Indian Journal of Biochemistry & Biophysics Vol. 37, December 2000, pp. 395-404

--,

Greening of intermittent-light-grown bean plants in continuous light: Thylakoid com onent\ in relation to photosynthetic performance and

capacity for photoprotection 2~o ~~/~ W S how*·a/ Funk"·b, A. rHopec and Govindjee*·d ~ , (

. ~

*·· Photobioenergetics Group, Research School of Biological Sciences, AustJ]lian National University, GPO"B' 75-;---"--- Canberra, A.C.T. 260 I, Australia )

"School of Biological Sciences, Flinders Uni versi ty, GPO Box 2loo, Adelaide, SA 500 I . Australi a dDepartments of Biochemistry and Pl ant Biology, Universi ty of Ill inois at Urbana-Champaign, 265 Morrill Hall , 505 South

Goodwin Avenue, Urbana, IL 61801-3707 , USA

Received 1 February 2000; accepted 30 May 2000

l Phaseolus vulgaris <~ Wi-ndsor-longped-and nap bean) plants, etiolated duri ng germination , were exposed to inter­mi ti'eilt light (2 min light every 2 hr) for up to 68 hr and then transferred to continuous white li ght. On transfe r of the plants to continuous light (100 photons J,.lmo l m·2

S· I, 24°C), the quantum yie ld o f oxygen evolution increased two-fo ld in about 30 hr. The chlorophyll content per unit leaf area or unit fresh weight increased dramaticall y, but the fresh weight per unit leaf area was relati vely constant. The changes were expressed on the basis of fresh weight or leaf area. On thi s basis, the contents of photo system (PS) I and II increased in continuou s light , by a factor o f 3 and 8, respectively. While the chlorophyll b con­tent and the contents of apoproteins of li ght-harves ting chlorophyll-protein complexes (LHCllb, CP29, CP26 and CP24) in­creased markedly, neither the total carotenoid content nor the de-epoxidation state of the xanthophylls [rati o of zeaxan­thin(Z) + antheraxanthin(A) to ( Z+ A + violaxanthin) was about 0.4)] responded significantly on transfer to continuous ' light. The fast rise of the flash-induced e1ectrochromic signal (L'1A518) was well correlated with the increases in PS I and PS II reaction centres, and with chlorophyll b and total carotenoid contents. The increase in the quantum yield of oxygen evolu­tion during greeni ng in continuous light is attributed to a more balanced di stribution of excitation energy between the two photosystems, facilitated by the increased number of PS II units, the increased antenna size of each unit and the enhance­ment of grana formation . The chloroplast in intermittent light was found to contain abundant xanthophyll cycle pigments and the psbS gene product , presumably adequate for photoprotection in conti nuous ligh t as soon as chlorophyll a/b- prote in complexes are synthesized. The results suggest that greening in continuous li ght is accompanied by adjustments that include enhanq:d quantum efficiency of photosynthesis and development of a capacity for harmless di ssipati on of excess excitation energy. __ "l '

.,-Introduction

Argyroudi-Akoyunoglou and Akoyunoglou l first showed that bean plants developed under intermittent light (cycles of 2 min light and 98 min dark) preferentially accumulated Chi a in the green plastids. Subsequently, Armond et al.2 observed that pea plants grown under cycles of 2 min light and 118 min dark developed similarly. They further showed that the photochemical effici ency of light-limited whole-chain

*.a Authors for correspondence Email : chow@rsbs . anu.edu . au~ Fax: +61 662798056 hpresent address: Department of Biochemistry, University of Stockholm, Stockholm S-10691. Sweden.

Abbreviatiolls ({ sed - A, antheraxanthin : ChI. chlorophyll : cyt. cytochrome ; 01 , product of the psbA gene; DBM IB , 2,5-dibromo-3-methyl-6-i sopropyl-p-benzoquinone; NPQ, non­photochemi cal qucnching; P700, special chlorophyll pair in the PS I reaction centre ; PII"", light- and CO2-saturated rate of oxygen evolution ; PS, photosystem; V. vio laxanthin ; Z, zeax anthin .

e lectron transport in isolated thylakoids was low, due to a lack of regulation of energy distribution between the two photosystems, but that continuous light induced grana formation and an mcrease m photosynthetic unit size. Intermittent-light-grown plants are thought to have fully active photosynthet ic reaction centres which are served by the core antennae that are devoid of most of the peripheral light-harvesting antenna2

.6

.

