Plant Physiol. (1990) 92, 891-8980032-0889/90/92/0891/08/$01 .00/0

Received for publication July 18, 1989and in revised form October 12, 1989

Adaptation of the Photosynthetic Apparatus toIrradiance in Dunaliella tertiolectal

A Kinetic Study

Assaf Sukenik, John Bennett, Anne Mortain-Bertrand, and Paul G. Falkowski*Israel Oceanographic and Limnological Research, P.O.B. 8030, Haifa 31080, Israel (A.S.); and Biology Department

(J.B.) and Oceanographic Sciences Division (A.M.-B., P.G.F.), Brookhaven National Laboratory,Upton, New York 11973

ABSTRACT

The time course of adaptation from a high to a low photon fluxdensity was studied in the marine chlorophyte Dunaliella terti-olecta. A one-step transition from 700 to 70 micromole quantaper square meter per second resulted in a reduction of doublingrate from 1.1 to 0.4 per day within 24 hours, followed by a sloweraccumulation of photosynthetic pigments, light harvesting an-tenna complexes, Photosystem II reaction centers and structurallipids that constitute the thylakoid membranes. Photoregulatedchanges in the biochemical composition of the thylakoid proteinsand lipids were functionally accompanied by decreases in theminimal photosynthetic quantum requirement and photosyntheticcapacity, and an increase in the minimal turnover time for in vivoelectron transport from water to CO2. Analysis of de novo synthe-sis of thylakoid membranes and proteins indicates that a highlight to low light transition leads to a transient in carbon metab-olism away from lipid biosynthesis toward the synthesis of thelight harvesting antenna protein complexes, accompanied by aslower restoration rate of reaction centers and thylakoid mem-branes. This pattern of sequential synthesis of light harvestingcomplexes followed by reaction centers and membranes, ap-pears to optimize light harvesting capabilities as cells adapt tolow photon flux densities.

Light regulates the development and organization of thephotosynthetic apparatus (1, 2, 10), yet the linkages betweenirradiance and molecular control mechanisms remain unclear(2, 20, 25, 34). Two basic model systems have been used tostudy the effects of light on chloroplast development, namelythe greening of etiolated chloroplasts (2, 19, 25), and analysisof mutants (16). During greening, reaction centers are synthe-sized prior to the light harvesting Chl proteins (25). Greeningis a developmental process and it is frequently difficult todistinguish between molecular events which are regulated bylight per se from those which are ontologically controlled.

'This research was supported by the U.S. Department of Energy,Division of Basic Energy Research, under contract No. DE-AC 02-76CH00016, by grant No. 86-00376 from the U.S.-Israel BinationalScientific Foundation, and by a Lavoisier grant to Anne Mortain-Bertrand from French Ministoire des Affaires Etrangeres.

Light adaptation, however, is an adjustment of preexistingcomponents of the chloroplast in order to optimize photosyn-thetic light utilization. Here we examine the sequence ofevents during the adaptation of the photosynthetic apparatusin a unicellular, eucaryotic green alga to changes in growthirradiance. The organism selected, Dunaliella tertiolecta, doesnot possess phytochrome, is a single cell type, and can phys-iologically acclimate to a wide range of growth irradiancelevels (20-1900 ,umol quanta m-2 s-') (7, 9-11, 31, 32).

In D. tertiolecta photoadaptation occurs on a time scale ofa few to tens of hours (3, 8). Following a one-step transitionfrom high to low growth irradiance levels, various molecularcomponents and physiological parameters change at differentrates. Establishing the sequence ofbiochemical alterations canclarify the mechanisms involved in photoregulation of pho-tosynthetic apparatus. For example, following a high to lowlight transition the rate of Chl synthesis surpasses the rate ofcellular growth by a factor of two, thereby increasing the lightharvesting capacity ofthe cell (8, 30, 31). Falkowski (8) found,however, that during such a light shift, the synthesis of PSIreaction centers lagged behind that ofbulk Chl, implying thatthe light harvesting complexes, encoded in the nucleus, aresynthesized prior to the chloroplast encoded reaction centers(19, 25).Here we examine in detail the time course of changes in

biochemical composition of the photosynthetic apparatus atthe protein level and the physiological responses these changesconfer in response to a decrease in growth irradiance. Changesin the pool size of cellular pigments, PSII reaction centers,the Chl a/b light harvesting protein complexes which formLHCII, and Rubisco2 were assayed and correlated with the

