Photosynthesis Research 41: 357-370, 1994. ~) 1994 Kluwer Academic Publishers. Printed in the Netherlands.

Regular paper

Non-photochemical fluorescence quenching and the diadinoxanthin cycle in a marine diatom

M i g u e l O l a i z o l a 1'3, Ju l i e L a R o c h e 2, Z b i g n i e w K o l b e r 2 and P a u l G. F a l k o w s k i 2 l Marine Sciences Research Center, State University of New York at Stony Brook, Stony Brook, NY 11794, USA; 2Brookhaven National Laboratory, Oceanographic and Atmospheric Sciences Division, Upton, NY 11973, USA; 3present address (for correspondence and reprints): Joint Research Center, Institute for Remote Sensing Applica- tions, TP 272, 1-21020 lspra (VA), Italy

R e c e i v e d 5 January 1994; accepted in revised form 2 May 1994

Key words: diadinoxanthin, diatoxanthin, fluorescence-quenching, photoprotection, phytoplankton, protein turn- over, xanthophyll-cycle

A b s t r a c t

The diadinoxanthin cycle (DD-cycle) in chromophyte algae involves the interconversion of two carotenoids, diadi- noxanthin (DD) and diatoxanthin (DT). We investigated the kinetics of light-induced DD-cycling in the marine diatom Phaeodactylum tricornutum and its role in dissipating excess excitation energy in PS II. Within 15 min following an increase in irradiance, DT increased and was accompanied by a stoichiometric decrease in DD. This reaction was completely blocked by dithiothreitol (DTT). A second, time-dependent, increase in DT was detected ,,~ 20 rain after the light shift without a concomitant decrease in DD. DT accumulation from both processes was correlated with increases in non-photochemical quenching of chlorophyll fluorescence. Stern-Volmer analyses suggests that changes in non-photochemical quenching resulted from changes in thermal dissipation in the PS II antenna and in the reaction center. The increase in non-photochemical quenching was correlated with a small decrease in the effective absorption cross section of PS II. Model calculations suggest however that the changes in cross section are not sufficiently large to significantly reduce multiple excitation of the reaction center within the turnover time of steady-state photosynthetic electron transport at light saturation. In DTT poisoned cells, the change in non-photochemical quenching appears to result from energy dissipation in the reaction center and was associated with decreased photochemical efficiency. D1 protein degradation was slightly higher in samples poi- soned with DTI" than in control samples. These results suggest that while DD-cycling may dynamically alter the photosynthesis-irradiance response curve, it offers limited protection against photodamage of PS II reaction centers at irradiance levels sufficient to saturate steady-state photosynthesis.

Abbreviations: CAP - chloramphenicol; D1- PS II reaction center protein; DD - diadinoxanthin; DD - cycle- diadinoxanthin cycle; DT - diatoxanthin; DTT - dithiothreitol; FCP - fucoxanthin chlorophyll a-c protein; Fm - maximum fluorescence yield in the dark-adapted state; Fo minimum fluorescence yield in the dark-adapted state; F~m and F'o - maximum and minimum fluorescence yields respectively in some light adapted state; Fv - maximum variable fluorescence yield in the dark-adapted state; Ik - Irradiance at the intercept of the initial slope of the photosynthesis-irradiance curve and the maximum photosynthetic rate; kD - first order rate constant for non- radiative de-excitation of excitons in the PS II antenna; kd - first order rate constant for non-radiative de-excitation of excitons in the PS II reaction center; kF - first order rate constant for fluorescence; kT - first order rate constant for exciton transfer to the reaction center; kt - first order rate constant for exciton transfer from the reaction center to the antenna; Rubisco - ribulose bisphosphate carboxylase; SVm - Stern-Volmer quenching coefficient of the maximum fluorescence yield; SVo - Stern-Volmer quenching coefficient of the minimum fluorescence yield; aps n - apparent absorption cross-section of PS II; ~-arr -- average interval between exciton arrival to the PS II reaction center (ms); ~'rem - average interval between electron turnover during photosynthesis in the PS II reaction center (ms); Wd - the probability that an exciton is non-radiatively dissipated in the reaction center; WT -- the probability that an exciton in the antenna is transferred to the reaction center; ~ I J t - - the probability that an exciton is transferred back from the reaction center to the antenna

358

Introduction

Optimization of the realized quantum yield of photo- synthesis requires coordination between light harvest- ing and electron transport capacities. Because natu- ral light environments are inherently variable, organ- isms have developed numerous strategies to regulate both light harvesting and electron transport capacity on a variety of time scales (Falkowski 1983; Long et al. 1994). Phytoplankton, being single-celled organ- isms suspended in a turbulent medium, are not fixed in space and can undergo large, short-term (minutes to hours) changes in irradiance due to vertical trans- port through the euphotic zone (Denman and Gargett 1983; Falkowski 1983). Cells adapted to low irradi- ance, such as found deep in a water column, can be transported into the surface layers, where they may be briefly exposed to supraoptimal irradiance levels (Falkowski et al. 1994). A number of photoacclima- tion strategies have evolved to optimize light harvest- ing at low photon fluences while minimizing damage at high irradiance levels. On times scales of several hours to days, cells may synthesize or degrade elec- tron transport components or antenna pigment pro- tein complexes (reviewed by Falkowski and La Roche 1991). However, on shorter time scales of minutes to hours, changes in irradiance are often accompanied by changes in non-photochemical quenching, which may be associated, in part, with a xanthophyll cycle (Demers et al. 1991; Olaizola et al. 1992; Olaizola and Yamamoto 1994). Here we examine the effect of that cycle on the quantum efficiency of Photosystem II and its potential to reduce PS II photodamage.

