mean of two different leaves with twosubsamples each.

S

o 0

it X

0

I

19




its control value when the water content had decreased to 97and 92% of DW. With decreasing water content between 255and 205% of DW, FVIFM declined marginally to fall more stronglybelow 205% of DW. In severely stressed leaves exhibiting a watercontent of 97 and 92% of DW, FV/FM was decreased to about65% of the control value, the latter being typical of healthynonphotoinhibited leaves. Since measurements were taken at theend of the dark period, thereby allowing for recovery of rapidlyreversible effects on photon yield, this decrease in FVIEM rep-resents a sustained reduction in PSII photochemical efficiency.Changes in Pigment Composition in Sun Leaves of N. oleander

Subjected to Drought and in Shade Leaves of N. oleander Sub-jected to a Sudden Strong Increase in PFD. Figure 4 shows theeffect of a decreasing leaf water content on Chl and carotenoidcontents determined at the end of the respective 12-h dark pe-riods. Fluorescence characteristics had also been determined onthe same leaves (Fig. 3). Contents of Chl and total carotenoidsare expressed on a DW rather than on an area basis. The specificleaf DW increased slightly (Table I) during drought probablydue to a small degree of shrinkage of the leaves which led to an

End of dark period

ci0,

E

0,

5

3

2

1

60

0

4-

0L.au

4-'

0

CS

4-

0,

0o

In4-

._

40

20

0

10

5

0

250 200 150 100

Water content, 0/0 of DW

FIG. 4. Chl content, carotenoid content, and carotenoid compositionin leaves of N. oleander as a function of leaf water content. Symbols are

the same as in Figure 3. Samples were harvested at the end of therespective 12-h dark periods.

apparent increase in the pigment content per unit leaf area. Adecrease in leaf water content from 255 to 123% of DW wasaccompanied by no decrease in Chl b and only by a slight decreasein Chl a (Fig. 4A). When the water content decreased further,the Chl a content declined somewhat more strongly and thereappeared to be a small decrease in Chl b. There was a smallapparent increase in total carotenoids when the water contentdropped from 255 to 123% of DW (Fig. 4B). Between 123 and92% the sum of all carotenoids decreased slightly.

Figure 4C shows the three components of the xanthophyllcycle. The proportions of zeaxanthin, antheraxanthin, and vio-laxanthin were largely altered by the water-stress treatment. Leaveswith a high water content contained several times more violax-anthin than zeaxanthin. The zeaxanthin content, however, wasnot zero even in well-watered leaves. During the stress treatmentzeaxanthin and antheraxanthin increased whereas violaxanthindecreased. The decrease in violaxanthin was insufficient to ac-count for the increase in zeaxanthin and antheraxanthin. Thus,the sum of the three components of the xanthophyll cycle (zeax-anthin + antheraxanthin + violaxanthin) increased by a factorof 1.6 when the water content fell from 255 to 92% of DW (Fig.4B). There was a concomitant decrease in 83-carotene such thatthe sum of the three xanthophylls plus /3-carotene (in percent oftotal carotenoids) remained fairly constant over the whole rangeof leaf water contents. The proportion of lutein did not changeduring the stress treatment.The effect of the drought treatment on the magnitude of diur-

nal changes in pigment composition was determined. Table IIcompares the carotenoid composition at the end of the lightperiod (cf. also Fig. 5) with that at the end of the respectivesubsequent dark period (cf. Fig. 4) in leaves of well-watered andwater-stressed plants from experiments 1 and 2. In the controls,zeaxanthin exhibited only a small further increase during thelight period above the background level present in the dark. Inseverely water-stressed leaves the zeaxanthin content at the endof the light period was much higher than in control leaves anddecreased by 40% during the subsequent dark period, to a levelstill 2 to 3 times higher than the corresponding level in the well-watered controls. There were concomitant opposite light-darkchanges in violaxanthin and (-carotene content indicating thatzeaxanthin in the light is formed partly at the expense of vio-laxanthin and partly at the expense of 3-carotene.These sustained changes in pigment composition of N. olean-