2 Abbreviations: Rubisco, ribulose 1 ,5-bisphosphate carboxylase/oxygenase; a-, spectrally averaged in vivo absorption cross-sectionnormalized to Chl a; a, initial slope of photosynthetic versus irradi-ance curve; DGDG, digalactosyl diacylglycerol; HL, high light;LHCII, light harvesting Chl a/b protein complex of photosystem II;LL, low light; LSU, large subunit of Rubisco; MGDG, monogalac-tosyl diacylglycerol; 1 /¢,, minimum quantum requirement for oxy-gen evolution; Pmax, maximal photosynthetic rate at light saturation;PSUo2, photosynthetic unit size for 02 evolution; P versus I curve,photosynthetic irradiance response curve; T, minimal turnover timefor photosynthetic 02 evolution.

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Plant Physiol. Vol. 92, 1990

minimum quantum requirement (1/qm) the maximal pho-tosynthetic rate (Pmax) and the minimal turnover time, (T),for photosynthetic 02 evolution. Changes in the rate of bio-synthesis of LHCII apoproteins and thylakoid membraneswere followed by autoradiographic analysis of incorporationof 35S into proteins and "1C into glycolipids. A kinetic analysisof the changes in the various biochemical and molecularparameters indicates that LHCII accumulates more rapidlythan PSII reaction centers, and the protein complexes aresynthesized prior to membrane lipids.

MATERIALS AND METHODS

Culture Conditions

Dunaliella tertiolecta (Woods Hole clone DUN) was cul-tured in natural seawater enriched with f/2 nutrients (14).Cells were grown in a 3.2 1 turbidostat under steady-stateconditions by periodic dilution of the culture vessel with freshgrowth medium (8, 9). Dilution rates were recorded hourlyby an HP-85 computer interfaced to the media pump (8).Culture temperature was maintained at 18 ± 1°C and contin-uous light was provided by banks ofVHO fluorescent tubes.Growth irradiance level was varied in one step from 700 (HL)to 70 (LL) ,mol quanta m-2 s-' by changing the number oftubes and their distance from the culture vessel. The high tolow light transition experiment was run and analyzed twiceunder identical conditions.

Pigment Analyses

During steady state growth prior to the light transition, andat selected times thereafter, the cultures were sampled forpigment content and photosynthetic characteristics. Photo-synthetic pigments were extracted by homogenizing samplesfiltered on glass fiber filters in 90% acetone. Corrected spectrawere recorded on an Aminco DW-2a spectrophotometer, andChl a and b were calculated using the equations of Jeffreyand Humphrey (18). Total carotenoid concentration was

calculated using an extinction coefficient of 2500 mM-' cm-'at 480 nm. (4). Cells were counted with a haemocytometer.

Photosynthetic Parameters

Photosynthesis-irradiance relationships were measured in a

thermostated PVC chamber with Clark type electrode (YSI5331 ) and a multigain amplifier using a collimated light beamfrom a tungsten-halogen source filtered through a hot mirror(5). Irradiance was attenuated with neutral density filters andmeasured with a Lambda-185S quantum sensor (5). Theinitial slope (a) of the P versus I plots, and the light saturatedphotosynthesis rate (Pmax), were derived from least square fitsof the data to an hyperbolic tangent function (17).

In vivo corrected absorption spectra were measured inoptically thin cell suspensions with filtered culture mediumas a reference. To reduce scattering effects, cuvettes were

placed close to the 2 inch window photomultiplier tube, andboth the reference and measuring beams were diffused by a

frosted quartz glass filter placed in front of the PMT.The in vivo, spectrally averaged, optical absorption cross-

section normalized to Chl a, (a ), was measured and calculatedaccording to Dubinsky et al. (5). The minimum quantumrequirement for oxygen evolution, l/0k..ax (quanta/02), wascalculated from a and a*:

1 a*fkmax a

Oxygen flash yields were measured with a Rank Brotherselectrode using single turnover flashes provided by three syn-chronized GenRad Stroboslave 1539A xenon flash tubes.Oxygen evolution was measured at flash frequencies of 10,20, 30, and 40 s ', and flash yields were calculated from linearregression analysis of flash frequency and the correspondingrate of oxygen production. Oxygen flash yields were normal-ized to Chl concentration to estimate photosynthetic unit size(PSUo2, the so-called Emerson and Arnold number). Thecellular concentration of PSII reaction centers was calculatedfrom cellular concentration of Chl a and the flash yield,assuming that each oxygen produced required four light-driven one-electron oxidation steps, each with a quantumyield of unity (1 1).The minimal turnover time for 02 evolution was calculated

from oxygen flash yields normalized to Chl a (PSUo2), andlight saturated, steady state, photosynthetic rates (5, 8):