In the ocean, chromophyte algae (those classes containing chlorophyll c) are often extremely abun- dant. The diadinoxanthin cycle (DD-cycle) of chro- mophyte algae converts the monoepoxide carotenoid, DD into the de-epoxide form, diatoxanthin, DT, under high light, and DT into DD under low light or dark- ness (Hager 1980; Yamamoto 1985). Several investi- gators have reported that DT accumulation is corre- lated with in vivo fluorescence quenching (Mortain- Bertrand and Falkowski 1989; Demers et al. 1991; Olaizola and Yamamoto 1994). Furthermore, based on Stern-Volmer analyses of the components of variable fluorescence, Olaizola and Yamamoto (1994) suggest- ed that DT accumulation in high light was correlated with increases in thermal dissipation in the antenna of PS II in the diatom Chaetoceros mueUeri. Similar observations associated with the violaxanthin cycle in higher plants have led to suggestions of a photopro-

tective role for the cycle. It has been proposed that zeaxanthin (reviewed by Demmig-Adams 1990) and antheraxanthin (Gilmore and Yamamoto 1993) allow increased thermal de-excitation of absorbed light ener- gy in the antenna of PS II, thereby enhancing non- radiative dissipation of excitation energy that might otherwise damage the reaction center. It has not yet been established whether the DD-cycle similarly pro- tects the PS II reaction center of chromophytes from excess excitation energy.

An increase in non-radiative dissipation of absorbed excitation energy in PS II antenna implies a short-term decrease in the effective absorption cross section of PS II (Mauzerall and Greenbaum 1989; Gen- ty et al. 1990). If the changes in cross section were significant, they could, in principle, dynamically alter the photosynthesis-irradiance (P vs. I) curve without affecting the maximum quantum efficiency of photo- chemistry in PS II. This process could optimize short term photosynthetic efficiency by reversibly adjust- ing light harvesting to match photosynthetic capaci- ty. Here we describe the relationship between non- photochemical quenching, the DD-cycle and photoin- hibition in a marine planktonic diatom, Phaeodactylum tricornutum. Our results suggest that while DD-cycle activity is correlated with increases in thermal dissi- pation in the antenna ~,f PS II, light-induced decreases in the quantum efficiency of PS II photochemistry and net loss of D1 protein were only slightly affected when the DD-cycle was blocked with dithiothreitol.

Materials and methods

Growth conditions

Phaeodactylum tricornutum (clone CCMP 1327) was grown at 18 ° C in an artificial seawater medium (Gold- man and McCarthy 1978) enriched with f/2 nutrients (Guillard and Ryther 1962) under 120 #mol m -2 s - l continuous irradiance provided by cool fluorescent tubes. The ceils were grown in 1 L, water jacketed, cylindrical vessels with a 10 cm internal diameter and were kept in log phase growth (1.1 day-1 under these conditions) by daily dilution with media. To minimize shelf shading, cell density was kept between 4-40 × 104 cells L -1, as determined from hematocytometer counts. At this density the cultures were optically thin.

Pigment analysis

Cells (20 ml of culture) were collected by filtration onto glass fiber filters (Gelman, type A/E). The filters were wrapped in aluminum foil, frozen in liquid nitrogen, and stored at - 8 0 °C. Prior to analysis, the filters were disrupted with a tissue grinder in 4 mi 100% acetone and allowed to extract overnight at - 2 0 °C. For analy- sis we followed the method of Mantoura and LleweUyn (1983). The pigments were quantified according to their peak height. Standards were obtained from P. tr/- cornutum cultures (DD and DT, extinction coefficient 262 L g- i cm-1 at 446 and 449 nm, respectively, Stauber and Jeffrey 1988) via preparative chromatog- raphy. Chlorophyll-a was obtained from Sigma Chem- ical Co. (St. Louis, MO).

Fluorescence measurements

Fluorescence yields were measured on a custom built pump-and-probe fluorometer (Kolber et al. 1988). This instrument calculates the ratio of emission to excita- tion energy (i.e., the quantum yield of fluorescence not simply the fluorescence intensity) from single- turnover xenon flashes. Changes in the fluorescence yield induced by a weak probe flash (Fo, the minimum yield of fluorescence) and the yield induced by a sim- ilar flash 70 #s after a saturating pump flash (Fro, the maximum yield of fluorescence) were measured at a frequency of 1 Hz (see Kolber et al. 1988 for details). Changes in the photochemical efficiency of PS II were calculated from these values as Fv/Fm, where Fv = Fm-Fo (Butler 1978). A bellows pump circulated the algae between the culture vessel and a 200 #1 flow- through cell in the fluorometer. In this configuration, measurements of fluorescence were made on samples within 3 s after removal from the chamber, there- by allowing relaxation of photochemical quenching. Thus, constant monitoring of Fo and Fm was possible as conditions in the culture vessel were manipulated. By varying the intensity of the pump flash, the appar- ent functional absorption cross-section of PS II (trps u) can be estimated from

A ~ f = 1 - e -~psa*E (1) ~ F s

where Aq~F is the change in fluorescence yield induced by the variable pump flash, ~Fs is the maximum change in fluorescence yield induced by a saturating flash, and E is the pump flash energy (Falkowski et al. 1986).

359

trpsn is a measure of the relative effective photon target area of PS II. Changes in Ups n indicate alter- ations in light harvesting capacity by the antenna ser- vicing PS II as well as the efficiency with which that energy is delivered to the PS II reaction center. By multiplying trps tI by the irradiance, I, and taking the inverse of the product, it is possible to calculate the time interval between exciton arrivals at the reaction center (rarr=(trvs II*I)- l).

