Excitation energy partitioning and quenching during cold acclimation in Scots pine

Sum mary We stud ied the in flu ence of two ir radi an ces oncold ac cli ma tion and re cov ery of pho to syn the sis in Scots pine(Pinus sylvestris L.) seed lings to as sess mech a nisms for quen -ch ing the ex cess en ergy cap tured by the photosynthetic ap pa ra -tus. A shift in tem per a ture from 20 to 5 °C caused a greater de -crease in photosynthetic ac tiv ity, mea sured by chlo ro phyllflu o res cence and ox y gen evo lu tion, in plants ex posed to mod -er ate light (350 µmol m – 2 s –1) than in shaded plants (50 µmolm – 2 s –1). In re sponse to the tem per a ture shift, max i mal pho to -chem i cal ef fi ciency of photosystem II (PSII), mea sured as thera tio of vari able to max i mal chlo ro phyll flu o res cence (Fv/Fm)of dark-adapted sam ples, de creased to 70% in ex posed seed -lings, whereas shaded seed lings main tained Fv/Fm close to ini -tial val ues. Af ter a fur ther tem per a ture de crease to –5 °C, only8% of ini tial Fv/Fm re mained in ex posed plants, whereas sha -ded plants re tained 40% of ini tial Fv/Fm. Seven days af ter trans -fer from –5 to 20 °C, re cov ery of pho to chem i cal ef fi ciency was more com plete in the shaded plants than in the ex posed plants(87 and 65% of the ini tial Fv/Fm value, re spec tively).

In re sponse to cold stress, the es ti mated func tional ab sorp -tion cross sec tion per re main ing PSII re ac tion cen ter in creased at both ir radi an ces, but the in crease was more pro nounced inex posed seed lings. Es ti mates of en ergy par ti tion ing in theneedles showed a much higher dissipative com po nent in theexpoesd seed lings at low tem per a tures, point ing to stron gerdevelop ment of non-pho to chem i cal quench ing at mod er ate ir -ra di an ces. The de-epoxidation state of the xanthophyll cy clepig ments in creased in ex posed seed lings at 5 °C, con trib ut ingto the quench ing ca pac ity, whereas sig nif i cant de-epoxidationin the shaded plants was ob served only when tem per a tures de -creased to –5 °C. Thermoluminescence (TL) mea sure ments of PSII re vealed that charge recombinations be tween the sec ond

ox i da tion state of Mn-clus ter S2 and the semireduced sec ond -ary elec tron ac cep tor quinone QB

– (S 2QB–) were shifted to

lower tem per a tures in cold-ac cli mated seed lings com paredwith con trol seed lings and this ef fect de pended on irradiance.Con com i tant with this, cold-ac cli mated seed lings dem on stra -ted a sig nif i cant shift in the S2 re com bi na tion with pri mary ac -cep tor QA

– (S 2QA–) char ac ter is tic TL emis sion peak to higher

tem per a tures, thus nar row ing the re dox po ten tial gap be tweenS 2QB

– and S 2QA–, which might re sult in in creased prob a bil ity

for non-ra di a tive rad i cal pair re com bi na tion betweem the PSIIre ac tion cen ter chlo ro phyll a (P680+) and QA

– (P680+QA–)

(re ac tion cen ter quench ing) in cold-ac cli mated seed lings. InScots pine seed lings, mech a nisms of quench ing ex cess lighten ergy in win ter there fore in volve light-de pend ent reg u la tionof re ac tion cen ter con tent and both re ac tion cen ter-based andan tenna-based quen ch ing of ex cess light en ergy, en ablingthem to with stand high ex ci ta tion pres sure un der north ernwin ter con di tions.

Keywords: an tenna quench ing, cold stress, elec tron trans port,Pinus sylvestris, re ac tion cen ter quench ing.

In tro duc tion

North ern win ter cli mate im poses a dual stress on ev er greenplants: not only is met a bolic ad just ment to low tem per a tures in green tis sues re quired, but re ar range ment of the photo syn -thetic ap pa ra tus to with stand high ir radi an ces ab sorbed in ex -cess of bio chem i cal con sump tion is also nec es sary (Öquist and Huner 2003). The long-term (i.e., weeks) re sponse of co ni fersto low tem per a tures con sists of two ma jor phases: frost hard -en ing, which oc curs at tem per a tures slightly above zero and

Tree Phys i ol ogy 26, 325–336© 2006 Heron Pub lishing—Vic to ria, Can ada

Excitation energy partitioning and quenching during cold acclimationin Scots pine

DMITRY SVESHNIKOV,1–3 INGO ENSMINGER,4–6 ALEXANDER G. IVANOV,5 DOUGLASCAMPBELL,7 JON LLOYD,4,8 CHRISTIANE FUNK,2 NORMAN P. A. HÜNER5 and GUNNARÖQUIST1

1 Department of Plant Physiology, University of Umeå, S-901 87 Umeå, Sweden2 Department of Biochemistry, University of Umeå, S-901 87 Umeå, Sweden3 Corresponding author ([email protected])4 Max-Planck-Institut für Biogeochemie, Postfach 100164, 07701 Jena, Germany5 Department of Biology and Biotron, University of Western Ontario, London ON, N6A 5B7, Canada6 Max-Planck-Institut für Molekulare Pflanzenphysiologie, 14424 Potsdam, Germany7 Mount Allison University, Sackville NB, E4L 1G7, Canada8 School of Geography, University of Leeds, West Yorkshire, LS2 9JT, U.K.

Re ceived De cem ber 20, 2004; ac cepted Au gust 5, 2005; pub lished on line De cem ber 15, 2005

does not af fect the ef fi ciency of pho to syn the sis un der light- lim ited con di tions, and win ter stress, which oc curs at subzerotem per a tures and is char ac ter ized by sup pres sion of photo -synthetic ef fi ciency be cause of deeper changes within thephoto syn thetic ap pa ra tus (Öquist et al. 1980).

Cold ac cli ma tion in co ni fers in au tumn is trig gered by de -creas ing day length and tem per a ture and in volves down-reg u -la tion of pho to syn the sis, in duc tion of pro tec tive pro teins andpig ments and ad just ments of an tenna size and com po si tion tofa vor de creased light ab sorp tion and in creased dis si pa tion ofex cess en ergy by non-pho to chem i cal quench ing (NPQ) or bydi vert ing ab sorbed en ergy from photosystem II (PSII) func -tion to photosystem I (PSI) (Huner et al. 1998, Ivanov et al.2001, 2002, Savitch et al. 2002, Öquist and Huner 2003).

Af ter the ini tial cold-stress re sponse, pro tec tive mech a -nisms are de vel oped dur ing the cold ac cli ma tion pro cess.Frost- hard ened nee dles ex hibit lower photosynthetic ac tiv ityat mod er ately low tem per a tures be cause of loss and pho to -chem i cal down-reg u la tion of PSII re ac tion cen ters (Ottanderet al. 1995). The im bal ance be tween the ab sorbed en ergy andmet a bolic ac tiv ity re sults in ex cess ex ci ta tion pres sure andphoto inhibition of PSII (Strand and Öquist 1985a, 1985b,Huner et al. 1998, Öquist et al. 2001). High ir radi an ces en -hance the neg a tive ef fect of low tem per a tures on pho to syn the -sis (Vogg et al. 1998, Yamazaki et al. 2003, Ensminger et al.2004), whereas at mod er ate ir radi an ces photosynthetic ca pac -ity is less af fected by low tem per a tures (Ad ams et al. 2002).