Early studies of intermittent-light-grown pl ants generally employed ill vitro isol ation techniques to investigate the re lationships between the composition , structure and function of pl ast ids. The results of such in vitro studies could be influenced by isolation artefac ts in preparations of frag ile pl astids from intermittent-light-grown plants. With the availability of modern ins trumentati on, attent ion has shi fted to in vivo measurements, mainly of chlorophyll a fluorescence6

.9 and P700 photo-ox idation9 m

396 INDIAN J. BIOCHEM . BIOPHYS., VOL. 37, DECEMBER 2000

intermittent-light-grown plants. Thus, it has been demonstrated in vivo that pea plants grown in intermittent light (cycles of 2 min light and 118 min dark) but not in flash light (cycles of 1 ms-f1ash every 15 min dark) have fully developed reaction centres with functional oxygen "clocks", and that upon transfer to continuous light, the number of PS II units and their connectivity increase9

.

In the present investigation of bean plants transferred from intermittent light to continuous light, we aimed to measure (1) the development of the quantum efficiency of oxygen evolution in vivo; (2) the changes in the contents of functional photosystem (PS) I and II reaction centres in vivo; (3) the functionality of PS I and PS II in vivo during gTeening, by measurement of the electrochromic signals associated with stable charge separation in the two photosystems in leaves; and (4) the capacity for non-photochemical quenching of chlorophyll fluorescence in relation to the pigments and the proteins to which the pigments are bound.

Materials and Methods

Plants Seeds of Phaseolus vulgaris (cvv. Windsor

longpod and snap bean) were germinated in darkness at 21 QC in a seeding mix covered with vermiculite. Seven days after sowing, the seedlings were exposed to 2 min intermittent light (50 Jlmol photons m'2 s" ) every 2 hr for 66 hr (sometimes 68 hr). At a time indicated by a vertical line in the figures, the seedlings were transferred to continuous light in a growth cabinet (100 Jlmol photons m'2 s", 24 QC). When seeds of cv. Windsor longpod became unavailable, we used Phaseolus vulgaris cv. snap bean which gave comparable responses during greening. Results obtained with both cultivars were pooled whenever appropriate.

Oxygen measurements The number of functional PS II complexes was

determined by using a leaf disc oxygen electrode (Hansatech, King's Lynn, Norfolk, UK) and the method of Chow el al. 10. A leaf segment was dark treated for about 10 min in moist air enriched with 1 % CO2 and illuminated with a train of single-turnover, saturating white light flashes (10 Hz, 2.5 Jls full-width at half-peak height; xenon lamp FX 200, EG & G Electro Optics, USA) in the presence of background far-red light'o. The flashes were delivered for 4 min, followed by 4 min darkness, and this alternation was

repeated. A small heating artefact was taken into account, while any limitation to linear electron flow due to PS I was removed by the background far-red illumination. After determination of the functional PS II content, the leaf segments were illuminated with continuous white light from a projector (900 ' Jlmol photons m'2 s") to estimate the maximum capacity for gross oxygen evolution (P max). The gross rate of oxygen evolution was estimated after taking the dark drift (mainly due to dark respiration) into account. Functional PS II contents and P max rates were expressed on the basis of either fresh weight or chlorophyll content; the latter was determined in 80% buffered acetone". The results from up to four batches of seedlings were pooled.

The quantum yield of oxygen evolution on an absorbed light basis was determined by measuring rates of oxygen evolution in limiting light, the irradiance of which was varied by neutral density filters. Leaf absorbance was calculated from the transmittance and reflectance of a leaf segment obtained with an integrating sphere. In this experiment, only the cultivar snap bean was used.