der leaves subjected to progressively developing water deficitwere similar to those observed previously in leaves of other spe-cies subjected to a sudden strong increase in PFD (10). FigureS compares the effect of an abrupt increase in PFD on a shadeleaf of N. oleander with the effect of slowly developing waterstress at a constant high PFD on a sun leaf. Data presented inFigure 5 refer to samples taken at the end of the 12-h light period(at the beginning and at the end of the water stress treatment)in the case of sun leaves, and to samples taken at the end of thesecond 10 h light period at a high PFD in the case of shadeleaves. Samples were kept in the dark for 5 min prior to meas-

urements. Chl and total carotenoid content on a DW basis were

similar in leaves of N. oleander grown at the low and the highlight level (Fig. SA), as were the relative proportions of (-car-otene, lutein and the sum of the three components of the xan-

thophyll cycle (Fig. 5B). In sun leaves of N. oleander there was,however, more zeaxanthin and less violaxanthin present at theend of the light period even when plants were supplied withample water (and nutrients) than in shade leaves kept at theirgrowth PFD (Fig. SC). This is consistent with the observationthat these nonstressed leaves of N. oleander grown at a PFD of800 and 1000 umol m-2 s-1 exhibited lower fluorescence yields(FO = 1.88, FM = 7.05) and PSII photochemical efficiency (FvlFM = 0.733) than the shade leaves (FO = 2.01, FM = 13.1, FvlFM = 0.847). During the two different stress treatments similar

Chi a a

Car,ot.1

| - n0_0Chl b

AI a. a

- ZAsV-P-Car

Lutein ,A

p3-Car

Z.A+V

B0

v

z

cA

20 DEMMIG ETAL.




Table II. Carotenoid Composition in Light and Dark in Leaves of Well-Watered and Water-Stressed Plants of N. oleander.Pigments were determined in samples collected at the end of the 12-h light period (L), and at the end of the dark period (D). Leaves in experiment

1 had water contents of 252 (well-watered) and 97% of DW (water-stressed), and in experiment 2 water contents of 205 (well-watered) and 92%of DW (water-stressed).

Well-watered Water-stressedPigment Exp. L D L/D L D L/D

umol g- ' DWTotal Car 1 2.46 2.42 1.02 2.31 2.24 1.03

2 2.28 2.20 1.04 2.04 2.07 0.99

% of total carotenoids/3-Car 1 39.7 36.8 1.08 28.7 32.2 0.89

2 41.7 38.1 1.09 26.4 30.1 0.88Lutein 1 42.0 40.1 1.05 45.9 42.2 1.09

2 42.7 43.3 0.99 42.4 41.0 1.03Z 1 4.3 4.1 1.05 19.4 12.4 1.57

2 6.5 5.9 1.10 20.1 13.2 1.52

A 1 2.9 2.5 1.16 3.3 5.0 0.662 2.6 2.4 1.08 6.7 7.0 0.96

V 1 9.8 13.9 0.71 1.8 6.6 0.272 5.7 8.3 0.69 3.0 5.0 0.60

Neoxanthin 1 1.9 2.6 0.73 1.1 1.7 0.652 1.1 2.0 0.55 2.6 2.2 1.18

Z + A + V 1 17.0 20.5 0.83 24.5 24.0 1.022 14.8 16.6 0.89 29.8 25.2 1.18

Z + A + V + P-Car 1 56.7 57.3 0.99 53.2 56.2 0.952 56.5 54.7 1.03 56.2 55.3 1.02

changes in pigment composition were observed irrespective ofwhether photoinhibition was caused by the sudden strong in-crease in PFD or by slowly developing water stress at a constantPFD. The sum of the three components of the xanthophyll cycleincreased markedly and (-carotene decreased to the same extentsuch that the sum of zeaxanthin + antheraxanthin + violax-anthin + /3-carotene as well as the sum of all carotenoids re-mained unchanged. In both treatments some decrease in Chlcontent, particularly of Chl a, was observed.The effect of growth PFD on the pigment composition was