In con trast to de cid u ous trees, ev er greens re tain sig nif i cantamounts of chlo ro phyll (Chl) dur ing win ter. In ad di tion, theamount of light-har vest ing Chl a/Chl b pro tein com plexes isrel a tively re sis tant to win ter stress in Scots pine (Öquist andMar tin 1980), fa cil i tat ing rapid re cov ery of pho to syn the sis inspring (Ottander et al. 1995, Ensminger et al. 2004). To pro tect the win ter-pre served Chl a/Chl b pro tein com plexes in Scotspine nee dles from photooxidation, a high and sus tained ca pac -ity for NPQ of light ab sorbed in ex cess of bio chem i cal con -sump tion de vel ops and is as so ci ated with sus tained high con -cen tra tions of xanthophyll cy cle pig ments in a lowepoxi da tion state (Ad ams and Demmig-Ad ams 1994,Ottander et al. 1995, Krivo sheeva et al. 1996, Savitch et al.2002). Ac cu mu la tion of lutein has also been re ported inoverwintering plants (Tausz et al. 1999, 2001, Matsubara et al.2003). Re cently, an in creased prob a bil ity for non-ra di a tivecharge re com bi na tion within the PSII re ac tion cen ter (re ac tion cen ter quench ing) was sug gested as an ad di tional mech a nismfor dis si pa tion of ex cess light in over wintering ev er greens(Ivanov et al. 2002, 2003, Öquist and Huner 2003).

Irradiance also af fects the re cov ery of pho to syn the sis in ev -er greens af ter cold stress, which is im por tant both dur ingwarm spells in win ter (Vogg et al. 1998) and dur ing spring(Ensminger et al. 2004). Un der lab o ra tory con di tions, re cov -ery of PSII photochemistry oc curs rap idly when nee dles aresub jected to mod er ate light and tem per a ture (Pharis et al.1970, Neilson et al. 1972, Ottander and Öquist 1991, Ivanov etal. 2001). How ever, un der field con di tions, the light-ex posedspruce and pine nee dles dem on strate slower re cov ery fromwin ter stress than shaded nee dles, prob a bly be cause of the

com bined ef fects of much higher ir radi an ces un der cloud lessskies to gether with cool or freez ing tem per a tures at night(Bauer et al. 1975, Lundmark and Hallgren 1987, 1988, Strand and Lundmark 1987, Ensminger et al. 2004).

Thus, the ev i dence in di cates that the harm less quench ing ofen ergy ab sorbed in ex cess by win ter-ac cli mated nee dles ofScots pine is an im por tant photoprotective mech a nism. Thispro cess is ev i dently ac com plished through mul ti ple dissi pa -tive path ways. To gain fur ther in sight into these accli ma tionalchanges, Scots pine seed lings grown in con trolled cli matecham bers under low or mod er ate irradiance (50 and 350 µmolpho tons m – 2 s – 2) and sub jected to cold ac cli ma tion and freez -ing fol lowed by re cov ery, were mon i tored to determine: (1)the ef fect of irradiance on pho to syn the sis at low and freez ingtem per a tures; (2) the ef fect of growth irradiance on the par ti -tion ing and quench ing of ab sorbed light en ergy; and (3) therel a tive con tri bu tions of an tenna quench ing and re ac tion cen -ter quench ing to photo-pro tec tion of PSII at low tem per a tures.

Ma te ri als and meth ods

Plant material

Scots pine (Pinus sylvestris L.) seeds were ger mi nated on wetver mic u lite at a day/night tem per a ture of 25/15 °C, 75% hu -mid ity and a 16-h photoperiod. Af ter 2 weeks, the seed lingswere trans ferred to liq uid cul ture on dense foam rub ber(Öquist et al. 1980) and cul tured for 6 weeks un til the pri marynee dles were de vel oped. The seed lings were then grown for12 months in a green house at 20 °C at an irradiance of350 µmol m – 2 s –1 in a 16-h photoperiod.

Experimental design

One-year-old seed lings were trans ferred from the green houseto a growth cab i net (2.5 × 2 × 2 m high), here af ter re ferred to as the con trol growth cham ber, set to pro vide a tem per a ture of20 °C and an irradiance of 350 µmol m – 2 s –1 dur ing a 16-hphotoperiod. Il lu mi na tion was pro vided by eight metal ha lidelamps (HQI-BT 400W day light) mounted above the trans par -ent ceil ing. Rel a tive hu mid ity was main tained at 75%. Eigh -teen seed lings were as signed to the ex posed treat ment andplaced 70 cm from the lamps, re sult ing in an irradiance atseed ling height of 350 µmol m – 2 s –1. The re main ing 18 seed -lings were as signed to the shaded treat ment and po si tioned140 cm from the lamps and ad di tion ally shaded with two lay -ers of metal mesh of 1 mm cell size, re sult ing in an irradianceat seed ling height of 50 µmol m – 2 s –1. Plants were ro tateddaily to en sure a uni form growth irradiance.

Af ter the seed lings had ac cli mated to the con trol growthchamber con di tions, they were trans ferred to treat ment growth cham bers that con sec u tively pro vided an au tumn-like en vi -ronment (15/10 °C, 8-h photoperiod), cold hard en ing (5 °C,8-h photoperiod) and win ter-like freez ing (–5 °C, 8-h photo -period) while main tain ing the seed lings in their re spec tivemod er ate and low irradiance treat ments. Con di tions in thetreat ment growth cham bers were set and equil i brated be forethe seed lings were trans ferred. For the re cov ery phase, theseed lings were re turned to the con trol growth cham ber (20 °C,

326 SVESHNIKOV ET AL.

TREE PHYS I OL OGY VOL UME 26, 2006

16-h photoperiod). Seed lings were kept at each tem per a turefor at least 3 weeks, ex cept dur ing the re cov ery phase, whichlasted only 7 days. Sam pling was per formed in the morn ings,just be fore the light was turned on. Each pooled sam ple con -sisted of two to four healthy nee dles taken from cur rent-yearshoots of each of four to six plants cho sen con sec u tively fromeach group.

Photosynthetic pigments

About six pairs of nee dles (2.5 g fresh mass) were taken fromthe stem above the first node of each of five seed lings, frozenin liq uid N2 and stored at –80 °C. Later on, sam ples wereground in liq uid N2 and freeze-dried. Pig ments were ex tractedfrom the nee dle pow der in ac e tone (100%) buf fered withNaHCO3 for 2 h at 4 °C in the dark. Pig ments were sep a ratedon a re versed phase C-18 col umn (Knaur, Berlin, Ger many) by high per for mance liq uid chro ma tog ra phy (HPLC) (Xyländeret al. 1996). To tal chlo ro phyll and to tal ca rot en oids were es ti -mated spec tro pho to met ri cally (Lichtenthaler 1987).