Measurement of P700 absorbance changes at 820 nm

Photo-oxidizable P700 (in relative units on a leaf area basis) was measured with a modified . pulse modulation fluorometer (PAM 101, 102, Walz, Effeltrich, Germany) fitted with an 820 nm light emitting diode to provide the modulated measuring light. The modulated signal was measured in the reflectance mode' 2. To photo-oxidize P700, leaves were first illuminated for 19 s with far-red light (l70 Jlmol photons m'2 S'I) from a projector (Schott KL 1500), transmitted through an RG 9 filter (Schott). To ensure complete oxidation of P700, it was necessary to superimpose strong actinic light which was admitted for 1 s (after 19 s of far-red light) by an electronic shutter'3. The white actinic light (6500 Jlmol photons m'2 s"), filtered through a Cal flex C heat reflection filter, momentarily oxidized P700 completely. Then both far-red light and white light were turned off, and P700 relaxed to a completely reduced state within about 1 s. The modulated signal from the PAM fluorometer was off-set, then amplified 20-fold and recorded by a digital storage oscilloscope (Gould Type 1421, Hainsault, England) before being plotted on a chart recorder.

The quantification of P700 content was performed essentially as described previously'4, by the measurement of photo-oxidisable P700 in isolated

CHOWel al. : GREENING OF INTERMITTENT-LIGHT-GROWN BEAN PLANTS 397

thylakoids in the presence of a detergent and electron donors and electron acceptor, methyl viologen. To obtain consistent and maximal P700 content, a cocktail of protease inhibitors (Ieupeptin, 1 mglml in water; pepstatin A, 2 mM in methanol; phenylmethanesulfonyl fluoride, 200 mM in ethanol; aminocaproic acid, 100 mM in water; and 5' -p­fluorosulfonylbenzonyl adenosine, 1 mglml in water) consisting of 0.50 ml of each stock solution was included in 100 ml of isolation buffer I 5.

Electrochromic signals Laser-flash-induced absorbancy changes

(electrochromic shifts) at 518 nm were measured by directing the measuring beam at 45° to the leaf surface; the actinic laser flash was also directed at 45° to the leaf, and at 90° to the measuring beam. Usually 16 flashes at 0.2 Hz were given to leaf segments that had been dark adapted for 20 min to stabilize the conductance of the A TP synthase complexes; otherwise, the relatively fast decay of the 518 nm signal, which depends on thylakoid ionic conductance, tended to obscure the slow rise in the signal. Data were digitised and stored in computer files, as previously described 16.

Measurement of non-photochemical quenching, NPQ

Non-photochemical quenching of chlorophyll fluorescence (NPQ) in leaves was measured using a PAM fluorometer (101 ED, Heinz Walz, Effeltrich, Germany). NPQ was determined (see eg., Gilmore et at. 17) as (F,JFm' - 1), where Fm' was determined at the end of the five minute light period before the light was extinguished and Fm after a dark period of five minutes following the five minute light period. The irradiance used to drive NPQ was 1000 Jlmol photons m-2

S- I of white light (passed through a heat absorbing Walz DT Cyan filter). The saturating (l s) light pulse intensity used to close the PSII traps and determine Fm and Fm' was 10 000 Jlmol photons m-2

S-I of white light, also passed through a Walz DT-Cyan filter. Prior to the light treatments, the leaf pieces were dark adapted for 30 min with background illumination by a weak far-red LED source (PAM 102, A = 700 nm, irradiance = 15 Jlmol photons m·2

S·I). The far-red background source remained actuated during and following the light period to ensure that all PSI! traps were open. All dark adaptation and light treatments were performed in a humidified leaf disk chamber at approximately 20°e. Chloroplast pigments from

matching leaf pieces, both before and following the light treatments, were extracted and analyzed by high performance liquid chromatography according to Gilmore and Yamamoto l8

.

Isolation and assay of proteins Thylakoid membranes were isolated as described

by Adamska et ai. 19• Sodium dodecyl sulfate­

polyacrylamide gel electrophoresis was performed according to Laemmli20 using the Hoeffer mini-gel system. Samples were loaded on an equal protein basis. Gels were either stained with silver nitrate according to Oakley et al.21 or immunoblotted according to Towbin et al.22 using polyvinylidene difluoride (PVDF) membrane. Blots were incubated with various antibodies, and the antigen-antibody complex detected by the enhanced chemiluminescence method (Amersham Corp.).

Results

During intermittent illumination, the Chi content per unit fresh weight remained low, but increased by an order of magnitude over the subsequent 100 hr of continuous illumination (Fig. lA), whereas the Chi a/Chi b ratio decreased drastically in a few hours (Fig. IB). In contrast, the fresh weight per unit leaf area was relatively constant in both intermittent and continuous light (Fig. lC). Hence, expressing changes in photosynthetic parameters on the basis of either fresh weight or leaf area gives a more accurate measure of the increases in contents of photosynthetic components than on the basis of chlorophyll.