further analyzed. Data shown in Table III represent samplestaken at the end of the dark period from either shade leaves orleaves developed at 800 (experiment 1) and 1000 (experiment 2),umol photons m-2 S-I of well-watered plants. Chl contents weresimilar on a leaf area basis but, as a consequence of the lowerFW and DW per unit leaf area in the shade leaves, decreasedwith increasing growth PFD when expressed on a FW or DWbasis. The ratio of Chl a/Chl b increased with increasing growthPFD (Table IV) as expected from previous studies (1). The sumof all carotenoids as well as the /3-carotene content per total Chlcontent increased with increasing growth PFD by a factor of 1.5between 200 and 1000 ,umol m-2 s-1. The sum of zeaxanthin,antheraxanthin, and violaxanthin per total Chl also showed anincrease, albeit less pronounced.

Relationship between Zeaxanthin Content and Rate of Radia-tionless Energy Dissipation. Zeaxanthin content and fluorescenceof high light grown N. oleander leaves at different stages duringthe drought cycle were compared. Data represent samples takenat the end of the light (cf. Fig. 5) and at the end of the darkperiod (cf. Figs. 3 and 4). The relationships between zeaxanthincontent and FV/FM as well as between zeaxanthin content andthe rate constant of radiationless dissipation (KD) are shown inFigure 6. Figure 7 depicts the relationships between zeaxanthincontent and variable fluorescence emission, F., and between

zeaxanthin content and a quenching coefficient qD (= 1 - FV'IF,) which relates the decreased fluorescence emission in stressedleaves, F.', to the higher fluorescence of a control leaf, Fv,The increase in zeaxanthin content was linearly related to changes

in KD (Fig. 6) over the entire range of pigment changes inducedby severe drought. The calculation of KD from the simple modelby Kitajima and Butler (13) is based upon the assumption thatradiationless dissipation occurs in the antenna Chl. The linearrelationship between the putative fluorescence quencher zeax-anthin and KD supports this assumption.Upon a decrease of F. to about 3, or upon an increase in

fluorescence quenching, qD, to about 0.7, respectively, changesin these two fluorescence parameters appeared to be linearlyrelated to the increase in zeaxanthin content (Fig. 7). However,strong further increases in zeaxanthin were no longer quantita-tively matched by further decreases in FV nor by increases in qDsince F. asymptotically approaches zero and qD approaches un-ity. Thus, FV and qD overestimate initial changes in zeaxanthinand therefore in the rate of radiationless dissipation, and arerelatively insensitive to further changes. FVIFM, however, is lin-early related to the increase in the rate of KD and zeaxanthincontent in N. oleander (Fig. 6).

DISCUSSIONThe study presented here provides further evidence of the

remarkable drought tolerance of N. oleander (6). The plant doesnot show osmotic adjustment (Fig. 2) but it survives periods ofprolonged severe drought undamaged and its leaves are capableof recovering from water stress exhibiting little loss of Chl orprotein (6). Even so, the plants show a strong and sustainedreduction in PSII photochemical efficiency during drought asindicated by a decrease in the ratio FVIFM (Fig. 3; see also Ref.3). It was suggested previously that the decrease in FV/FM is

21




0

7

0)

E

In

a)