Chlorophyll fluorescence and photosynthetic oxygenevolution

Mea sure ments of ox y gen evo lu tion (Clark-type elec trode,Han satech, Nor folk, U.K.) and pulse-mod u lated chlo ro phyllflu o res cence of PSII (PAM-101, Heinz Walz GmbH, Effel -trich, Ger many) were re corded si mul ta neously (Walker 1990,En sming er et al. 2004). For each mea sure ment, freshly col -lected nee dles were aligned side by side on ad he sive tape and a 2-cm di am e ter disk was ex cised. The disk was placed in anLD2/3 leaf cham ber (Hansatech) at 20 °C with 100% in ter nalhu mid ity and in cu bated in the dark for 20 min. The PSII ac tiv -ity was mon i tored as the ra tio of vari able (Fv) to max i mum(Fm) chlo ro phyll flu o res cence (Fv/Fm = (Fm – Fo) /Fm, whereFo is the min i mum yield of chlo ro phyll flu o res cence at openPSII cen ters) of dark-adapted sam ples (Schreiber et al. 1994).The nee dles were then ex posed to a se ries of 5-min ex po suresto ac tinic light of in creas ing pho ton den sity, each fol lowed bya 15-s pulse of weak far-red light from a light- emit ting di ode(LED) source (102FR, Heinz Walz GmbH) to de ter mine themin i mum flu o res cence at open PSII cen ters dur ing il lu mi na -tion (Fo′). The cor re spond ing max i mum flu o res cence value atclosed PSII cen ters (Fm′) was mea sured at the end of each ac -tinic light ex po sure af ter ap ply ing a sat u rat ing light pulse. Par -ti tion ing of light en ergy ab sorbed by PSII was es ti matedas sug gested by Demmig-Ad ams et al. (1996), by mea sur -ing pho to chem i cal uti li za tion of the ex ci ta tion en ergy (P) as(Fv′/Fm′)qp (Fv′ = Fm′ – Fo′ , vari able flu o res cence dur ing il lu -mi na tion; qp = (Fm′ – F )/(Fm′ – Fo′), pho to chem i cal quench -ing co ef fi cient; F = steady state flu o res cence yield) ther maldis si pa tion (D) as 1 – Fv′/Fm′, and the ex cess en ergy (E), po -ten tially dam ag ing for the PSII, as (Fv′/Fm′)(1 – qp)(Demmig-Ad ams et al. 1996, Kato et al. 2003, Kornyeyev etal. 2003). The dis tri bu tion of the ab sorbed en ergy in nee dles of ex posed and shaded seed lings was cal cu lated from the data ob -tained at the cor re spond ing growth irradiance (i.e., 350 or50 µmol m – 2 s –1).

Estimation of PSII and PSI functional absorption crosssections

Nee dle disks (12 mm in di am e ter) were dark-adapted for atleast 30 min at the growth tem per a ture. The disks were thenplaced in a cuvette (mod i fied Hansatech leaf-disk cuvette)main tained at 20 °C with 100% in ter nal hu mid ity. The Fo chlo -ro phyll flu o res cence was re corded with a PAM-101 pulse-mod u lated fluorometer, op ti cally cou pled to the cuvette with afi ber op tic. The 655 nm mea sur ing beam was mod u lated at100 kHz with a mean in ten sity of 1.66 µmol m – 2 s –1 at the nee -dle disk sur face. We then ap plied a se ries of five sin gle-turn -over flashes (1.5 µs peak width at 50% of max i mum) with axe non-dis charge flash lamp (XST103, Heinz Walz GmbH).The flu o res cence trace was cap tured by a PC-based data ac qui -si tion sys tem (In sight V 3.0, Intelligente Mess technik GmbH,Backnang, Ger many) at a rate of 40 points per µs. This pro cesswas re peated over a se ries of in creas ing flash in ten si ties from0.12 to 23.6 pho tons nm – 2 flash –1. Fi nally, each leaf was ex -posed to a mul ti ple-turn over sat u rat ing flash of 8700 µmolpho tons m – 2 s –1 for 1–2 s (KL1500, Heinz Walz GmbH).

The traces were plot ted and smoothed by ad ja cent averaging over a three-point win dow us ing the Or i gin soft ware pack age(Ori gin Lab, Northampton, MA), and mean vari able flu o res -cence in duced by each sin gle-turn over flash in ten sity was ex -tracted from the trace. The vari able flu o res cence val ues werenor mal ized to the max i mal vari able flu o res cence in duced bythe mul ti ple-turn over flash and plot ted against flash in ten sity.Func tional ab sorp tion cross sec tion per PSI was as sessedthrough changes in absorbance at 820 nm (∆A820) mea suredwith an ED800T Walz emitter/de tec tor. The de creases in A820

were nor mal ized to the max i mal value ob tained by the mul ti -ple- turn over flash and plot ted against flash in ten sity. Theslopes of the ex po nen tial curves of max i mal vari able flu o res -cence and ∆A820 against flash in ten sity rep re sent the func -tional ab sorp tion cross sec tions for PSII and PSI, re spec tively(Mauzerall and Greenbaum 1989). Sig nif i cance of dif fer encesbe tween the 20 and 5 or –5 °C treat ments was es ti mated byone-way anal y sis of vari ance (ANOVA) fol lowed by pairwisemul ti ple com par i sons us ing Scheffe’s method (Neter et al.1996) as im ple mented in the Or i gin soft ware pack age.

Thermoluminescence measurements

Thermoluminescence (TL) of in tact Scots pine nee dles wasmea sured at a heat ing rate of 0.6 °C s –1 with a TL data ac qui si -tion and anal y sis sys tem (Ivanov et al. 2001). Sam ples of nee -dle discs (2 cm in di am e ter) com posed of nee dles from threeplants were used for all mea sure ments. A flash lamp as sem bly(Type FX200, EG&G Elec tro Op tics, Sa lem, MA) was used toex pose the sam ples to two sin gle-turn over flashes (2.5 µs halfband width at 10 Hz fre quency). Dark-adapted nee dle discs(10 min in dark ness at 20 °C) were cooled to 0 °C be fore ex po -sure to the flashes. Af ter the flash, sam ples were quicklycooled in liq uid ni tro gen. For S 2QA

– re com bi na tion stud ies,nee dles were vac uum in fil trated with 20 µM 3-(3′,4′-di chloro -phenyl)-1,1dimethylurea (DCMU) in dark ness be fore flash il -lu mi na tion. De com po si tion anal y sis of the TL glow curves wasmade with a nonlin ear, least squares al go rithm that min i mizes

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EN ERGY PAR TI TION ING AND QUENCH ING IN SCOTS PINE 327

the χ2 func tion us ing the Or i gin software pack age. The no men -cla ture of Vass and Govindjee (1996) was used to char ac ter izethe TL glow peaks.

Statistical analysis

The Or i gin soft ware pack age Ver sion 6.0 was used for allgraphics and sta tis ti cal cal cu la tions.