Photosynthetic activities In plants grown under intermittent light, the

quantum yield of O2 evolution of leaf discs , in limiting light but saturating CO2, was less than 0.04 mol O2 per mol photons absorbed. Upon illumination with continuous light, it increased steadily to almost 0.10 after about 30 hr of continuous light (Fig. 2). That is, the quantum efficiency of light-limited photosynthesis improved with development of the photosynthetic apparatus during greening. The minimal quantum requirement reached a value of 10 per O2 evolved (see a historical perspective23

) . Our observations in vivo agree with those of Armond et ai.2 who showed that the quantum efficiency of whole-chain electron transport in isolated thylakoids differed by a factor of 2 between intermittent-light­grown peas and those exposed to 24 hr of continuous light.

398 INDIAN J. BIOCHEM. BIOPHYS., VOL. 37, DECEMBER 2000

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Fig. I-Changes in chlo rophyll content on a fresh weight basis (A) ; ChI a/Chi b (B) ; in fresh weight per unit leaf area (C) after transfer of intermittent-light-grown bean plants to continuous light. Plants were transferred after 66 hours of intermittent light (2 min every 2 hr), at the time indicated by a vertical line.

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Fi g. 2-lmprovement in the quantum yield of O2 evolution on an absorbed light basi s following transfer of intermittent- light-grown bean pl ants to continuous light. The quantum yield was measured in leaf discs in limiting li ght and I % CO2,

The light- and CO2-saturated rate of O2 evolution (Pnulx, the photosynthetic capacity) on a fresh weight basis was also low in plants grown in intermittent light; however, it increased relatively abruptly by a factor of about 3 within about 30 hr in continuous light (Fig. 3A). When expressed on a ChI basis, Pmax

was high in plants grown under intermittent light; it decreased steadily in continuous light as the Chi content increased (Fig. 3B).

The two photosystems The number of functional PS II complexes on a

fresh weight basis was low in plants grown under intermittent light. It increased by about 5-fold after 30 hr and about 8 fold after 90 hr of continuous light (Fig. 3C). Owing to the low Chi a content per unit fresh weight and the near-absence of Chi b, the functiona l PS II content per un it ChI was generally high (4 nmol per mol ChI) in plants grown under intermittent light. However, it decreased to a va lue of

about 2.7 mmol per mol Chi (l PS II per 370 Chi molecules) after 100 hr of continuous light (Fig. 3D) .

The absorbance change at 820 nm, induced by a combination of continuous far-red light and a brief flash of strong white light, gives a measure of photo­oxidisable P700, the primary electron donor in PS 11 3.

This signal is a relative measure of the P700 content in the leaf area (1.2 cm2

) defined by the combined end of the multifurcated light gu ide used in conjunction with the PAM fluorometer. Fig. 3E shows that the signal on a leaf area basis increased abruptly in the early period (4 hr) of continuous light , and relatively slowly for the next 50 hr or so. On a Chi basis, the P700 signal declined on illumination with continuous light (Fig. 3F).

In a separate batch of plants (cv. Windsor), the content of P700 in thylakoids, isolated from plants fully greened under 100 f.i mol photons m-2 s - I , was determined to be 0 .62 ).lmol m-2 [or 1.41 mmol P700 (mol Chi) -I], while the functional PS II content was 1.34 ).lmol m-2

, i.e. PS II1PS I = 2.2, as measured by the methods used in this study.

The electrochromic shift When a transmembrane electric field is established

at PS II and PS I reaction centres following charge separation, pigments such as ChI band carotenoids undergo an electrochromic shift in response to the subsequently de localized electric fie ld, giving the fast-rise phase of M518 after a flash (applied at t = 0 in Fig. 4A). The magnitude of this fast phase depends on both the numbers of PS II and PS I capable of undergoing stable charge separation and the number of sensing pigments (Chi band carotenoids). It is seen in Fig. 4A that the fast phase was very small (~ 1 unit) at or before the beginning of conti nuous illumination (zero hr) . After 5 hr of continuous illumination ,

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big. :I- Changes In IIght- and COrsalLllatcd pholosynthcli l capacity CA, lh and COnLcnts of functiona l PS II (C, D) and PS I (E. F) unel transfer of intermillcnt- li ght-brown bcan plants to continuous light. Valuc of the parametcrs in the left panels are plolled on eithe a f-c,h weight or leaf arca basis, while thosc In th right panels are expressed on a ch lorophyll basi s. The vertical lines indicate the time of trans­fe r rrol11 intermittent to conti nuous light.