Quenching of the Fo and Fm signals was calcu- lated using the Stern-Volmer coefficients SVo (which quantifies quenching in the Fo signal as Fo/F'o-1) and SVm (which quantifes quenching in the Fm signal as Fm/F'm-1), where the prime superscript indicates the value of the respective variable in some light- adapted state. Stern-Volmer quenching coefficients (Demmig-Adams 1990; Gilmore and Yamamoto 1991; Krause and Weis 1991; Olaizola and Yamamoto 1994) were chosen because they allow direct comparisons of increases in thermal dissipation of absorbed light energy (i.e., k'D--kD, the change in the first order rate constant for non-radiative de-excitation of excitons in the PS II antenna) with the concentration of the puta- tive quencher, DT. The bipartite model was used to derive SVo and SVm (Butler, 1978; see Olaizola and Yamamoto 1994, for details) as

and

Fo 1 (2) S V ° = Foo -- 1 = (k~ - k d ) k F "st- KD "Jr- kT

SVm = Fm _ 1 = SVo + WT(W~ -- ~Pd) (3) Elm 1 - - ~PTItPt

where kD, kF and kT are the first order rate constants for thermal dissipation of excitation energy in the antenna, for fluorescence and for transfer to the reaction center, respectively. The quantities ~'IJT, ~'I/a and ~ut represent the probabilities that an exciton in the antenna will be transferred to the reaction center, that an exciton in the reaction center will be thermally dissipated or that it will be transferred back to the antenna, respectively.

Protein characterization

Two hundred and fifty ml of culture were harvested by centrifugation for protein analysis. Total protein con- tent was determined using bicinchoninic acid (Smith et al. 1985) on aliquots of extracts obtained as described previously (Geider et al. 1993). The remainder of the extract was diluted with an equal volume of 0.2 M

360

D'I"F and two volumes of 4% SDS,1.5% glycerol, and 0.05% Bromthymol Blue, and boiled for 2 min. At this point, the samples were quick frozen in liquid nitrogen and stored at - 8 0 °C until further analysis. Proteins were analyzed by 15% SDS-PAGE slab gels with a 7% stacking gel (15 #g of protein per lane) using Laemm- li's (1970) buffer system. The gels were stained with Coomassie Blue or electrophoretically transferred to nitrocellulose (Towbin et al. 1979). The proteins on nitrocellulose were probed, using the western blot pro- cedure (Bennett et al. 1984), with polyclonal antisera raised against the purified fucoxanthin chlorophyll a/c protein (FCP) complex of the haptophyte lsochrysis galbana, ribulose bisphosphate carboxylase holoen- zyme (Rubisco) of L galbana (Falkowski et al. 1989) or the Photosystem II reaction center protein (D1) of Amaranthus hybridus. The blots were quantified using laser densitometry (Molecular Dynamic Densitometer running Imagequant).

Experimental protocol

Three sets of experiments were conducted. In each experiment, aliquots of the main culture were subject- ed to a 2 h shift in irradiance, from 120 #mol m -2 s -1 (the growth irradiance) to 415, 750 or 1500 #mol m -2 s -1. Each subculture was subjected to four treat- ments: (i), a non-manipulated culture (control); (ii), a culture pre-incubated for 10 min with DTT (100 #M final concentration) ; (iii), a culture preincubated with chloramphenicol (CAP, 100 #M final concentration); and (iv), a culture incubated with both DTF and CAP (100 #M each final concentration). Dithiothreitol is a known inhibitor of the violaxanthin cycle of high- er plants (Yamamoto and Kamite 1972), but it does not inhibit the formation of the photosynthetic pH gra- dient across the thylakoid membrane (Sokolave and Marscho 1976). Chloramphenicol is an inhibitor of chloroplast-encoded protein synthesis (such as reac- tion center protein, D1) although it can also directly inhibit photosynthesis at concentrations above 100 #M (Okada et al. 1991). Monitoring of Fo and Fm during the preincubation period indicated lack of an effect due to inhibitor addition. Samples for pigment anal- ysis were collected just before the light switch (T = 0) and 1, 3, 5, 10, 15, 30, 60 and 120 mins after the light switch. Changes in DD and DT concentrations were used to estimate DD-cycle activity. Fluorescence yields, Fo and Fm were continuously monitored during the same period. At the end of the two hour irradiance

._o0.10

0 E 0.05 f . -

C~ q Initial () OnnF 10 50 100 200 500

DTT concentration (uM) Fig. 1. Effect of DTT (dithiothreitol) concentration (pM) on DD-cycle activity as determined from changes in the concentra- tion of diatoxanthin (DT) per chlorophyll (mol:mol). The cells were exposed to 1000/~mol rn -2 s -1 for 12 rain. Initial = diatoxanthin concentration before exposure to the 1000/zmol m -2 s -1.

treatments samples were collected for D1, FCP and Rubisco determinations.

Results

Kinetics of diadinoxanthin-cycle components

Phaeodactylum tricornutum grown at 120 #mol m -2 S - 1 a r e light limited but not stressed by very low growth irradiance (Geider et al. 1986). The growth rate was 1.1 d - l, while the maximum specific growth rate is 1.5 d - l (Greene et al. 1991). DT concentration in P tricornutum grown under these conditions (120 #mol m -2 s -1) was low, about 0.02 mol DT (mol Chl-a) -1 . DD concentration was an order of magnitude higher, about 0.24 mol DD (mol Chl-a) -1. Following a shift up in irradiance, DT increased and DD decreased dur- ing the first 10--15 min. This rapid increase in DT was stoichiometrically inverse to the loss of DD, and this rapid interconversion between the two xanthophylls was completely blocked by DTT (Figs. 1 and 2). A second increase in DT concentration occurred 20-30 min following the light shift to 750 and 1500/zmol m -2 s -1. This second increase was insensitive to DTT additions in the 1500 #mol m -2 s -1 treatment. In the 750 and 415 #mol m -2 s -1 treatments there was an increase in DD (Fig. 2). A separate experiment was conducted in which a second addition of 100 #M DTT was made to the culture 60 rain into the incubation. The results (not shown) indicated that this second increase in DT was independent of D'I"F and not an artefact. In all treatments there was a pool of DD which was not de-epoxidized. This pool represented a minimum of 0.12 mol of DD (mol Chl-a) -1, or about 40--60%

361

DT

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n A V . : : : . O "

0.1 . . . . '~ . . . . u . . . . ~&,: : -g ' . . . .D .... D ....