Re sults

Responses of exposed and shaded seedlings to changes ingrowth temperature

Photosynthetic ef fi ciency The con trol plants had Fv/Fm val -ues of about 0.8, dem on strat ing a good func tional state of PSII(Ta ble 1). At 5 °C, Fv/Fm of ex posed nee dles dropped to 70% of the con trol value, whereas Fv/Fm of shaded nee dles re mained at not less than 95% of the con trol value. When seed lings weretrans ferred to –5 °C, Fv/Fm in the ex posed seed lings droppedfur ther to only 5% of the con trol value, whereas Fv/Fm ofshaded seed lings was main tained at about 40% of the con trolvalue. Re cov ery of Fv/Fm was slower and less com plete in ex -posed seed lings than in shaded seed lings (78 ver sus 90% of thecon trol value af ter 7 days of re cov ery).

Photosynthetic ca pac ity of the nee dles was as sessed by thelight re sponse of ox y gen evo lu tion mea sured at 20 °C. There -fore, this pa ram e ter does not in di cate the achieved photo -synthetic ac tiv ity of the nee dles un der growth con di tions, butrather the po ten tial ac tiv ity, re flect ing the func tion al ity of thephotosynthetic ma chin ery in gen eral, and PSII cen ters in par -tic u lar. Nee dles of the ex posed and shaded seed lings showedsim i lar pro gres sive in hi bi tion of ox y gen evo lu tion rate fol low -ing ac cli ma tion to lower tem per a tures. Ex posed and shadedseed lings showed sim i lar rates of ox y gen evo lu tion at growthtem per a tures of 20 and 5 °C; how ever, at –5 °C, the rate of ox -y gen evo lu tion in ex posed seed lings was com pletely sup -pressed, whereas it re mained de tect able in shaded seed lings(Fig ure 1, up per panel).

En ergy par ti tion ing Pho to chem i cal ef fi ciency of open PSIIcen ters, mea sured as Fv′/Fm′ at 20 °C (Fig ure 1, lower panel),de creased in ex posed seed lings fol low ing trans fer to 5 °C, withlit tle ac tiv ity de tect able at –5 °C. In con trast, PSII photo -

chemical ef fi ciency in shaded seed lings did not change un tilthe tem per a ture fell to –5 °C.

Es ti mates of the par ti tion ing of light en ergy ab sorbed byPSII (Fig ure 2) in di cated a no tice able shift of en ergy frompho to chem i cal uti li za tion to dis si pa tion fol low ing trans fer ofex posed seed lings to 5 °C. A fur ther de crease in tem per a ture to –5 °C re sulted in com plete loss of the frac tion of light usedpho to chem i cally with vir tu ally all en ergy ab sorbed in ex posed seed lings be ing dis si pated as heat. In the shaded plants, en ergy par ti tion ing did not change on trans fer to 5 °C, but the freez ing treat ment re sulted in a strong de crease in photochemistry withcon sid er able gain in ther mal dis si pa tion. Af ter 7 days of re -cov ery at 20 °C, the pho to chem i cal frac tion in shaded plantswas re stored al most to the ini tial value, whereas in the ex posed plants, photochemistry reached only about 30% of the ini tialvalue. The ex cess en ergy in ex posed plants de creased with de -creas ing tem per a ture and com prised about 12% of the to tal en ergy ab sorbed at the be gin ning of the re cov ery pe riod and18% of the to tal en ergy ab sorbed at the end of the re cov ery pe -riod. In shaded plants, ex cess energy was al most com pletelyquenched at all tem per a tures.

An tenna cross sec tion Func tional ab sorp tion cross sec tionper re ac tion cen ter was es ti mated from PSII vari able fluor -escence (Fv′ ) or PSI absorbance re sponses (Mauzerall andGreen baum 1989). Val ues of Fv′ plot ted against in creas ingflash in ten sity (Fig ure 3) were lower in ex posed nee dles than in shaded nee dles at all tem per a tures, con firm ing that PSII wasdown-reg u lated in the ex posed plants. Ef fec tive cross sec tion(SigmaPSII) val ues ex tracted from the plots (Ta ble 2) re vealedthat at 20 °C, PSII func tional ab sorp tion cross sec tions weresmaller in ex posed nee dles than in shaded nee dles. A de creasein tem per a ture re versed the sit u a tion: at 5 °C, ex posed nee dlesdem on strated an in crease in PSII func tional ab sorp tion crosssec tions serv ing their re main ing PSII cen ters, whereas nochanges were ob served in shaded nee dles at 5 °C. A tem per a -ture of –5 °C caused a large drop in Fv′ in ex posed plants, so thenu meric es ti ma tion of PSII func tional ab sorp tion cross sec -tions per PSII be came prob lem atic, but nev er the less gave highval ues (data not pre sented). The small re sid ual Fv′ in ex posedplants at –5 °C was nearly sat u rated by flashes of low pho tondose (Fig ure 3), in di cat ing large ef fec tive an tenna cross sec -



Ta ble 1. Photosynthetic char ac ter is tics of ex posed (350 µmol m– 2 s–1) and shaded (50 µmol m– 2 s–1) nee dles of Scots pine seed lings sub jected tothe fol low ing treat ments: con trol (20 °C, 3 weeks), cold ac cli ma tion (5 °C, 3 weeks), freez ing stress (–5 °C, 3 weeks) and re cov ery from freezingstress (20 °C, 7 days). Max i mal pho to chem i cal ef fi ciency (Fv/Fm) of photosystem II was cal cu lated from flu o res cence mea sure ments made at20 °C. Chlo ro phyll (mg g DM

– 1 ) and carotenoid (mg g DM– 1 ) con cen tra tions were de ter mined in 80% ac e tone ex tracts of freeze-dried nee dles. Each

value rep re sents the mean (± SE) of 3–5 rep li cates. Ab bre vi a tion: Chl = chlo ro phyll.

Pa ram e ter Con trol (20 °C) Cold ac cli ma tion (5 °C) Ar ti fi cial win ter stress (–5 °C) Re cov ery (20 °C)Ini tial ma te rial

Ex posed Shaded Ex posed Shaded Ex posed Shaded(Ex posed)

Fv/Fm 0.81 ± 0.01 0.57 ± 0.03 0.79 ± 0.03 0.04 ± 0.03 0.31 ± 0.04 0.63 ± 0.03 0.73 ± 0.03Chlorophyll 4.02 ± 0.17 1.38 ± 0.03 2.76 ± 0.10 1.18 ± 0.09 2.65 ± 0.21 1.34 ± 0.03 2.50 ± 0.22Carotenoid 0.98 ± 0.03 0.61 ± 0.01 0.80 ± 0.01 0.53 ± 0.01 0.80 ± 0.06 0.51 ± 0.04 0.79 ± 0.06Chl a/b 3.02 ± 0.09 2.86 ± 0.03 2.68 ± 0.06 2.66 ± 0.01 2.62 ± 0.06 2.21 ± 0.04 2.47 ± 0.02

tions rel a tive to the few re main ing ac tive re ac tion cen ters.Shaded nee dles also showed a larger PSII func tional ab sorp -tion cross sec tion at –5 °C than at higher tem per a tures (Ta -ble 2), but the in crease was less pro nounced than in ex posednee dles.

The es ti mated PSI func tional ab sorp tion cross sec tionshow ed lit tle dif fer ence be tween the ex posed and shaded seed -lings (Ta ble 2). Some in crease in SigmaPSI seemed to be pres -

ent in both groups of plants when tem per a tures were lowered,al though the es ti mated dif fer ences were sig nif i cant only forshaded plants at –5 °C (Ta ble 2).