400 INDIAN J. BIOCHEM. BIOPHYS .. VOL. 37, DECEMBER 2000

NOllphotocliemical quenching and the pigments

A measure of the non-radi ative di ssipation of excess exc itat ion energy is given by NPQ, the non­photochemica l quenching of chlorophyll a fluorescence. As can be seen in Fig. SA, NPQ (measured at 1000 ~lIno l photons m':! S· I) was very low in plan ts grown under intermittent light; however, it increased rap idl y by an order of magnitude upon transfer of pl ants to contin uous li ght (100 )..tmol photons m·2

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S· I) for S min and which was already at a high value (-0.4) in plants . from intermittent light (Fig. SB ). Rather, the rapid increase in NPQ on transfer to continuous li ght was matched by a rapid decline in the Chi a/Chi b ratio, shown by a correla tion between NPQ and the Chi a/Chi b ratio in Fig. Sc.

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CHOW el al. : GREENING OF INTERMITTENT-LIGHT-G ROWN BEAN PLANTS 40 1

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Fi g. 5-Changcs in (A) NPQ and (B) the de-epox idation status after trans fer of intermittent-li ght-grown bean plants to cont inuous light. Pl ant s were transferred after 66 hours of intermittent light (2 min every 2 hr), at the time indicated by a vert ical line. In (C). the con'ela­tion of NPQ with the ChI a/ChI b ratio is shown.

Table I-Relati ve increases in the contents of sO l11e thylakoid proteins in PS II and carotenoids (Car) before and after transfer of bean plants fro m intermittent light (IML) to continuous light (CL). PSlI-S is the psbS gene product, beli eved to playa direct role in

~pH- and xanthophyll-dependent nonphotochemical quenching.

Apoprolein Etiolated 24 hr IML 43 hr IML 66 hr IML I hr CL 2 hr CL 4 hr CL 6 hrCL 30 hr CL

LHC li b ±

CP29 + + ++

CP26 :t +

CP24

PSII-S + + ++ +++

Z + A + V (\l11101 m-2) 22.7 2 1.5 24 5 20.2

Total Car (\lmol m-2) 67.6 74.2 88.6 74.4

Nm, not measured

Proteins As shown in Table 1, the apoprotein of LHClIb, the

major light-harvesting chl orophyll alb-protein complex associated with PS II, was scarcely detectable in leaves fro m plants grown under intermittent light. Upon transfer of plants to continuous light, it increased rapidly on a total thylakoid prote in bas is, reaching a maximum in approx . 4 hr. Of the three minor chlorophyll-protein complexes, the apoprotein of CP 29 and CP 26 increased even more rapidly, reaching a maximum at I to 2 hr of continuous light. The apoprotein of CP 24, in contrast, increased more Slowly. Interestingly, PS Il-S (psbS gene product), a pigment-binding protein essential for the regulation of photosynthetic light-harvesting24

, increased gradually during development in intermittent light, reaching a max imal amount by 66 hI' of intermittent light; thereafter, its content seemed to increase little on transfer of the plants to continuous light (Table 1).

Table I al so shows that the total content of the xanthophyll cycle pigme nts did not change signifi­cantly on transfer of pl ants from intermittent to con-

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tinuous light; the total carotenoid pigments increased by about 45 %.