I-

0

0

04-0

Shade leafHigh light stress

6.0 -a Chi-. Chlia

4.0Cartot.l

2.0 -- --

A Chl bnv

60

40

20

0

20

10

0

Z- A. V+P-Car

Lutein

p-Car*'*

Z+ A+V-B

Sun leafWater stress

* .Chl a-*_,\

Cartot0lO--- -_ _ __ o

Chi b

- Z- A. V+p-Car

Lutein

/X-Car-

Z.A.V

0 2 0 10

Time, days

FIG. 5. Changes in pigment composition of a shade leaf of N. oleandersubjected to a sudden increase in PFD (200-2000 ,umol protons m- 2 - 1)and a sun leaf of N. oleander subjected to water deficit at a constanthigh PFD. The shade leaf was kept for 2 d at 2000 ,umol photons m-2s- I for 10 h/d in a leaf chamber at 25°C and a vapor pressure differenceof 10 mbar bar- '. Samples were taken at the end of the respective lightperiod and were darkened for 5 min prior to fluorescence measurementsrespective to freezing of the samples for pigment analysis. The data forthe water stress treatment at high PFD are mean values of data on well-watered and water-stressed leaves from experiment 1 and 2 (Table II).In the shade leaf fluorescence changed from FO = 2.01, FM = 13.1, FvIFM = 0.84 to FO = 2.10, FM = 5.35, FlvFM = 0.608, and in the sun

leaf from Fo = 1.88, FM = 7.05, F/1FM = 0.733 to FO = 1.78, FM =

3.16, FV/FM = 0.437.

Table IV. Effect of Growth Light Regime on Ratios of VariousPigments in N. oleander

Determinations were done at the end of a 12-h dark period.

PFD during GrowthPigment Ratio (,umol m-2 S-1)

200 800 1000

ratioChl a/Chl b 2.56 2.95 3.03Cartotai/Chl a + b 0.29 0.32 0.38f3-Car/Chl a + b 0.10 0.14 0.15Z + A + V/Chl a + b 0.045 0.047 0.055

caused predominantly by increased radiationless dissipation ofexcitation energy (3, 8) and that such dissipation is mediated bythe carotenoid zeaxanthin (10). In this study, the zeaxanthincontent of leaves was indeed found to increase as a water deficitdeveloped (Figs. 4 and 5; Table II). This sustained increase inzeaxanthin content was linearly related to the increase in therate of radiationless dissipation processes over the whole range

of changes (Fig. 6). Therefore, zeaxanthin may mediate the dis-sipation process as proposed earlier (10). In this previous study,zeaxanthin content and fluorescence were measured in leaf sam-ples (predarkened for 5 min) which were taken from leaves keptin the light. In the light, the xanthophyll cycle and fluorescencequenching processes are governed by the same parameters, ApHand a redox component (11, 21, 25), whether or not this may becausal. It is remarkable that the correlation between zeaxanthinand fluorescence still exists after 12 h of darkness in water-stressedN. oleander leaves, i.e. long after the above factors have ceasedto exert their direct influence on fluorescence and on the de-epoxidase engaged in the formation of zeaxanthin. Therefore,the relationship between zeaxanthin and fluorescence does notseem fortuitous. The exact nature of the interaction betweenzeaxanthin and Chl molecules in vivo is not known. Beddard etal. (2) have reported the quenching of Chl a fluorescence bycarotenoids in vitro which they interpret in terms of an electrontransfer process:

IChl + °Car -> Chl + Car +

Reversible electron transfer reactions involving Chl and caro-

tenoids have been demonstrated (18-20).The linear correlation between zeaxanthin and the increase in

radiationless dissipation, as calculated from the simple model by

Table III. Chl and Carotenoid Content of N. oleander Leaves which Had Developed at Three Different PFDsSun leaves had been kept at either 800 or 1000 umol photons m-2 s-1 and had a water content of 255% (experiment 1) or 205% of DW

(experiment 2), respectively. Shade leaves had developed at a PFD of 200 umol m-2 s- . The specific leaf weights at 200, 800, and 1000 ,umolphotons m-2 s-1 were 122, 134.8, and 132.9 g DW m-2, respectively. Samples were taken at the end of the 12-h dark period.