PSUo2* Pmax

LHCII Apoproteins and Rubisco Content

The cellular levels of LHCII and Rubisco were determinedby Western blots of whole cell protein extracts separated bySDS-PAGE on 10% polyacrylamide gel slabs (2, 30). Thelarge subunit (LSU) of Rubisco and LHCII apoproteins weredetected by radioimmune blots using polyclonal antibodiesraised against pea proteins (30, 31). Autoradiograms wereoptically scanned with a densitometer to provide quantifica-tion. For LHCII quantification the densitometric data werenormalized to the LHCII values of HL grown cells and aregiven as relative numbers. For Rubisco quantitation, thepurified enzyme from D. tertiolecta was used to establish anabsolute calibration (30).

Synthesis of LHCII Apoproteins

The kinetics of LHCII apoprotein synthesis, following ashift from HL to LL were determined by monitoring 35SO4incorporation into proteins. For these experiments, cells weregrown in artificial seawater (13) with a reduced concentrationof sulfate (200 uM), fortified with f/2 nutrients and microele-ments (14). The reduction in external sulfate allows a muchhigher incorporation of "SO4 into proteins than can be ob-tained in natural seawater, but does not affect the growth rateof D. tertiolecta. Several hours before the light transition, andat selected times thereafter, 100 mL samples from the 3.2 Lculture vessel were removed and incubated in situ with 1 mCiof 3'SO4. After a 1 h incubation at 18°C at either 700 Mmolquanta m-2 s-' or following transfer to 70 umol quanta m2s-1, cells were harvested by centrifugation, and thylakoidmembranes were prepared as previously described (31). Ra-

892 SUKENIK ET AL.

PHOTOADAPTATION OF THE PHOTOSYNTHETIC APPARATUS

dioactivity was measured on 10 ,uL aliquots of the thylakoidpreparation. Aliquots containing 40,000 cpm were separatedon a 15% polyacrylamide SDS gel using the buffer ofLaemmli(23), and the proteins were electrophoretically transferred tonitrocellulose. 35S-Labeled proteins were identified by auto-radiography of the nitrocellulose blots. Samples were alsoloaded on SDS gels at equal Chl concentration. LHCII apo-proteins were identified using polyclonal antibodies (2) andvisualized using the horseradish peroxidase system.

Synthesis of Glycolipids

De novo synthesis of thylakoid membranes was assessed byfollowing the assimilation of NaH'4C03 into glycolipids in anexperimental setup similar to the one described above for15SO4 incorporation; the only difference was that cells weregrown in natural sea water supplemented with f/2 nutrients.At each time interval, 50 mL samples were withdrawn fromthe turbidostat and incubated in situ (1 8°C and the corre-sponding light intensity) with NaH'4CO3 (1 ,uCi/mL, 4.5 mCi/mmol) for 1 h. At the end of the incubation, cells werecollected, total 14C incorporated was measured and the cellcontents were separated into three fractions: lipids, small molwt metabolites, and a carbohydrate/protein fraction (33). Thelipid fraction was chromatographed by TLC on SilicaG plates(Merck, W. Germany); the solvent system for polar lipids waschloroform:acetone:MeOH:acetic acid:H20 (50:30:10:3:0.3)(21). Lipids were visualized by reaction with iodine vapor,and glycolipid spots were semiquantified by running a dilutionseries of purified standards. The glycolipids, MGDG andDGDG, were identified by using authentic standards (SigmaCo., St. Louis, USA). The plates were autoradiographed toidentify de novo synthesis of specific lipid classes.

RESULTS

Growth and Pigmentation

Following a transition from 700 to 70 ,umol quanta m-2 s-'the cellular doubling rate of D. tertiolecta was reduced by afactor ofthree and stabilized at a new steady-state value withinabout 24 h (Fig. 1 a). The downshift in light was accompaniedby an increase in cellular Chl a content (Fig. la). The cellularcontent of Chl b and total carotenoids increased dispropor-tionally to Chl a, consequently, Chl a/b and carotenoid/Chla ratios decreased (Fig. lb).