Discussion Quantification of the two photosystems in vivo

Since the chlorophyll content rapidly increased on a fresh weight basis on transfer fro m intermittent to continuous light , quantification of the two photosystems on the basis of chlorophyll does not re flect the increases in the reac tion centres. On the other hand , expressing the contents of the two photosystems on e ither a leaf area bas is or a fresh weight bas is gives a good measure of the build up of reaction centres during illumination with continuous light. Thus, while PS II reaction centres in the case of intermittent growth li ght were capable of oxygen evolution in repetiti ve fla shes, the number of such centres 011 a fresh weight basis was low. Only after transfer of plants to continuous light did the functional PS II content increase several fold (Fig. 3C). Similarly, PS I, as measured by the P700 photo­oxidation signal at 820 nm on an equal leaf area basis,

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;,,1Lt:d 1'\Lil:lI i llll \'1 t" ,I, I 1',' IlJ \ 'kl'l". (}, 1 ':' :om,d, ,lIlk l1l1 d ~1/t: : (,( ; ! ,( lit' b"ill" ao

1. I ) 'J I It' It til 'I cell l rc:;, alsl (():jli.l,"c<, t. tk' l' .', l' ,; c!lcrg: distribution 01 :11 ~ itl,Ii" ~-:,:l i'l c ).It' ',;(,ll li~ht

at'tlcad, stall'. PI.) 1:11 ,,1 7/':-. ", I ~ " ~ r.: .. ~ ( '. iE. :--11,1]1 bean). bUI ill int ',I).lliCIII 11:-:!-t f', Ii'" - jiJ ,3 "­U arbitral) unit" (f ,1, I ;~. iC, ~I~). n' the !)~

II: p~ I stoich iomctl J' i<., lo(.,!' if' illl II1li' ,_ 11 !h l'\

cc si\ d y f:l 'ourinS l .. h, ahsorptill', [ t.> l'~ I

The small extent of (}d' fast rise oj the e[(c{ro,'llI'f)f:l;C signaL in plants grm 'll 1II illtelllllttnit light

The fast phase of th,:: r ise in lhe e/cctlOcilrn!11ic signal is a gooe' measur of the combined contributions of beth photosystems to electrogenic charge trdl1sfer acros~ the thylakoid Il1cmbnlllc. Thus, if PS Il is se lectivel :; inhib ited, thc remJlI1inf" fast nse is due to the PS J reac tion centres. and the l ciali\' comributions from ~he twO photo<;ys tems agree we ll with the photosystem stoi ' hiometry determi ned independent l l 7

, In intelmittent-l ight-gro\\'Il beims, the fast rise was scarcely detec table (solid circles, time 0 hI' in Fig. 4B). Based on our quantification or the rhotosystems and measuremen ts of the pigments which are likely to give the electrochromic response.

it :~, p()s~,lb l e tn pn:did tl ' 'II,' (If Ih' 1::,[ \i~ll, tI, ,I~

l ullOI\ S.

I I' ,01:(111' l" I '!'ll ,I,.' 'lIl'Ll il n:.1 P'J II ('till' ',1

Il'~lt he.i :TprtI': , ~; ~l' 1(il (c' I'\V rl I Fig, ~ \ \ 1,.1 .. 1

Ihe fll',1l \.,:' tellt In I L 1"(),2 m ' ~; 'i.~' B; tl'l' PI,: ~I '(I ! t 'l1 I:; I )~ ~l!li,d 11~- !1:' c1 ett'nnlninr lltc ~)-()' . LI'iltelll \'1 i~ )l,lllt! til) I:d,(li,j< llil a c'd olnph:. 11 h l"j, ,llll! !I " :, ,1" phyll -:ollll'1l1 (lCI ullit ie,If' :m',l II, I~,':

sall1-: ka\( '> IIs1'd f,t!' tilyl ,lf.;uid iSl I lli()II. till' P7()\l l JIlIl'11l ,)f '" I) -~I\ L'lled ka'I" II 'IS ml'a~lI 'cd to h," I I (, ~ Jill'ol n~ ,- 'h. II fr rl' 'h,' tllia l content of' Il'.Il'lion lcntrcs ill Liliit lItH) J:-, he.hl !:'I11 II [l lal t ~ wa~ 1.35 ,!