0 5 10 15 30 60 120

DD

:.:jco,,,o, . . . . . , , , : = - - " :

°i'oI ,o. . . . .

t '0 ,d., 0.3 t 0.2 DTT .=

0.1 ""'"

00 5 10 15 30 60 120

0.4

0.3

0.2

0.1

0

DTT , . ::~::::1

0 5 10 15 30 60 120

T IME (min) T IME (min)

Fig. 2. Changes in concentration of DD-cycle components DD and DT following shifts in growth irradiance from 120 #mol m -2 s-1 to 415 (squares), 750 (circles) and 1500 #mol m -2 s -1 (triangles) in the CONTROL and DTT experiments. Results obtained in the CAP and CAP+DTT treatments paralleled those obtained in the CONTROL and DTT treatments (not shown).

of the total DD+DT pool. Chlorophyll-a concentra- tion did not change during the incubations (regression of chl concentration against t ime were never signif- icantly different from 0, Student 's t-test, p > 0.05) so that the changes in DD and DT concentrations are not ascribed to bleaching of chlorophyll by the irra- diance treatments. Results obtained with cultures pre- incubated with CAP (CAP and CAP+DTT treatments) paral leled those obtained in the CONTROL and DTT treatments (not shown); i.e., on the time scale of these experiments CAP had no observable effect.

The changes in DT levels following a shift to a higher growth irradiance, resulting from DD de- epoxidation, were fit to a first order kinetic model of the form

(DTt -- DToo) - k t = In (-D-~o= D - - - ~ ) (4)

where k was the first order rate constant, DTo was the initial concentration, DToo was the concentration at the end of DD-cycle activity, and DTt was the concentra- tion at t = 1, 3, 5, 10 and 15 min after the light switch. Data from the CONTROL and CAP experiments were used to calculate the rate constant, k; cultures incu- bated with DTT and C A P + D ~ were not included because DTT blocked the light dependent intercon- version of DD to DT. The values for k ranged between 0.170 min -1 and 0.071 min - l (Table 1 ). There was no linear effect of irradiance on the rate constant in

Table 1. First order rate constants (rain -1) for the increase in DT/Chl-a (mol/mol) following an increase in growth irradiance. Pigment concentrations were lin- earized (log) and linearly regressed against time. The regression coefficients (slopes of the linear regres- sions) were pooled and tested for homogeneity. The hypothesis that all slopes were homogeneous was rejected (P < 0.001, one tailed F-test; Sokal and Rohlf 1981). Numbers in parenthesis are the correlation coef- ticient (z a)

Increase in irradiance from 120/zmol m -2 s - t to 415 750 1500

CONTROL 0.131 0.089 0.105 (0.934) (0 .967) (0.943)

CAP 0.170 0.125 0.071 (0.982) (0 .990) (0.910)

the CONTROL experiments but a decrease in k with increasing irradiance in the CAP experiments.

In all cases where cells were shifted to higher irra- diances, the sum of DT and DD increased linearly with time. To estimate the rate of increase of DD-cycle components, DD+DT data from all treatments were fit to a zero order model (linear regression with time). The regression coefficients ranged between 0.863 and 1.398 mmol DD+DT (mol Chl-a) - l min -1 and were pooled and tested for homogeneity. The hypothesis that

362

E In

I.L,

CONTROL

1

0.8

0.6

0.4

0.2

0

• t~, . ' ,r~:: .:~:, . , . . :~.: . .~.:~.~;- ,~, , , . ,5.~ . . . . . .

0 20 40 60 80 100 120

1

0.8

0.6

0.4

0.2

0

DTT

0 20 40 60 80 100 120

o > ¢..0 0.5

,~lt ~ 6~'~ *¢1 "I

.~.~ ! , . /: . . . . . .

0 20 40 60 80 100 120

1

0.5 "v'llf~"" ""

o . . . . .

0 20 40 60 80 100 120

E >

0 20 40 60 80 100 120 TIME (min)

t

0 20 40 60 80 100 120

TIME (rain) Fig. 3. Light-induced changes in Fv]F; m and the non-photochemical quenching parameters SVo and SVm in the CONTROL and DTT treatments following shifts in growth irradiance from 120 #mol m -2 s - l to 415 (solid line). 750 (dotted line) and 1500 #mol m -2 s -1 (dashed line) in the CONTROL and DTT treatments. The Fv/Fm data has been normalized to the initial Fv/Fm value (0.64). Similar results were obtained in the CAP and CAP+DTT treatments (not shown).

all slopes were homogeneous was accepted (P > 0.10, one tailed F-test, Sokal and Rohlf 1981). The average rate of increase was 1.127 (+ 0.151 standard deviation) mmol DD+DT (mol Chl-a)-1 min-1. Because the rate was similar for the 415, 750 and 1500 #mol m -2 s -1 treatments, chlorophyll photobleaching is not believed to be a factor in these measurements.

Fluorescence yield and the origin o f non photochemi- cal quenching

Changes in the Chl-a normalized maximum (Fro) and minimum (Fo) fluorescence yield were observed in all treatments following the light shift. Average values for Chl-a-normalized Fo and Fm were 1.17 and 3.26 (nM Chl -a) - I respectively for P. tricornutum grown at 120 #mol m -2 s -~. Thus, the maximum value of

Fv/Fm was 0.64, lower than that commonly report- ed for higher plants but a typical maximum value for eukaryotic algae measured by single turnover saturat- ing flash techniques (Kolber et al. 1988). Following a 120 min exposure to 415, 750 and 1500 #mol m -2 s - ] , Fo decreased in the CONTROL and CAP treat- ments. The decrease was either less pronounced, or there was actually a small increase in yield (in three cases), in the D ' I~ and CAP+DTT treatments (Table 2). Fm decreased in all treatments following a light shift. The decrease in Fo (CONTROL and CAP) and Fm depends on irradiance with Fm decreasing more than Fo, resulting in a decrease in Fv/Fm in all treat- ments. Most of the change in Fv/Fm occurred during the first 30 min of incubation for all cases (Fig. 3).