Pigment Content

200 800 1000 200 800 1000 200 800 1000(PFD during Growth, umol m - Is - 1)

limol g FW ,umol M-2 ,tmol g IDWChl a 1.94 1.60 1.50 699 764 607 5.73 5.67 4.57Chl b 0.76 0.54 0.50 273 259 201 2.24 1.92 1.51Cartota, 0.77 0.69 0.75 277 330 303 2.27 2.45 2.28,-Car 0.27 0.29 0.30 98 141 119 0.80 1.04 0.90Z + A + V 0.12 0.10 0.11 44 48 44 0.36 0.35 0.33

Ln

Ecm

Ca:

0_

22 DEMMIG ET AL.




0 0.2 0.4 0.6

FV / FM

0.8

0

0

0B

0 10 20 30 40 50 60

0.5

0.4

0.3

0.2

0

0EM._C

Ccaxa0L)N

0.1

0

0.5

0.4

0.3

0.2

0.1

0

A

0

A

A0

..t

0

Ar0

0

A

2 4 6 8 10 12

0 0.2 0.4 0.6 0.8

KDFIG. 6. Relationship between zeaxanthin content and (A) the ratio

of variable to maximum fluorescence from the upper leaf surface and(B) the rate of radiationless dissipation processes (KD) in leaves of N.oleander subjected to water stress in strong light. (A, A), Plants fromexperiment 1; (0, 0), plants from experiment 2. Open symbols refer tosamples taken at the end of the light period and closed symbols to samplestaken at the end of the dark period.

Kitajima and Butler (13) supports the interpretation that pho-toinhibition arising from a combination of high light and waterstress in N. oleander is caused to a large extent by an increasein radiationless dissipation in the antenna Chl and not by photo-inhibitory damage. In the experiments presented here, there wasno net decrease in Fo during the stress treatment as reported inan earlier study using low temperature fluorescence (3). Evi-dently, a second process, resulting in a rise in Fo, contributes tothe decrease in FVIFM under the conditions used by us. Furtherinterpretation of these findings has to await direct quantitativecomparisons between room and low temperature fluorescence.While the rate constant KD is a good measure of the increase

in radiationless dissipation and the concomitant decrease in pho-tochemical efficiency in N. oleander, neither variable fluores-cence, Fv, nor the fluorescence coefficient qD can be used tofully describe the increase in zeaxanthin content or changes inthe rate of radiationless dissipation in a linear fashion (Fig. 7).Both variables become relatively insensitive at high rates of ra-diationless dissipation.

N. oleander has a strong capacity to increase its zeaxanthincontent not only at the expense of violaxanthin but also at theexpense of p-carotene (Figs. 4 and 5; Table III). This capacityis independent of the growth PFD, since it was observed both

FIG. 7. Relationship between zeaxanthin content of N. oleander and(A) variable fluorescence and (B) the quenching coefficient qD (=1 -

F,'IFv). Symbols as in Figure 6.

in sun leaves of N. oleander subjected to slowly developing droughtand in shade leaves subjected to a sudden strong increase in PFDunder favorable conditions of turgor and temperature (Fig. 5).Thus, this capacity is probably genetically determined. Otherspecies like Hedera helix, with a similar capacity for rapid for-mation of additional amounts of zeaxanthin from 8-carotene,also exhibit strong increases in radiationless dissipation as a ma-

jor photoinhibitory response (10). Evidently the potential forrapid interconversions between 8-carotene and zeaxanthin +antheraxanthin + violaxanthin is related to the resistance of a

plant to photoinhibitory damage. In Populus balsamifera, a spe-cies which did not increase the sum of the three xanthophylls,photoinhibitory damage became apparent upon stepwise in-creases in PFD as soon as all of the violaxanthin had been con-verted to zeaxanthin (10).