OF:' -:. !) 7 11I'1111 Ill', !II inrcllnillelll liQhl-!2f1)\11l I ...... ,_

plant:,. Illl' flillcti 1Il~" ]> • II LCtntcnt \1, 1<; aholl' I nmol (g n,' )-' (h~ ') ('1. Ta ~ jllr tilt' rl'c ~h \ 1 clghtlC' hl' J ~()

g 111 (h~, jel. lilt. J S II • (1lIll'llt \\~I<' i) I, ~ln11l111l '. f he I") I '_'Jill :d "1 il (' l" ll"I1 ! li

c,1l1 ~rol\l' l'lant~

(',m hL' _"llIll_IICd (1'('111 I ';;.. "L 1<) k I (ll x (1 .. '/':;) =

() [(1 ~lIlltll 111'- fill II i~ l11alk:' 11 1.111 ,1::.:1 i:, cllnlindlll!<; Ilg!.t ') ,I 1.10.1111 .1' :Ik'dl !.?,r, TIll' IOU

(,I(1tCl1 t or IC.l\.11011 '-l"~lll':-. :n ill ~'IJl,ill('l! t !i,;llt \\,1<-

t'lC'~ !CI I, " 'i :h 1If '

= 5 ti:"l' J t I' ·'- ....... 11 I ,'J ' If: ,,' I I' !. : r

.11 I, I.Ul11' L' _ "f !II ( I

\:~·i~'. 413,"'1 I r l illl i,.!1. t.11..11~ , .iI t. ,I H.,

111 Illl'l,1 ! III , I' I" 1,1, I,,, i( /. I 'I ,

'\ ,I

"'~'·'Ii"S. iI,'. '["l: I dl'"" 'I~, ... ( ,', ~I _ ::::,', l,"'I<'

il~ :Lt~rr·1I1ft..'''f~. l:~h'. '1 "I "\'C' •. : ... II t,'rpl it~J:~!' ,,0 hI

l(lldl Ci'!!{CIl: ui L'I:ol 'I~' ;,j, /fj ~II I, ' 111-' \ r :hL

,!Ill' Chl', '" (; ()'l II .e '1(!1 , ',lIlJ. 1'1 ~'·.l!I;Il[ll' ... ~;_ 'I

11 lit~ ~,,,J ~I HL. 1,1'.:' II 1I'\,'!1 t), l,llotCIl I);L ! '

I) P 1110' 111-- til. I' '101' Ill. addlll :2, lop ;u ~, t, ,..:! ot 2'+·1 f U1I I.'! ill

'\'\UIl!in}' tl ,11 each Ldl\Y'_'IOid ~l,r)IcCllk ~~ "

Lri'l.~lti'L as c'il.h Chi III tl~c t'iec!:()Llt;OI1lIi..

le~r(lIiSC , we expect to I11C<l~lIIe 2, 8 '>( (791244) ,lI1l l~

for (l; , fast ri :,c phase ill intermit tent ·llght leJ\'e ~, i c , 0,9 unite., appro>.imately \\hat wa ' ob\clved (Fig, 4Bj T'HIS, \\~ concl ude that tile fa st lise ph,,<; c; oft! C 11ash induced electrochromic signal i ' a f:ood Il1ca~ure of the: stable, electrogenic charge sCpar<l l lOIl a(l'o:-,::. Ihe thylakoid membrane in PS I and PS II rcacti ull centres, in both light regi:nes,

Th e slow rise oJtlte electrochrolllic signal The slow I'ise in the clectrochromic signal i: most

easi ly interpreted as being due to elec trogenic election transfer in the cytochrome bI complex as part of a Q­cycle or a similar cyc le, Hope and R ich2R have shown

CI IOW ('I a/" GREEN ING or Il\TERM 11 TEj\T U(d IT-G ROWN BEAN PLANTS 403

in ch loroplasts that both the slow ekctrocl !"Omic signJI a ill I cyt b'(11 reduc tion arc ;lccompanicJ by prott 11

LipI.lke sensi: \ e to cytochrome hf inhibi ttlrs <. uch ,I.

1 P:'-.1 fB. Fi~ 4 sho\\ ~ thaI the .,ic)\.\ ri~'L' ph-l''''­II ",a~cd gradually lelalive 10 the fa~t pkl<'C \.>Ce Irj,lngics). sll:;gcsting thai acti 'it) of a Q-cyck or d