The changes in fluorescence yield were observed in dark adapted cells and, hence, reflect changes in non-

363

Table 2. Changes in chlorophyll-normalized minimum and maximum fluorescence yields (Fo and Fro) and photochemical efficiency (Fv/Fm) following 120 rain exposure to 415, 750 and 1500/zmol m -2 s -1 under the four experimental treatments. The values shown are the fractional change after 120 min of exposure to the irradiance indicated

415 750 1500 Fo Fm Fv/Fra Fo Fm Fv/Fm Fo Fm Fv/Fra

CONTROL -0 .29 -0 .47 -0.19 -0.42 -0 .65 -0.34 -0.55 -0 .76 -0.52 CAP -0.12 -0 .41 -0.28 -0.23 -0 .58 -0.47 -0.36 -0 .74 -0.80 DTT +0.02 -0 .25 -0.20 -0.08 -0 .38 -0.27 -0.45 -0 .69 -0.44 CAP+DTr +0.07 -0 .21 -0.21 +0.31 -0 .32 -0.53 -0.23 -0 .68 -0.76

"o 2.0 .¢_ >,,,

1.5

~_ 1.0

~0.5 1-

o 0.0 0.1 1.0

• + D l - r

• :'., .."' o,

• :..- -DTT

10 100 1000

1.0

'10 0~

. N

0.5 O

z

0.0 0.1 1.0 10 100 1000

Pump Flash Energy

Fig. 4. Flash saturation curves used for the determination of ~rps n before (DARK, solid line) and after 2 hr incubation under 750/~mol m -2 s -1 in the absence (-DTT, dotted line) and presence (+DTT, broken line) of dithiothreitol. Panel A shows the changes in fluores- cence yield, AOF, produced by the pump flash. Panel B shows the fit of Eq. (1) to the same data but normalized to the maximum change in fluorescence yield, ~ s -

photochemical quenching processes. To attempt to elu- cidate the origins of the quenching we first analyzed the kinetics of the changes in the quenching coefficients, SVo and SVm. The increases in SVo following a shift in irradiance were biphasic. The first, rapid, increase occurred in parallel with the observed increase in DT and was el iminated in the DTT and CAP+DTY treat- ments (Fig. 3). At the highest light intensity, 1500 #mol m -2 s - l , a second increase in SVo was detected 20-30 minutes fol lowing the light shift and was present in all treatments. This second phase of increase in SVo was

correlated with the slow, DD-independent accumula- tion of DT. Fol lowing a light shift, SVm also displayed biphasic kinetics, however the first, rapid phase was not totally eliminated by DTT (Fig. 3), suggesting a source of fluorescence quenching independent of DD- cycle activity. The second increase in SVm occurred in all treatments but only under 1500 #tool m -2 s -1.

Increases in SVo suggest changes in thermal de- excitation within the antennae pigment serving PS II (Eq. (2)). I f the fluorescence quenching does, in fact, occur in the antennae, then the effective absorption cross section of PS II should decrease as quenching increases (Mauzerall and Greenbaum 1989; Genty et al. 1990). I f the decrease in effective absorption cross section were not accompanied by large changes in the optical absorption cross section of the pigment bed, a corresponding decrease in quantum yield of oxy- gen evolution would ensue without necessarily any change in the quantum yield of photochemistry within the reaction center (Ley and Mauzeral l 1982; Kolber et al. 1988). Typical flash-saturation curves for the deter- mination of the apparent absorption cross section of PS II (ores II) are presented in Fig. 4. The calculated values of aPs n indicated that there was a decrease in aps n of up to 14% following a light shift which could be inhibited by DTI' . These results suggest that DD- dependent quenching affects the transfer of absorbed excitons by PS II antenna, but the change in cross sec- tion is relatively small (Falkowski et al. 1986; Genty et al. 1990).

Correlation between changes in SVo and SVm and DT concentration

The initial rise in DT concentration following the light shifts was quantitatively correlated with SVo and SVm. The results of the DTT and CAP+DTT (no Fo

364

Table 3. Linear regression coefficients (SV (DT/Chl-a) -1) between DT concentration and non-photochemical quenching of fluorescence in P. tri- cornutum during the initial rise in DT (first 15 minutes following a light shift). The fluorescence yields obtained in the experiments under DTT poi- soning provide an estimate of non-photochemical quenching by processes other than the DD-cycle (DT-independent). Those results were subtract- ed from the CONTROL experiments to estimate the fraction of quenching apparently dependent on DT accumulation (DT-dependent) and are present- ed in the -CAP columns. This was repeated for the CAP and CAP+DTT treatments (+CAP columns). I is the irradiance of the light shift in #mol m-2 s-1

SVo TOTAL DT-DEPENDENT DT-INDEPENDENT

I -CAP +CAP - C A P + C A P -CAP +CAP 415 5.67 5.40 5.67 6.04 0.00 -0.64 750 7.06 6.41 6.53 7.37 0.65 -0.96 1500 6.13 6.02 6.03 5.80 0.11 0.22

SVm TOTAL DT-DEPENDENT DT-INDEPENDENT

I -CAP +CAP - C A P + C A P -CAP +CAP 415 11.46 12.00 8.92 10.38 2.48 1.62 750 19.01 16.89 14.32 14.50 4.69 2.39 1500 18.92 20.52 12.98 11.26 5.94 9.26

quenching occurred in the absence of DT increases) experiments suggest a common mechanism relating the changes in fluorescence yield and DT concentra- tion. Linear regression analysis was thus used to esti- mate the dependency of SVo and SVm on DT con- centration during the first 15 min following the light shift. By subtracting the results of the experiments under DTT poisoning from the CONTROL experi- ments, the fraction of quenching apparently dependent on DT accumulation and the fraction that occurred in the absence of changes in DT were determined. The data from the CAP and CAP+DTT treatments were treated similarly to obtain an estimate of DT- dependent and DT-independent quenching under CAP poisoning. The increase in DT concentration was asso- ciated with nearly all of the change in SVo (Table 3), i.e., there was no appreciable quenching of Fo in samples poisoned with DT]" (Fig. 3). The regression coefficients ranged between 5.67 and 7.37 SVo (mol DT/mol Chl-a) -1. Unlike SVo, SVm was associated with both DT-dependent and DT-independent process- es. DT-dependent SVm ranged between 8.92 and 14.50 SVm (mol DT/mol Chl-a) - l . The DT-independent fraction increased with irradiance, especially under

¢n 2.0 s ~ ~

~ 1.0

2, ~.o.995 0 .0 . . . . . . . . . . . . . . . . . . . 0.0 0.05 0.10 0.15 0.20

DT Fig. 5. Changes in non-photochemical quenching and DT con- centration in a culture poisoned with DTT following a shift to 1500 #mol m -2 s - l (data from Figs. 2 and 3). This figure shows the linear dependency of non-photochemical quenching on the DTl'-independent increase in diatoxanthin 15-120 rain after the light shift. Note that a fraction of SVm developed without an increase in DT.