Light energy quenching mechanisms

Pig m ents Thylakoid pig ments re sponded dif fer ently to chan -ges in growth tem per a ture. Ac cli ma tion to low tem per a turescaused a de crease in to tal chlo ro phyll and to tal ca rot en oids per



Fig ure 1. Light re sponsecurves of photosynthetic netox y gen evo lu tion (O2, up perpanels) and pho to chem i cal ef -fi ciency of open photosystemII cen ters (Fv′/Fm′, lowerpanels) in ex posed (350 µmolm– 2 s–1) and shaded (50 µmol m– 2 s–1) nee dles of Scots pine seed lings sub jected to the fol -low ing treat ments: con trol(20 °C, 3 weeks), cold ac cli -ma tion (5 °C, 3 weeks), freez -ing stress (–5 °C, 3 weeks)and re cov ery from freezingstress (20 °C, 7 days). Eachvalue rep re sents the mean re -sponse (± SE) of three disks of nee dles, one mea sure ment perdisk.

Fig ure 2. En ergy par ti tion ing inphotosystem II (PSII) in ex posed(350 µmol m– 2 s–1) and shaded(50 µmol m– 2 s–1) nee dles of Scotspine seed lings sub jected to the fol low -ing treat ments: con trol (20 °C,3 weeks), cold ac cli ma tion (5 °C,3 weeks), freez ing stress (–5 °C,3 weeks) and re cov ery from freezingstress (Rec; 20 °C, 7 days). Ab bre vi a -tions: P = frac tion of light en ergy ab -sorbed by PSII an ten nae that is usedfor pho to chem i cal charge sep a ra tion((Fv′/Fm′)qp); D = ther mally dis si pated en ergy frac tion (1 – Fv′/Fm′); and E =

ex cess en ergy, de fined as the frac tion of ab sorbed light go ing to nei ther P nor D, i.e., ex cess en ergy trapped or dis si pated, or both, by PSII re ac tioncen ters with QA in a re duced state ((Fv′/Fm′)(1 – qp)). Mea sure ments were taken at 20 °C. Each value rep re sents the mean re sponse (± SE) of three disks of nee dles, one mea sure ment per disk.

unit dry mass at both 5 and –5 °C (Ta ble 1). Ex posed plants atlow growth tem per a tures had about 70% less chlo ro phyll com -pared with plants grown at 20 °C, and the cor re spond ing valuefor shaded plants was about 30%. Com pared with the de creasein chloro phylls, the de cline in the carotenoid pool size wassmaller. The mass-based carotenoid con cen tra tion of ex posed,cold-treated plants was about 45% of that of plants grown at20 °C, whereas low tem per a tures caused a 20% de crease incarotenoid con cen tra tion in shaded plants (Ta ble 1). The ra tioof Chl a/Chl b de creased with de creas ing tem per a ture in seed -lings in both light treat ments (Ta ble 1), and af ter 7 days of re -cov ery at 20 °C the Chl a/Chl b ra tio was still lower than the ini -tial con trol value, how ever, the shaded plants had higher ra tiosthan the ex posed plants (Ta ble 1). Al though to tal carotenoidcon cen tra tion per unit dry mass de creased dur ing cold ac cli ma -tion, chlo ro phyll-based carotenoid con cen tra tion in creased byabout 40% in the ex posed seed lings, but by only 5–10% inshade plants (Fig ure 4). Ex po sure to low tem per a tures not onlyin creased the rel a tive size of the carotenoid pool, but also in -creased the chlo ro phyll-based lutein con cen tra tion three- tofour fold in ex posed plants and two- to three fold in shaded

plants (Fig ure 4). There was a sim i lar sub stan tial in crease inthe pool of the xanthophyll cy cle pig ments viola xanthin, an -ther axanthin and zeaxanthin (VAZ) per mole of chlo ro phyll,es pe cially in ex posed plants where the ap par ent size of thexanthophyll cy cle pig ment pool dou bled dur ing the cold hard -en ing pe riod. In agree ment with the en ergy par ti tion ing data,shaded seed lings de vel oped high de-epoxidation state val ues(DEPS) only un der win ter con di tions (–5 °C), whereas ex -posed seed lings achieved about 60% of their max i mum DEPSat 5 °C (Fig ure 4).

Thermoluminescence Light-in duced TL emis sion pro videdin for ma tion on the ac ti va tion en er gies as so ci ated with thecharge re com bi na tion re ac tions of the pri mary and sec ond aryquinone elec tron ac cep tors (QA and QB, re spec tively) with theelec tron do nors (S2 and S3) of PSII. Ex po sure of dark-adaptedsam ples to sin gle-turn over flashes of white sat u rat ing light incom bi na tion with DCMU to block elec tron flow be tween QA

and QB yielded TL glow curves with peak emis sion tem per a -tures char ac ter is tic for the S 2QA

–, S 3QA–, S 2QB

– and S 3QB–

charge recombinations (Inoue 1996, Sane 2004). The char ac -



Fig ure 3. Func tional an tenna cross sec -tion of a photosystem II (PSII) re ac tion cen ter in ex posed (350 µmol m– 2 s–1)and shaded (50 µmol m– 2 s–1) nee dlesof Scots pine seed lings sub jected to the fol low ing treat ments: con trol (20 °C,3 weeks), cold ac cli ma tion (5 °C,3 weeks), freez ing stress (–5 °C,3 weeks) and re cov ery from freez ingstress (Rec; 20 °C, 7 days). The curvesrep re sent vari able flu o res cence re -sponses in ex posed and shaded Scotspine nee dles. Val ues of PSII vari ableflu o res cence (Fv′ , mV) are plot tedagainst ac tinic flash in ten sity. The ex -po nen tial slope of a Pois son-fit of thedata (nor mal ized to max i mal re sponse)

rep re sents the func tional ab sorp tion cross sec tion per PSII re ac tion cen ter (pre sented in Ta ble 2). Each value rep re sents the mean re sponse (± SE)of three disks of nee dles, 3–5 mea sure ments per disk.

Ta ble 2. Func tional an tenna cross sec tion of photosystems II and I (PSII and PSI) in ex posed (350 µmol m– 2 s–1) and shaded (50 µmol m– 2 s–1)nee dles of Scots pine seed lings sub jected to the fol low ing treat ments: con trol (20 °C, 3 weeks), cold ac cli ma tion (5 °C, 3 weeks), freez ing stress(–5 °C, 3 weeks) and re cov ery from freez ing stress (20 °C, 7 days). For PSII: the num bers were ob tained as the ex po nen tial slope of a Pois son-fitof the vari able flu o res cence re sponses of the nee dles (Fv′, mV) plot ted against ac tinic flash in ten sity (as in Fig ure 4) and nor mal ized to max i malre sponse. For PSI: the num bers were ob tained as the ex po nen tial slope of a Pois son-fit of the ab sorp tion changes at 820 nm in the nee dles (∆A280,mV) plot ted against ac tinic flash in ten sity and nor mal ized to max i mal re sponse. Mean val ues (± SE) were cal cu lated from the re sponses of threedisks of nee dles, 3–5 mea sure ments per disk. Sta tis ti cal sig nif i cance of the dif fer ences in 5 and –5 °C treat ments com pared with con trol val ues isin di cated by as ter isks (* = P < 0.0003, ** = P < 0.01 and *** = P > 0.1).