Photosynthetic Proteins

RCII/cell increased following the light shift, while the ap-parent size of PSUO2 underwent a transient increase (Fig. I c).To examine this transient in more detail we used polyclonalantibodies raised against LHCII apoproteins, to probe westernblots. We observed that the cellular content ofLHCII apopro-teins began to increase shortly after the light transition (Fig.1d), while PSII reaction centers accumulated more slowly (seebelow). Consequently, there was a rapid temporary increasein the relative abundance of LHCII per PSII reaction centerimmediately following the light transition. This ratio de-creased as the adaptation process continued until a new steady

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Plant Physiol. Vol. 92,1990

state value, about 25% higher than that originally calculatedin HL, was obtained, 72 h after the transition. No significantchanges in the cellular content of Rubisco were measuredduring the adaptation process.

Variations in Thylakoid Membrane Lipids

The cellular pool size of the main lipid constituents of thethylakoid membranes, MGDG and DGDG, were analyzed atvarious time intervals (Fig. 2). MGDG and DGDG were atrelatively low levels when cells were grown at 700 ,umol quantam-2 s I, and began to accumulate 24 h after the light transi-tion, reaching a new steady state level after 72 h. The finalsteady state value was about threefold greater than that underHL conditions (Fig. 2).

Photosynthetic Properties

Photosynthesis-irradiance curves for HL and LL adaptedD. tertiolecta are presented in Figure 3. When normalized toChl a (Fig. 3a), the maximum rate of photosynthesis in HLcells was about threefold higher than in LL cells. Whennormalized to cell number, however (Fig. 3b), the maximum

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Figure 2. Thin layer chromatogram of total lipids extracted from D.tertiolecta before and at indicated time intervals following a shift from700 to 70 ymol quanta m-2 s-'. Lipids were loaded on the basis ofconstant cell numbers in each lane and visualized by exposure toiodine vapors. DGDG and MGDG were identified using authenticstandards.

Figure 3. Photosynthesis irradiance curves for HL (700 Mmol quantam-2 s-1) and LL (70 Amol quanta m-2 s-1) adapted cultures of D.tertiolecta. Cultures were adapted to the given growth irradiance levelfor at least 5 d. Photosynthetic rates were normalized to (a) Chl aand (b) cell numbers.

photosynthetic rates were virtually identical. Nevertheless, theinitial slope of the P-I curve was greater for LL cells than forHL cells.

*The minimal photosynthetic quantum requirement de-creased rapidly after the light transition from 26 to 12 molesof photons per mole of oxygen at the new steady state (Fig.4). The minimal turnover time, r, for photosynthetic electronflow from water to CO2 increased after a 24 h lag (Fig. 4).Changes in T were accompanied by proportional variations incellular content of PSII reaction centers, but not with totalcell Chl a (Fig. 5). The correlation between r and RCII/cellis similar to that found in steady state cultures ofD. tertiolectawhere changes in r were linearly correlated with the cellularcontent of PSII reaction centers (30).Net photosynthesis, at the growth irradiance, as measured

by 14C02 incorporation, was sharply reduced immediatelyfollowing the light transition (Fig. 6). Such a reduction ispredicted from the gross photosynthesis-irradiance curve of asteady-state HL culture (Fig. 3). Using an irradiance level of70 ,imol quanta m-2 s-' to extrapolate the new gross photo-synthesis rate of HL cells shifted to LL, a six-fold reductionis predicted, and a comparable change was measured (Fig. 6).The sharp decline in the net photosynthesis was followed by

894 SUKENIK ET AL.

PHOTOADAPTATION OF THE PHOTOSYNTHETIC APPARATUS

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Figure 6. Effect of a shift down in growth irradiance level from 700to 70 Amol quanta m-2 s-' on photosynthetic carbon fixation and theflow of fixed carbon into lipid compounds in D. tertiolecta.

1 2 3 4 5 6

Figure 5. Relationships between the minimum turnover time of thephotosynthetic apparatus (T) and the cellular content of Chl a andPSII reaction centers in D. tertiolecta following a transition in growthirradiance from 700 to 70 ,mol quanta m-2 S-'. PSII reaction centersare linearly correlated with T (r = 0.912).

a slow recovery until a new steady state value was establishedat 15 x 10-18 limol C cell-' s-1.