CAP poisoning (Table 3). The ratio of DT-dependent SVm/SVo also changed with irradiance. This ratio was similar for the shifts to 750 and 1500 #mol m -2 s - ] but lower for the shift to 415 #mol m -2 s - l . The dif- ference was less pronounced in the CAP treatments.

The slow increase in DT concentration, observed 20-30 min following the light shift to 1500 #mol m -2

s- 1, was not affected by D T r or CAP (Fig. 2) but was associated with quenching of Fo and Fm (Fig. 3). The relationships between DT increases and both SVo and SVm were linear during this period (Fig. 5). The regres- sion coefficients (6.00 SVo(mol DT/mol Chl-a)- 1 and 11.74 SVm(mol DT/mol Chl-a)- l) are within the range of those calculated from the initial rise in DT following the light shifts (Table 3). Thus, increases in DT concen- tration have the same effect on fluorescence quenching whether arising from the initial, rapid, increase follow- ing the light shift (correlated with DD de-epoxidation) or arising from the second, slow, increase (presumably associated with de novo synthesis of DT).

Changes in D1 protein concentrations

Following shifts in irradiance, the relative abundance of D1 compared to Rubisco decreased in all treat- ments (Fig. 6). The results are similar when compared to FCP (data not shown). The duration of the incu- bations is short enough that both FCP and Rubisco remain relatively constant. Thus, changes in the ratios reflect changes in D 1. The decrease in D 1 was most pronounced in cultures undergoing the largest light shifts (i.e., up to 1500 #mol m -2 s - l ) and paralleled the decrease in Fv/Fm (Fig. 7). Although most of the change in Fv/Fm occurred within the first 30 min fol- lowing the light shift (Fig. 3) our data does not provide information on the kinetics of the D1 changes. There was little difference in the CONTROL and DTT sam- pies. On average, the concentration of D 1 protein was only 3% higher in CONTROL than in D'I*F samples. The decrease in D1 protein was more pronounced in CAP poisoned samples in all cases and still more pro- nounced in samples poisoned with both DTF and CAP. On average, the concentration of D1 protein was 36% higher in CAP than in CAP+DT'I" samples. Thus, under CAP poisoning, DD-cycle blockage by DT'I" appeared to have an effect in the extent of D1 decrease.

Discussion

Light-induced DT accumulation in P. tricornutum

The results of this study suggest that there are two light- induced processes affecting DT levels in Phaeodacty- lum tricornutum. The first is a rapid, light-induced, stoichiometric conversion of DD to DT, which is in agreement with DD-cycle activity reported pre- viously (Hager 1980; Yamamoto 1985; Demers et

365

al. 1991; Olaizola and Yamamoto 1994). This rapid, light-induced, DD-cycle activity is inhibited by DTT. While it is well known that DI"T inhibits violaxanthin de-epoxidase in the higher plant violaxanthin cycle (Yamamoto and Kamite 1972), this is the first report on the use of DTT to block the DD-cycle. Prolonged exposure to higher irradiance levels beyond the first 30 min led to further increases in DT without an apprecia- ble decrease in DD in the 1500/~mol m -2 s -1 experi- ments. In the 415 and 750 #mol m -2 s - l experiments DD increased instead of DT. This second process was not inhibited by DTT. These results imply the presence of two functionally distinct pools of DD, only one of which can be quickly de-epoxidized. The second pool of DD may serve a different function or may not be available to the de-epoxidase (Siefermann-Harms 1984). Similar results (a non-de-epoxidized pool of DD) have been obtained with the diatom Chaetoceros muelleri (Olaizola and Yamamoto 1994).

Based on the similar rates of increase in DD for the 415 and 750 #mol m -2 s -1 experiments and DT for the 1500 #mol m -2 s -1 experiments 15-20 min after the light shifts it is presumed that de novo pig- ment synthesis occurs following a shift in irradiance. We propose that the de-epoxidized pigment (DT) is synthesized first, as is the case in the higher plant vio- laxanthin cycle (Spurgeon and Porter 1983). Thus, DT accumulation would occur without the need for de- epoxidation of DD. Although de novo DT synthesis may have occurred in all treatments we would only expect it to accumulate if epoxidase activity was hin- dered. In our experiments, DT accumulated only at the highest irradiance tested. Previous experiments have shown an apparent inhibition of epoxidase activity fol- lowing high irradiance treatments. Olaizola and Mcln- tyre (unpublished data) found that a 30 min, 1600 #mol m -2 s- 1, incubation was sufficient to inhibit DT epox- idation in P. tricornutum. However, it is not known if those earlier results were a direct effect on the epoxi- dase, which has a limited pH optimum, or may have been indirectly mediated by other mechanisms such as ATP hydrolysis (i.e., Gilmore and Yamamoto 1992) or chlororespiration (Ting and Owens 1993). In the experiments reported here, epoxidase activity resulted in increases in DD at the expense of DT in the 415 and 750 #mol m -2 s -1 treatments. Thus, we propose that the accumulation of DT measured in DTT poisoned cells must be the result of de novo pigment synthesis triggered by the irradiance shift and hindrance of the epoxidase at 1500 #mol m -2 s -1.