Func tional Con trol Cold ac cli ma tion Ar ti fi cial win ter stress ab sorp tion cross (20 °C) (5 °C) (–5 °C)sec tion (nm2)

Ex posed Shaded Ex posed Shaded Ex posed Shaded

PSII 0.17 0.23 0.35* 0.23*** – 0.31**PSI 0.10 0.09 0.12*** 0.11*** 0.13*** 0.14*

ter is tic tem per a ture max ima (TM) of the TL peaks re lated to there com bi na tion of these charge pairs re flect the ac ti va tion en er -gies and hence a mea sure of the re dox po ten tials of the par tic i -pat ing ox i dized and re duced do nors (DeVault and Govindjee1990). In a pre vi ous study, TL emis sion peaks around 45 °C incon trol and 2 °C in DCMU-poi soned Scots pine nee dles wereas cribed to S 2QB

– and S 2QA– recombinations, re spec tively

(Ivanov et al. 2002). Ex po sure of non-hard ened Scots pine nee -dles grown at a mod er ate irradiance (350 µmol m – 2 s –1) to twoflashes of white sat u rat ing light yielded a TL glow curve thatcould be sep a rated into three peaks with the char ac ter is tic peakemis sion tem per a ture (TM) of the ma jor peak cen tered around45 °C and two mi nor bands ap pear ing around 2 °C and above60 °C (Fig ure 5A, Ta ble 3). Plants grown in shade (50 µmolpho tons m – 2 s –1) ex hib ited sim i lar TL pat terns as the ex posedplants, al though the over all TL in ten sity of the glow curve waslower (Fig ure 5B). The re com bi na tion of S 2QA

– charge pairses ti mated in the pres ence of DCMU (traces not shown) in di -cated only a min i mal dif fer ence be tween the ex posed andshaded plants (Ta ble 3).

Ac cli ma tion of ex posed and shaded plants to a tem per a tureof 5 °C caused a large de cline in the TL emis sion of the S 2QB

–

band (Fig ures 5C and 5D) and a 7 and 12 °C shift to lower tem -per a tures in shaded and ex posed nee dles, re spec tively (Ta -ble 3). In ad di tion, cold ac cli ma tion caused a shift of the S 2QA

–

band to a slightly higher TM. Ex posed nee dles frost hard ened at –5 °C ex hib ited a fur ther de crease in the TL emis sion (Fig -ure 5E) with no change in the S 2QB

– and S 2QA– peak po si tions

(Ta ble 3). In con trast, shaded nee dles at –5 °C showed no fur -ther changes in TL emis sion, but the S 2QB

– peak shifted to alower TM, whereas S 2QA

– shifted to a higher TM (Ta ble 3).The TL glow curve pat terns of frost-hard ened ex posed and

shaded pine nee dles af ter re cov ery at room tem per a tures (Fig -ures 5G and 5H) closely re sem bled those of the cor re spond ingcon trol ex posed and shaded plants. In both cases, the TL emis -sions re cov ered al most com pletely to the val ues ob served incon trol sam ples and re cov ery was ac com pa nied by a shift inS 2QB

– peak emis sion to higher tem per a tures. Si mul ta neously,the S 2QA

– band shifted to tem per a tures close to that in con trolsam ples (Ta ble 3).

Dis cus sion

Light enhances the effect of cold treatment on photosynthetic efficiency

Ac cli ma tion of Scots pine seed lings to low (5 °C) and freez ing(–5 °C) tem per a tures at two ir radi an ces fol lowed the an tic i -pated pat tern in terms of photosynthetic ef fi ciency, quench ingpa ram e ters and pig ment con tent ad just ments based on the con -cept of en ergy bal ance (Huner et al. 1998). Un der win ter fieldcon di tions, cold-in duced photoinhibition has been re portedpre vi ously in var i ous plant spe cies (Ball et al. 1991, Holly etal. 1994), with a higher photoinhibition and a slower rate of re -cov ery in pine nee dles ex posed to high ir radi an ces (Bauer etal. 1975, Lundmark and Hallgren 1987, Strand and Lundmark



Fig ure 4. Tem per a ture-in duced changes in carotenoid con cen -tra tions in ex posed (350 µmolm– 2 s–1) and shaded (50 µmol m– 2 s–1) nee dles of Scots pine seed lings sub jected to the fol -low ing treat ments: con trol(20 °C, 3 weeks), cold ac cli -ma tion (5 °C, 3 weeks), freez -ing stress (–5 °C, 3 weeks) and re cov ery from freezing stress(Rec; 20 °C, 7 days). Ab bre vi -a tions: Car/Chl = to tal ca rot -en oids per chlo ro phyll;Lut/Chl = lutein per chlo ro -phyll; VAZ/Chl = violaxanthin cy cle com po nents per chlo ro -phyll; and DEPS =de-epoxidation state of theviolaxanthin cy cle pig ments.Ex posed and shaded seed lingsgrown at 20 °C had sim i larval ues, rep re sented by a sin glefilled col umn in each panel.Val ues rep re sent means (± SE) of three mea sure ments frompooled sam ples of 10 nee dlescol lected from five plants (two nee dles per plant).

1987, Lundmark and Hallgren 1988, Ensminger et al. 2004).In nat u ral hab i tats, the in ter ac tive ef fect of irradiance and lowtem per a tures has im por tant eco log i cal im pli ca tions. For ex -am ple, Slot et al. (2005) found that Scots pine seed lings grow -ing on the south ern side of shel ter ing older Scots pine trees ex -pe ri ence more se vere photoinhibition and a lower rate ofre cov ery af ter win ter stress than seed lings grow ing to the north of the shel ter ing trees, and con cluded that low tem per a tures to -gether with a low so lar an gle dur ing the bo real spring and early sum mer cre ate dif fer ent light and shad ing for north ern-ex -posed com pared with south ern- ex posed seed lings, which ac -counts for much of the ob served spa tial regener a tion pat tern.

Our flu o res cence data in di cated a de crease in pho to chem i -cal ef fi ciency of the PSII re ac tion cen ters in Scots pine seed -

lings in re sponse to low tem per a tures (Ta ble 1, Fig ure 1) andalso re vealed a stron ger de crease in pho to chem i cal ef fi ciencyin ex posed seed lings com pared with shaded seed lings, cor rob -o rat ing ear lier find ings (Lundmark and Hallgren 1987, Ad ams et al. 2002). The rate of ox y gen evo lu tion de creased in both ex -posed and shaded seed lings fol low ing trans fer from a growthtem per a ture of 20 to 5 or –5 °C, but es pe cially in the ex posedplants (Fig ure 1), in di cat ing that, dur ing the acclimation ofnee dles to 5 °C, the ob served de crease in ox y gen evo lu tionwas mainly an ef fect of tem per a ture.