Synthesis of LHCII Apoproteins and Thylakoid Lipids

An autoradiogram of 35S-labeled thylakoid membrane pro-teins, taken at various time intervals after the light transition,reveals an increase in the fraction of newly synthesized poly-peptides in the range of 24 to 31 kDa (Fig. 7). These heavilylabeled bands were challenged with polyclonal antibodies toLHCII, and the blot was visualized using the peroxidasemethod. The polypeptides corresponding to LHCII apopro-teins comigrated with heavily labeled bands and were verifiedby two-dimensional gel electrophoresis (data not shown).These results suggest that a one-step transition from high tolow irradiance triggers a rapid synthesis in the major lightharvesting antenna complex, over the first 18 h, followed bya decrease in synthesis after 24 h. The synthesis ofthe reaction

Figure 7. Synthesis of thylakoid proteins following a shift from HL toLL. Lane 1 is nitrocellulose transfer decorated with LHCII antibodiesand visualized with peroxidase. Lanes 2 to 6: autoradiograms ofnitrocellulose transfers showng 35S incorporation into proteins at theinitial time for cells grown at 700 /smol quanta m-2 S-1 (lane 2), and 6h (lane 3), 12 h (lane 4), 18 h (lane 5), and 24 h (lane 6) after atransition to 70 Mimol quanta m-2 s-1. The four LHCII apoproteinswere identified by incubation of the nitrocellulose with polyclonalantibodies raised against pea LHCII (lane 1). Antibodies to D1, CP45,and CP47 (data not shown) were used to identify these respectiveproteins on the autoradiograms. Each lane in the autoradiogram wasloaded with an equal amount of radioactivity. Synthesis of all fourLHCII apoproteins (indicated with arrows) increased over the first 18h and then gradually decreased as cells approached a new steadystate.

center protein Dl initially lagged behind that of LHCII, butcontinued at a higher rate after 24 h.The fraction of total 14C incorporated into lipids rapidly

declined, and subsequently recovered to a new steady staterate (Fig. 6). The rate of lipid biosynthesis was calculatedfrom net photosynthesis and the percentage of carbon assim-ilated into lipids. An immediate 10-fold decrease in the rate

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Plant Physiol. Vol. 92, 1990

of lipid biosynthesis was calculated for cells after the lighttransition. This drastic reduction was followed by a gradualincrease in lipid biosynthesis until a new steady state rate,greater than the one measured in HL conditions, was estab-lished. A "4C labeling study of the lipid fraction indicated aninitial decrease in the biosynthesis of MGDG and DGDG(Fig. 8). The synthesis ofMGDG and DGDG increased 24 hafter the light transition and rapidly achieved a new steadystate rate that was about 2-fold greater than in the high lightconditions (Fig. 8).

Kinetic Analyses

Changes in several cellular parameters and physiologicalprocesses were fitted by nonlinear, least squares regressionanalysis to a first order kinetic model:

A, = (Ao - A.)ek + A.

where A, is a process or variable at time t; Ao and A. are theinitial and final levels of a process or a variable respectively;k, is the first order rate constant. This first order kineticanalysis was found to fit data better than an exponentiallogistic model (sigmoid curve).The calculated first order rate constants for changes in

various pools of the photosynthetic apparatus, photosyntheticparameters, and cellular doubling rate are given in Table I fora high to low light transition. The kinetic results show thatthe fastest cellular response to a shift down in light intensityis a drop in the growth rate (k = 0.130 h-'), followed bychanges in the cellular content of Chl a, Chl b, and LHCIIapoproteins (ks varying between 0.043 and 0.053 h-'). Inter-estingly, the change in cellular content of carotenoids wasfaster than that of other pigments with a rate constant of0.063 h-'. The change in MGDG, with a rate constant of0.022 h-', was slower than the other biochemical parametersexamined (Table I).Changes in several components of the photosynthetic ap-

paratus diverge from the first order kinetics. For examplePSUo2 size significantly overshot the final steady state value(Fig. lc). While the accumulation of RCII cell could bedescribed by a first-order model, the data suggests a slight lagphase following the light transition. In addition, the cellularcontent of Rubisco was not affected by the change in theirradiance level (data not shown).The photosynthetic characteristics of D. tertiolecta were

directly affected by changes in the photosynthetic apparatus.The minimal quantum requirement, 1/Amax, was reduced witha rate constant of 0.072 h-', and the maximal photosyntheticcapacity per unit Chl a was reduced at a slower rate (k =0.038 h-') (Table I). Maximal cellular photosynthetic capac-ity, Pmax/cell (data not shown) was unchanged.