366

Fig. 6. Western blot showing the differences in D1 and Rubisco concentrations produced under the three actinic lights (415 #mol m -2 s -1, 750/zmol m -2 s -1 and 1500/zrnol m -2 s - t ) for tbe four treatments (CONTROL, DTr, CAP, and CAP+DTT).

1.0

0.8

E 0.6 LL

0.4

0.2

0.0 0.0 0.2 0.4 0.6 0.8 1.0

D1/Rubisco Fig. Z Linear regression between Fv/Fra and D1/Rubisco in P tricornutum for the control (open circles), +DTT (closed circles), +CAP (open triangles) and +CAP+DTT (closed triangles) treat- ments. Light-induced changes were measured after two hour expo- sure to three actinic lights (415 /zmol m -2 s -1 , 750 /zmol m -2 s -1 and 1500/zmol m - 2 s - l ) . The Fv/Fm and D1/Rubisco values shown are normalized to initial values, before exposure to increased irradiances.

Correlation between D T accumulation and non photo- chemical quenching

Antenna quenching via increases in kD are mediated by DD-cycle activity. This is supported by lack of initial Fo quenching (low SVo values) in DTT poisoned sam- pies and the l inear relationship between changes in kD and DT concentration. Antenna quenching decreases the number of excitons which may be trapped by the reaction center, affecting both SVo and SVm, and is reflected in the larger decrease in the apparent absorp- tion cross-section in samples not poisoned with DTT than in those poisoned with DTT. We found that one mole of DT was associated with a 5.67 to 6.53-fold increase in SVo and an 8.92 to 14.32-fold increase in SVm. Similar values have been obtained for Chaeto- ceros muelleri (Olaizola and Yamamoto 1994) and for zeaxanthin in higher plant chloroplasts (Gilmore and Yamamoto, 1991). It is significant to note that slow DT accumulation (via de novo synthesis) occurring

: No change 30 : - - 1 5 % change

• . . . . . 5 0 % change

g 20 !X~ 10

°°°...,

" % . . . 0 500 1000 1500

Irradiance

(umol nl 2 s "~ )

Fig. 8. Effect of changes in ~PS n on the time interval between exciton arrivals at the reaction center (~'arr). The horizontal fine at 5 ms (~'rem) indicates the rate of exciton removal via photochemistry (Greene et al. 1991). When l"arr>~-rem, the cells are fight limited for photosynthesis. The solid, slashed and dotted lines represent the changes in the rate of exciton arrival to the reaction center as a function of irradiance assuming no change, 15 %, or 50% decrease in crps n. The three vertical arrows represent the values for Ik assuming no change, 15%, or 50% decrease in ~PSn.

under the highest light treatment in the presence of dithiothreitol (Fig. 5) had the same effect on Fo and Fm quenching than DT accumulated via DD-cycle activ- ity. This means that so called DTT-independent non- photochemical quenching is not necessarily DT inde- pendent (or zeaxanthin-independent in higher plants and green algae).

Changes in SVo are directly proportional to changes in the rate of thermal de-excitation in the functional antenna of PS II (kD, Eq. (2)). SVm is also proportion- al to changes in ko (Eq. (3)) but may be affected by changes in de-excitation in the reaction center (Wd, kd). These quenching mechanisms are not mutually exclu- sive. If there are no changes in ka (i.e., kd=k'd)

SVo SVm -- (5)

1 -- WT~t

367

and the relationship between DT-dependent SVo and SVm would be constant. A constant SVo/SVm ratio' would indicate a single quenching process in the anten- na. In these experiments, DT-dependent SVo was pro- portional to DT-dependent SVm under all three light treatments but the ratio changed with irradiance from 1.57 for the 415 #mol m -2 s -1 treatment to 2.19 and 2.15 for the 750 and 1500 #mol m -2 s -1 treatments respectively (the values were 1.72, 1.97 and 1.94 under CAP poisoning). According to Eq. (3), SVm/SVo will change if there are changes in reaction center quench- ing, i.e., kd#kld (note that WT and Wt are considered constants, reflecting the initial conditions before the light shift). Thus, there is an apparent change in lq associated with changes in DD-cycle activity which was roughly equal in the 750 and 1500 #mol m -2 s-1 (supersaturating for photosynthesis, Greene et al. 1991, Fig. 8) treatments compared to the 415 #mol m -2 s -1 treatment in both sets of experiments (i.e., with and without CAP additions). It is not clear whether this change in kd associated with DD-cycle activity is a direct effect of DT accumulation on reaction center quenching or a second, undescribed, process occurring in parallel and also inhibited by DTr. For example, damaged reaction centers may have a higher probabil- ity of transferring excitons back to the antenna, thus, increasing the probability of interaction between the exciton and the presumed quencher (DT).

DT-independent non photochemical quenching

A second type of quenching was observed in D T r poi- soned cells and, thus, was independent of DD-cycle activity. Since it quenched Fm but not Fo, it presum- ably occurred at the reaction center. This is in con- trast with 'constitutive' quenching described in high- er plant chloroplasts, which does affect the Fo signal (Gilmore and Yamamoto 1991). This quenching result- ed in increasing DT-independent SVm with increas- ing irradiances (Table 3). Quenching occurred within the first 15-20 min and was associated with a large decrease in PS II quantum efficiency (Fv/Fm). Deriv- ing Eq. (2) for DT-independent quenching (i.e., SVo = 0), we obtain

S V m - - ~IJT(~I'ld - - t l Jd ) ( 6 )

1 - W-rWt

o r

(~Ij ~ __ ~iid ) ~- SVm(1 - - ~ ' / T V t ) (7) WT

which shows the relationship between DT-independent SVm and increases in thermal deexcitation in the reac- tion center (lq). Bilger et al. (1989) and Adams et al. (1990) have also found DTT-independent high ener- gy quenching in higher plants. Dissipative processes around the reaction center, perhaps involving electron supply from the water splitting enzyme, may produce such quenching (Adams et al. 1990, Krieger et al. 1992). The same result would be obtained if lq was not changing per se but if a fraction of reaction cen- ters become quenching centers following the light shift (Weis and Berry, 1987). Giersch and Krause (1991) have proposed the occurrence of damaging events that allow damaged reaction centers to trap energy with the same efficiency as functional open reaction centers and just dissipate it as heat. Thus, the unit's fluorescence yield will be Fo, lowering the bulk Fro.