In ad di tion, a de creased rate of ox y gen evo lu tion in ex posedplants at 5 °C sug gests a stron ger de cline in the pho to chem i -cally al lo cated frac tion of the en ergy ab sorbed by PSII in these seed lings. The en ergy par ti tion ing data (Fig ure 2) con firm that



Fig ure 5. Thermoluminescence (TL) glow curves and math e -mat i cal de com po si tion insub-bands af ter il lu mi na tionwith two sin gle turn overflashes of white sat u rat inglight in ex posed (350 µmolm– 2 s–1) and shaded (50 µmol m– 2 s–1) nee dles of Scots pine seed lings sub jected to the fol -low ing treat ments: con trol(20 °C, 3 weeks) (A, B), coldac cli ma tion (5 °C, 3 weeks)(C, D), freez ing stress (–5 °C,3 weeks) (E, F) and re cov eryfrom freez ing stress (20 °C,7 days) (G, H). The TL glowcurves pre sented are meansfrom 5–7 mea sure ments in2–3 in de pend ent ex per i ments.

photochemistry is con sid er ably de creased in ex posed plants at5 °C, in di cat ing that the frac tion of ab sorbed light not used forphotochemistry must be con sid er ably larger at higher ir radi an -ces, putt ing the re ac tion cen ters un der se ri ous stress. At thesame time, the dis si pated frac tion of the ab sorbed en ergy washigher in the ex posed plants at 5 °C (Fig ure 2), thus min i miz -ing the damaging potential of the excess light energy.

Trans fer of shaded plants to –5 °C re sulted in some re ten -tion of ox y gen evo lu tion and the pho to chem i cally used frac -tion of PSII ab sorbed light en ergy. In con trast, ex posed plantsshowed com plete in hi bi tion of pho to syn the sis at –5 °C, withal most com plete loss of photochemistry (Fig ure 2). Thus, de -spite ther mal dis si pa tion mech a nisms de vel oped in the ex -posed seed lings, we con clude that freez ing and ex po sure tomod er ate irradiance to gether led to in creased dam age to PSII.This con clu sion is sup ported by the low ef fi ciency of photo -synthetic re cov ery in ex posed seed lings af ter return to 20 from–5 °C. In con trast, nee dles of shaded plants re tained someoxygen-evolv ing ac tiv ity af ter freez ing, en abling the plantsto quickly relaunch pho to syn the sis when tem per a tures in -creased.

Protective mechanisms and irradiance

The en ergy chan nel ling and quench ing pa ram e ters de rivedfrom room tem per a ture flu o res cence mea sure ments in di cateda strong de crease in photochemistry in pine nee dles at lowtem per a tures. The changes in pho to chem i cal quench ing dif -fered be tween ex posed and shaded plants. The ex posed seed -lings were more af fected, with photochemistry de creas ing toless than 50% at 5 °C and vir tu ally dis ap pear ing at –5 °C. Thether mally dis si pated frac tion of ab sorbed light en ergy wasmuch higher at low tem per a tures, es pe cially in the ex posedplants (Fig ure 2), point ing to in creased de vel op ment of NPQmech a nisms at low tem per a tures.

In both irradiance treat ments, to tal chlo ro phyll con cen tra -tion in the nee dles de creased with de creas ing tem per a ture, and the de creases were greater in the ex posed plants than in theshaded plants (Ta ble 1). Retaining chlo ro phyll dur ing win ter(Öquist and Mar tin 1980) re quires an in crease in carotenoidcon tent to in crease radiationless dis si pa tion of ex cess en ergyto pro tect the re main ing PSII re ac tion cen ters when pho to syn -

the sis is in hib ited (Gilmore and Ball 2000, Matsubara et al.2002). The rel a tive in crease in ca rot en oids per mole of chlo ro -phyll at low tem per a tures was higher in ex posed seed lingsthan in shaded seed lings (Fig ure 4). Nev er the less, there was anet loss in the ab so lute con cen tra tion of ca rot en oids per unitdry mass in both seed ling types, which was ac com pa nied by an even higher net loss in total chlorophyll per unit dry mass inthe exposed seedlings (Table 1).

Sus tained high con cen tra tions of lutein (Tausz et al. 1999,Tausz et al. 2001, Matsubara et al. 2003), zeaxanthin andantheraxanthin in Scots pine in win ter are re spon si ble forphotoprotective ther mal en ergy dis si pa tion (Ad ams and Dem-mig-Ad ams 1994, Ottander et al. 1995), and are as so ci atedwith main te nance of low PSII ef fi ciency at low tem per a tures(Barker et al. 2002). Changes in the con cen tra tions of thexanthophyll cy cle pig ments (VAZ) and their de-epoxidationstate (DEPS) dur ing the hard en ing pe riod cor re lated pos i tively with in creased irradiance and de creased tem per a ture in bothex posed and shaded seed lings. How ever, the irradiance treat -ments re sulted in dif fer ent quan ti ta tive re sponses as a re sult ofthe dif fer ent ex ci ta tion pres sure im posed on these plants (Fig -ure 2). A com bi na tion of mod er ate irradiance and low tem per -a ture forced the for ma tion of DEPS in the ex posed seed lingsdur ing cold ac cli ma tion to lower tem per a tures. Carotenoidsyn the sis in the ex posed seed lings there fore in creased dur ingthe cold-hard en ing pe riod, the xanthophyll cy cle be came fullyfunc tional and high lev els of NPQ were de vel oped. Thesechanges cor rob o rate the ob served in crease in ther mal dis si pa -tion of light en ergy in ex posed seed lings at 5 °C (Fig ure 2). At–5 °C, al most all ab sorbed en ergy be came dis si pated in the ex -posed nee dles. In con trast, the shaded plants dem on strated ahigher ex cess en ergy frac tion, and also some par ti tion ing tophotochemistry even at –5 °C.

Remaining light harvesting antenna complexes and reactioncenter quenching

Low tem per a tures lead to a de crease in pho to chem i cal re -sponses, al though a sub stan tial frac tion of the an tenna pro teinis pre served in tact af ter the ac cli ma tion. This de mands a largeredis tri bu tion of ex ci ta tion en ergy from pho to syn the sis toquen ch ing, through re ar range ments and tun ing in both an -



Ta ble 3. Thermoluminescence de ter mi na tion of photosystem II charge re com bi na tion ther mo dy nam ics. Peak emis sion tem per a tures (TM) ofS2QA

– and S2QB– glow peaks in ex posed (350 µmol m– 2 s–1) and shaded (50 µmol m– 2 s–1) nee dles of Scots pine seed lings sub jected to the fol -

low ing treat ments: con trol (20 °C, 3 weeks), cold ac cli ma tion (5 °C, 3 weeks), freez ing stress (–5 °C, 3 weeks) and re cov ery from freez ing stress(20 °C, 7 days). The TM of glow peaks rep re sented by char ac ter is tic sub-bands were es ti mated by a nonlin ear least-squares fit ting of the ex per i -men tal glow curves ob tained af ter il lu mi na tion with two sin gle turn over flashes (see Fig ure 5). Mea sure ments for es ti ma tion of S2QA

– were per -formed in the pres ence of 20 µM 3-(3′,4′-di chloro phenyl)-1,1dimethylurea. Mean val ues (± SE) are based on 3–11 in de pend ent ex per i ments.