Zeaxanthin increased to a similar extent in N. oleander trans-ferred from low to high light or subjected to drought in high light(Fig. 5) as well as in H. helix during short term treatment in 2%02, zero CO2 in weak light (10). Whereas zeaxanthin disappearedwithin about 60 min upon return to control conditions after theshort-term treatment in H. helix, the increase in zeaxanthin dur-ing the water stress treatment in N. oleander was virtually irre-versible (Fig. 4). Thus, the water stress treatment specificallyslowed down the reconversion of zeaxanthin to its precursors.

In conclusion, the present study shows that the carotenoidzeaxanthin probably mediates the process of radiationless energydissipation in the antenna Chl and is responsible for a substantial

0.5

0.4

0.3

0.23:0

cm

0EM._C

C-

x

a,N

0.1

0

0.5

0.4

0.3

0.2

0.1

0

I -I a

23

L




part of the reduction in photochemical efficiency arising from a

combiriation of high light and water stress in N. oleander. Thisfinding corroborates previous conclusions, based exclusively uponchanges in fluorescence characteristics, that leaves of N. oleanderwhich experience high light levels during a period of droughtvirtually shut down their photochemical apparatus by allocatinglarge poftions of the trapped light energy to radiationless dissi-pation processes rather than to photochemistry. The resultingphotoinhibition is primarily a reflection of an avoidance strategywhich probably allows for survival of the photosynthetic struc-tures. Other species which, for example, show osmotic adjust-ment and maintain some photosynthetic activity during periodsof drought, may experience, if at all, a different kind of pho-toinhibition involving damage and repair.

Acknowledgments-We thank Maria Lesch for skillful technical assistance andDrs. Ulrich Heber and William W. Adams for critically reading the manuscript.

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photochemical fluorescence quenching and their response to photoinhibition.Aust J Plant Physiol. In press

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16. KRAUSE GH, JM BRIANTAIS, C VERNOTTE 1983 Characterization of chloro-phyll fluorescence quenching in chloroplasts by fluorescence spectroscopyat 77K. I delta pH-dependent quenching. Biochim Biophys Acta 723: 169-175

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19. LAFFERTY J, TG TRuscoTr, EJ LAND 1978 Electron transfer reactions in-volving chlorophylls a and b and carotenoids. J Chem Soc Faraday 74: 2760-2762

20. MCVIE J, RS SINCLAIR, D TAIT, TG TRUSCOTT 1979 Electron transfer re-actions involving porphyrins and carotenoids. J Chem Soc Faraday 75: 2869-2872

21. OXBOROUGH K, P HORTON 1986 Characterization of the effects of antimycinA upon the yield of chlorophyll fluorescence in spinach chloroplasts. Re-search Institute for Photosynthesis Annual Report (Sheffield) pp 10-12

22. POWLES SB 1984 Photoinhibition of photosynthesis induced by visible light.Annu Rev Plant Physiol 35: 1544

23. SCHREIBER U, U SCHLIWA, W BILGER 1986 Continuous recording of photo-chemical and nonphotochemical fluorescence quenching with a new type ofmodulation fluorometer. Photosynth Res 10: 51-62

24. SIEFERMANN, D 1972 Kinetic studies on the xanthophyll cycle of Lemna gibbaL.-Influence of photosynthetic oxygen and supplied reductor. In G Forti,M Avron, A Melandri, eds, lInd International Congress on Photosynthesis,Stresa 1971, Vol 1. W Junk, The Hague, pp 629-635

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26. WEBER A, FC CZYGAN 1972 Chlorophylle und Carotinoide der Chaetophor-inae (Chlorophyceae, Ulotrichales). I. Siphonoxanthin in Microthamnionkuetzingianum (Naegeli). Arch Mikrobiol 84: 243-253

27. WEIs E, JT BALL, J BERRY 1987 Photosynthetic control of electron transportin leaves of Phaseolus vulgaris: evidence for regulation of photosystem 2 bythe proton gradient. In J Biggins, ed, Progress in Photosynthesis Research,Vol 2. Martinus Nijhoff, Dordrecht, pp 553-556

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24 DEMMIG ET AL.



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