Changes in O'ps n and the question of photoprotection

In higher plants, the violaxanthin cycle is believed to serve as a photoprotectant. Most of the evidence for protection has been provided by studies that have shown a correlation between violaxanthin cycling and non-photochemical quenching of fluorescence under, for example, high-light stress (Demmig et al, 1987; Demmig-Adams and Adams, 1990; Adams et al, 1990). Furthermore, Bilger and Bj6rkman (1990) found that the violaxanthin cycle effectively decreased the degree of photoinhibition (measured as a decrease in light-limited oxygen evolution and Fv/Fm) in plants, but only when the plant's capacity for zeaxanthin accu- mulation was enhanced (i.e., 'sun' plants). The rela- tionship that we have found between DT-cycling and non-photochemical quenching suggests a similar role for the cycle in chromophyte algae, confirming earlier reports (Demers et al. 1991; Olaizola and Yamamoto 1994).

Changes in ~rvs n occurring in parallel to DD-cycle activity reflect changes in the efficiency of exciton delivery to the reaction center, not in light absorption (Olaizola and Yamamoto 1994). The changes in tres It measured here indicate that typical decreases in trps It (DT-dependent) are on the order of only 15% although larger changes in ~rps n (up to 3-fold) are common in unicellular algae as they photoadapt to different growth irradiances over a period of days (Ley and Mauzerall 1982; Kolber et al. 1988). Such acclimation does not lead to any changes in the quantum efficiency of PS II

368

(Kolber et al. 1988). Thus, changes in ~rpsn do not a priori alter the quantum yield of photochemistry within PS II reaction centers, although such changes may alter the quantum yield of oxygen evolution by altering the ratio of absorbed light to aps n (Mauzerall and Green- baum 1989; Falkowski 1992). We have calculated the effect of changing aps a on the rate of exciton arrival at the reaction center (~-m) to determine the effective- ness of the DD-cycle in dissipating excess excitation energy (Fig. 8), i.e., to dissipate enough excitons so that Tarr<7"rern, the rate of exciton removal via photo- chemistry (Greene et al. 1991). Our analysis indicates that a 15% decrease in ~rpsn can extend the range of irradiance under which the reaction center is not sat- urated from about 400 to 460 #tool m -2 s -1. A 50% decrease in ~rps n would be necessary to increase that range to 750 #mol m -2 s -1, and such large changes have been measured in natural phytoplankton commu- nities (Falkowski et al. 1994). The irradiance level at which the rate of exciton arrival begins to exceed that of electron transport is analogous to the Ik level that represents the intercept of the initial slope of the P vs I curve and the maximum photosynthetic rate, where Ik=('rrem * O'pslI) -1 (Falkowski 1992).

The limited capacity of the DD-cycle to dissi- pate excess excitation energy, i.e., to photoprotect, is reflected in the small differences in D1 degrada- tion rates. The degradation of D1 is light-triggered and mediated by events occurring at both the donor and acceptor side of PS II (Andersson et al. 1992). We found only a 3% difference in D1 degradation between CONTROL and DTI" samples but a 36% difference between CAP and CAP+DTr samples. The combina- tion of both inhibitors is also known to cause stronger photoinhibition in higher plants than when used sepa- rately (Demmig-Adams and Adams 1993). We found a good correlation between D1 abundance and Fv/Fm (Fig. 8) suggesting, in part at least, a relationship between loss ofD 1 and photochemical efficiency. Such a relationship has also been described in P. tricornutum cultures when inhibition was induced by nutrient stress (Geider et al. 1993) and in higher plants (Ottander et al. 1993).

Our results suggest that the DD-cycle affords mod- est short-term adjustment in the effective absorption cross section of PS II. However, we can not discard that under specific situations, such as nutrient limita- tion when the DD pool is enlarged (Geider et al. 1993), the capacity for thermal dissipation of excitation ener- gy in the antenna may increase, as is the case for 'sun' plants. In nature, phytoplankton do not generally expe-

rience such large and abrupt changes in irradiance as we experimentally contrived to elucidate the role of the DD-cycle in the non-photochemical quenching pro- cesses. Rather, the changes in irradiance experienced by cells in the upper ocean are slow enough to per- mit dynamic adjustment of the P vs I curve, thereby modifying Ik tO optimize light harvesting to electron transport capacity (Falkowski et al. 1994). In the face of chronic (minutes to hours) exposure to high irradi- ance levels, however, this adjustment is not sufficient to prevent multiple excitation of the reaction center with- in the minimum turnover time of whole chain electron transport. Consequently, a second non-photochemical quenching process emerges. That quenching appears to originate from PS II reaction centers and is independent of the DD-cycle. To the extent that PS II photochem- istry can be restored without de novo protein synthesis, the impaired reaction center can effectively dissipate excitation energy (Demmig-Adams and Adams 1993). However, this so-called 'down regulated' or 'sulking center' (i.e., capable of charge separation but not of QB-reduction) constitutes an escape valve for excess excitation energy only if energy transfer between PS II reaction centers occurs.

Acknowledgements

This research was conducted in partial fulfillment of a doctoral dissertation by Miguel Olaizola. It was supported by the US Department of Energy, Office of Health and Environmental Research (contract DE- AC02-76CH00016) and the Environmental Protection Agency (IAG grant DW 899 35239-O1-0), both to P. Falkowski. The authors would like to thank K. Wyman for technical assistance and I. Davison, R. Geider, R. Greene, H. Yamamoto and three anonymous review- ers for critically reading the manuscript. The authors would also like to thank J. Hirschberg for providing the D1 antibody.

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