Treat ment Ex posed Shaded

S2QA– TM (°C) S2QB

– TM (°C) S2QA– TM (°C) S2QB

– TM (°C)

Con trol (20 °C) 2.6 ± 0.07 46.0 ± 0.02 3.4 ± 0.02 44.5 ± 0.02Cold ac cli ma tion (5 °C) 4.8 ± 0.06 32.9 ± 0.02 5.3 ± 0.04 37.4 ± 0.01Freez ing stress (–5 °C) 4.5 ± 0.30 33.6 ± 0.03 6.9 ± 0.20 36.4 ± 0.08Re cov ery (20 °C) 1.5 ± 0.06 43.7 ± 0.01 4.3 ± 0.07 39.2 ± 0.01

tenna and re ac tion cen ters. We found that the re main ing an -tenna com plexes are not com pletely de tached from the fewre main ing re ac tion cen ters un der win ter con di tions. The PSIIfunc tional ab sorp tion cross sec tion in creased in the re main ingre ac tion cen ters of both photosystems with de creas ing tem per -a ture, es pe cially at mod er ate irradiance (Ta ble 2). This en -hance ment might be the re sult of dam age to more re ac tioncen ters un der con di tions of mod er ate irradiance and low tem -per a ture. De creased ab so lute re sponse val ues of Fv′ (Fig ure 3)in cold-stressed ex posed seed lings are also in ac cor dance withlower pho to chem i cal ef fi ciency of PSII (as also in di cated bythe Fv/Fm val ues in Ta ble 1), sup port ing a pro posed in crease inre ac tion cen ter quench ing ac tiv ity served by the in creasedabsorbance cross sec tions.

Strong steady-state re duc tion of PSI re ac tion cen ters in re -sponse to low tem per a tures has been shown in Scots pine inwin ter (Ivanov et al. 2001). Be cause the func tional ab sorp tioncross sec tion of PSI also in creased in re sponse to cold treat -ment (Ta ble 2), PSI may serve in the dis si pa tion of ex ci ta tionen ergy for the remaining antennae.

Reaction center quenching is increased under coldacclimation

It has been re ported re cently that in creased ex ci ta tion pres sure fol low ing ex po sure of cyanobacteria and higher plants to lowtem per a tures is ac com pa nied by in creased prob a bil ity of non- ra di a tive ex ci ta tion en ergy dis si pa tion within the re ac tion cen -ter of PSII (re ac tion cen ter quench ing) me di ated by a shift ofthe re dox po ten tials of the pri mary (QA) and sec ond ary (QB)quinone elec tron ac cep tors of PSII (Sane et al. 2002, 2003,Ivanov et al. 2003). This was re vealed through a shift in the TM

of flash-in duced S 2QB– and S 2QA

– charge recombinations. Asim i lar mech a nism for dis si pa tion of ex cess light en ergy wasalso pro posed to sup ple ment the zeaxanthin-de pend ent NPQin Scots pine in win ter (Ivanov et al. 2001, 2002), but no sys -tem atic flash-in duced anal y sis of S 2QB

– and S 2QA– charge

recom binations was per formed.The TL glow curve pat terns dem on strated that the TM of the

S 2QB– band shifted to lower tem per a tures by 13 and 7 °C in

the ex posed and shaded seed lings, re spec tively, on ac cli ma tion to low tem per a ture (Ta ble 3). The TM of the DCMU-in ducedS 2QA

– band was up-shifted by about 2 °C. These shifts nar -rowed the gap be tween the TM val ues of S 2QB

– and S 2QA– by

15 and 10 °C in the ex posed and shaded seed lings, re s pec -tively. Ex po sure of the seed lings to freez ing tem per a turecaused no fur ther sig nif i cant changes in the S 2QB

– and S 2QA–

peak emis sion tem per a tures, re gard less of the growth irra -diance. How ever, these shifts were com pletely re vers ible and,af ter the re cov ery pe riod, the peak po si tions of both S 2QB

– and S 2QA

– charge re com bi na tion re sem bled those in con trol plants (Ta ble 3). In ad di tion, and in agree ment with a pre vi ous re port(Ivanov et al. 2001), cold-ac cli mated seed lings had lower TLemis sion. We found that growth irra diance ap pears to in flu -ence the TL re sponse pat tern (Fig ure 5, Ta ble 3). The ex posedseed lings showed much higher sen si tiv ity to the tem per a turede crease, los ing much of their TL in ten sity at +5 °C, whereas

the shaded seed lings main tained their TL sig nal through outboth cold treat ments, likely re flect ing the higher num ber of re -main ing re ac tion cen ters in these seed lings.

The change in the ac ti va tion en ergy of the re com bin ing ionpair re flected by a shift in TM is re lated to the mid point po -tentials of the re com bin ing spe cies (DeVault and Govind -jee 1990). Nar row ing the gap in TMs be tween S 2QA

– andS 2QB

– sug gests that the re dox po ten tials of QA and QB havecome closer, which should re sult in changes in the equi lib riumcon stant of the elec tron flow be tween QA and QB such thatthere is an in creased prob a bil ity of the elec tron be ing re tainedon QA. The in creased pop u la tion of re duced QA (QA

–) wouldin crease the prob a bil ity of non-ra di a tive charge re com bi na tion be tween QA

– and P680+ (Prasil et al. 1996, Krieger- Liszkayand Rutherford 1998, Vavilin and Vermaas 2000). It has beensug gested that in creased re duc tion of QA is a ma jor re quire -ment for ef fi cient re ac tion cen ter quench ing (Bukhov et al.2001). Fur ther more, the ac cu mu la tion of QA

– in hib its the for -ma tion of P680 trip lets (Schatz et al. 1988, Vass et al. 1992) re -duc ing the pos si bil ity of photodamage to PSII.

Con clu sions

Ex po sure to in creased growth irradiance in creased the in hib i -tory in flu ence of low tem per a tures on photosynthetic ac tiv ityin Scots pine nee dles and im paired re cov ery of pho to syn the sisaf ter trans fer to 20 °C. Light-ex posed plants lost more PSII re -ac tion cen ters than shaded plants, but kept a rel a tively largeran tenna and a rel a tively larger pool of photoprotective xantho -phyll cy cle pig ments, re spon si ble for the ther mal dis si pa tionof the ab sorbed light en ergy. We sug gest that the light-de pend -ent changes ob served in the re dox prop er ties of both the pri -mary (QA) and sec ond ary (QB) ac cep tors of PSII re ac tion cen -ters at low and freez ing tem per a tures en hance the prob a bil ityof non-ra di a tive en ergy dis si pa tion within the PSII re ac tioncen ter (re ac tion cen ter quench ing), thus sup ple ment ing an -tenna-based xanthophyll-de pend ent NPQ of ex cess light dur -ing win ter.

Ac knowl edg ments

This work was fi nan cially sup ported by grants from The Swed ish Re -search Coun cil, the Swed ish Foun da tion for In ter na tional Co op er a -tion in Re search and Higher Ed u ca tion (STINT) and by the Nat u ralSci ence and En gi neer ing Re search Council of Canada.

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Excitation energy partitioning and quenching during cold acclimation in Scots pine

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