DISCUSSION

Photoadaptation in unicellular algae can be interpreted interms of cellular energy balance (29). In D. tertiolecta, a one-step transition from HL to LL imposes an immediate ener-getic problem. The growth rate promptly decreases, but doesnot completely stop. Some external energy is still availableand can be used, though initially it is used with a relatively

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Figure 8. Autoradiogram of a thin layer chromatogram of 14C-labeledtotal lipids extracted from D. tertiolecta before and at indicated timeintervals after a shift from HL to LL. Labeling of lipids and extractionprocedures are described in "Materials and Methods." Each lane wasloaded with equal levels of radioactivity. N.L. = neutral lipid.

low efficiency, as indicated by the high quantum requirement.Furthermore, internal energy stores such as carbohydrates(28) and storage lipids (i.e. triacylglycerol) are consumed tosupport growth (A Sukenik, unpublished data).The photoadaptation process is associated with changes in

cellular carbon partitioning. Upon a transition from high tolow photon flux densities, the net carbon assimilation rate ispromptly reduced and initially less carbon flows into lipids(Fig. 6). At the same time, carbon assimilation into proteinsincreases and supports de novo synthesis of proteins thatincrease light utilization. Proteins such as LHCII apoproteins

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SUKENIK ET AL.896

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PHOTOADAPTATION OF THE PHOTOSYNTHETIC APPARATUS

Table I. Calculated First Order Rate Constants and Half-TimeValues for Changes of Various Physiological and BiochemicalParameters during One-Step Transition in Growth Irradiance Level(700-70 imol quant m-2 s-1) in D. tertiolecta

Values given in parenthesis are the correlation coefficients for alinear regression of log transformed data for the first order rateconstants and the 95% confidence interval for the half time values.

Transition Half-TimeParameter Rate2) (h; 95%CI)

(h'1 r)(h95C)Doubling rate 0.130 (0.915) 5.3 (2.7 to 9.4)ChI a/cell 0.053 (0.967) 13.1 (10.3 to 17.8)Chi b/cell 0.043 (0.995) 16.1 (12.2 to 23.9)Carot/cell 0.063 (0.944) 1 1.0 (8.2 to 16.5)LHCII/cell 0.052 (0.978) 13.3(11.2 to 16.5)RCII/cell 0.028 (0.894) 24.8 (18.2 to 38.5)Pm. (per unit Chi a) 0.038 (0.997) 18.2 (16.1 to 21.0)yo/fw, 0.072 (0.958) 9.6 (7.2 to 14.4)MGDG/cell 0.022 (0.967) 31.5 (24.8 to 38.5)

STAGE RCII LHCIII nV-Am lt 0k 0,A-

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Figure 9. Schematic presentation of the changes in the areal densityand relative size of PSII in thylakoid membranes in D. tertiolecta ascells shade adapt. High light adapted cells are characterized by fewthylakoid membranes and a relatively low concentration of PSII re-action centers (stage 1). When these cells are exposed to a one-steptransition to low photon flux density, light harvesting antenna com-ponents are rapidly synthesized increasing light harvesting capability(stage 2). This is associated with a relatively slower synthesis of PSIIreaction centers (stage 3). The PSII units undergo a reorganizationwith LHCII accompanied by a synthesis of thylakoid membranes(stage 4).

are rapidly accumulated in response to the light transitionchange at a slower rate or even remain unchanged.

Based on the results presented here and elsewhere (9, 31,32), we propose a schematic model which describes the re-

sponses of the thylakoid membrane to a downshift in light(Fig. 9). A change from high to low irradiance leads to thefollowing sequence of biosynthetic processes: (a) a transientdecrease in lipid synthesis followed by (b) a rapid increase inChl a, b, carotenoids and LHCII apoproteins, forming LHCII.(c) The newly synthesized LHCII temporarily associates withextant complexes, thereby leading to a transient increase in

the physical as well as functional cross-section of PSII. (d)The synthesis of LHCII is accompanied by a change in thesubpopulation ofthe four apoproteins which constitute LHCII(31) (see also Fig. 7). (e) There is a net synthesis of both PSIIand PSI (8) reaction centers at a slower rate, which (f) func-tionally associate with their respective LHCs. We note that inthis clone of Dunaliella there is no change in the ratio ofPSII/PSI reaction centers between HL and LL cells (12, 30).(g) Finally synthesis of reaction centers is accompanied by thesynthesis of structural lipids to increase the surface density ofthylakoid membranes, such that the average intermoleculardistances between reaction centers remains relatively constant(30). Interestingly, a transfer from light to dark (as opposedto low light), does not lead to a net synthesis of LHCII (27).

This sequence differs qualitatively from that found duringthe light-dependent development of the photosynthetic ap-paratus in etiolated chloroplasts of higher plants (15, 25, 34)and in greening mutants of chlorophyte algae (16), wherereaction centers are synthesized prior to light harvesting. Thedifference between the sequence observed during photoadap-tation in D. tertiolecta and chloroplast development suggeststhat photoreceptors which trigger the cascade of biosyntheticprocesses observed during photoadaptation differ from thosewhich control ontology. This difference is not surprising giventhat photoadaptation processes occur in mature cells withfunctional electron transport systems, while etiolated chloro-plasts must synthesize electron transport systems prior to lightharvesting systems in order to maximize photosynthetic en-ergy conversion.On a functional level the increases in both thylakoid mem-

brane proteins and lipids, leads to membrane stacking (3). InHL cells on average, two thylakoid membranes constitute astack, while in LL cells this number doubles. Increased stack-ing of thylakoids leads to decrease in the optical absorptioncross-section of the pigments, due to the package effect (3, 6,1 1), which normally would lead to a larger quantum require-ment. Concurrently however, there is an increase in absorp-tion cross-section of PSII. This latter phenomenon occurs,not simply by addition of LHCII, but by changes in thepigment composition ofLHCII (32). The minimum quantumrequirement can be defined as the ratio of functional PSIIcross-section to the optical absorption cross-section (24). Thetwofold decrease in l/Om. observed following the light shift,suggests that the functional cross-section of PSII increasedtwice as much as the optical absorption cross-section.

In spite of large increases in the abundance of light har-vesting pigment protein complexes following a reduction ingrowth irradiance, the cellular content of Rubisco remainedunchanged, implying that Rubisco does not serve as storagefunction for de novo protein synthesis. The ratio of RubiscoPSII reaction centers is linearly correlated with the maximalrate (1/i) ofphotosynthetic electron flow from water to carbondioxide in D. tertiolecta in steady-state growth (30). Ourresults suggest that this correlation also holds during a tran-sient photoadaptive period.The transduction mechanism that relates a change in pho-

ton flux density, to a message that causes a cascade of biosyn-thetic processes, and results in a photosynthetic apparatusthat is adapted to a new light intensity, remains unclear. In

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higher plants, light regulation of Rubisco has been associatedwith phytochrome (34), a system which has not been con-

vincingly demonstrated in unicellular algae. Photoregulationof LHCII has been demonstrated at the transcriptional (20),translational (12), and posttranslational (22) levels, and hasbeen associated with phytochrome (15, 34) as well as theprotochlorophyllide holochrome (BA Mortain, J Bennett, PGFalkowski, unpublished data). Our results suggest that in D.tertiolecta Rubisco synthesis is not regulated by light intensityper se, but LHCII is. These results clearly suggest that thelight transduction mechanism for these two protein complexesis different (2).A basic regulatory model that is controlled by the cellular

energetic status has been suggested for the accumulation ofChl in low light adapted cells (7). However, we have no

indication that such a metabolic model is applicable to othercomponents of the photosynthetic apparatus. Several studieshave suggested a posttranslational mechanism for the regula-tion of LHCII accumulation in higher plants (22, 25). Ac-cording to this mechanism, pigments are required for stabili-zation of the newly synthesized apoproteins from the existingmessenger. Studies on intermittently illuminated plants andon a Chl b-less mutant of barley have led to the concept thatLHCII apoproteins are degraded in the absence of Chl b.However, when Chl a biosynthesis is blocked in Dunaliellafollowing a HL to LL shift, LHCII apoproteins continue tobe synthesized and are relatively stable (BA Mortain, J Ben-nett, PG Falkowski, unpublished data). These results suggestthat the low light-dependent synthesis of LHCII apoproteinsin D. tertiolecta is translationally or transcriptionally con-trolled. Preliminary results with D. tertiolecta indicate thatthe LHCII messenger level increases several-fold within 9 hfollowing a downshift in light intensity (J LaRoche, unpub-lished results).

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