~, ' :1,~1 cycle might nol ha\'(: tllen constant. ArIel 2') ! 01 Ilillie ill cOlltinuous light. the latio or the slo\\ 1',h,hL 10 till fast pl.,lse reac hed ClI 0.7; thi ~ \ alue i,> I, ~lhcI than "1(, "alue or l ·a. 0.3 CUi control tobacco il'd\'e' (I ig 1.\ in ( hOI\' and 110p,,27), Q-cycle activlt~ ,tin'l,

d(c~ 'lOt seem to explain such a large ralip (,f () ,. A~clllll,ng a llla\ill1Ulll PS I1IPS I ratio uf 2 . .2 for fully ~'Il'L'lw'l beal s (see Results\ dllJ that one eicClr(111 IL "k·l.'~ cyt /"(" p CI' pail 01 e,,"Llmns originalin,£' lrolll P Ii : 1(' sin \ phase shuuld he 1.1 unit rdati\ e tn the 1(11ai 1,[ ~ pll,I''- 01 (2.2 + I) L:nit ~ dUl to '2.2 PS l[ 'md I ;lS 1. Ie 1.1;122+ I) = 0.3. Till.' higher rat io of slc)\\ i'hasl'/LI~1 pI )se = 0.7 could ha 'e had a contribution rr(1'1~ \)>.iJ,lli, '11. at the cyt b( complex. of plastoquinol fm'llL'd l'~ d Llrons and protons on the acceptor , ide of PS 1. Tal is. yc lic e lec ti on flow via PS I may result in lol(lre l ,Ll, )' transfers to cyt "50.1 Jnd an increased ,'II'.', rill',' the electroc hromic signal. If ~;o. given tl I~ \:"1 ;;d k ,,( i,) of slow pha sclfa~ t plnse during &1'('l'nll11:' '. ':'11~ tha t Q-cycle activity or cyc lic PS I ('ic ·t'·OI; flu )1' both. may vary according to growth conditions ,11' ,k"clopmcntal stages in pl ants.

Relations/lljJ ,,{ i\'PQ to tile ell! b contellt On IransiL ,I' ldnti nuous light, the increase in NPQ

(fig SA) \. a Ih)! \,,\~II correla teo with total carotenoid cnnlent or th, \d;lllwphytl cycle pigments (Table I) . 0"

the dc-expo'. ,,";Oil sta tus (Fig. SB). Rather. the increase in 1\'1 J \ a" well correlated with the Ch i alChl /; I :.lI IU (rig ~,('; OJ tile Chi h content itsel f (not shown). The lOlTcl.itipil of ' PQ with the Chi a/Ch! b ratio is consisknt \\ itll 01 sen ations that ma ximum NPQ in sun and shade lca\(~s is linearly cOIl'elated with photoconvert lhle vi o! ux:.l!1thin which is in turn linearl y correlated with Ihe Chi {[/Chl b ratio"<J.

In plants glO\ ' ll nl y in intermittent light , however. the de-epoxidation statc was alrC'Jdy at a high value of 0.4, and was little changed UP0!l trans fe r of pl ant s to continuous ligllf (Fig. SB). PrpsumJbly, v. hen the dL­epoxidat ion sla te wa~ at a high v,due. other l.11lnrs were limiti ng NPQ in leaves de ve loped undel intermit tent iighl. Indeed, .Iahn!' and Schweig7 have previously shown that p l ant~ grown in intermittent light are capable of formi ng large amounts of zeaxanthin, but energy .. dependent quenching IS

~uhslant iall y suppressed. Presuma bly, l11a xi l11~d . PQ i" dchie\ 'ed when violaxanth in de-epoxidation. , >tdhli~hIl1Cllt ur a ...\pJ 11-.10.11 , the pre~~nce of the JI.\h~'

cne proJLlcr'.l Jnd fo rmati oll of inner and pel ipheral , I :CII comple.\es (heIll" the dependence Oil Chi II)

all OCLLlI tO~ClhLr these rl'quirel11ents bl'in~'

c' )Ib:ti\(~ly dchi~\l-J onl) in full)-greened tissue. Signilil.,tIl tly. til, phULUS) ntiletic :qJparatlis during

[1('\\ th in inierl11illl' I light \\ as already equipped "itll ~(bll ndant \'<l nthoph) II c: c k pigments in a high de­cptl\iJa tioIl stale. The pI/;.) ,~~t'nc product. previously 5h O\\ n to be capable of billciing chlorophyll12

"'.1'>

found tu be stable evell ill lilL' ahSl'I~Ce of chloroph) II and carotl'noid:,.1·~ . anc! belieH J i'l he olrel~tl)' il1\ oh'ed in \{i 11- and x~lllthophyll·Jepc Ilde'lt q lienchi Ilg. ()f l?\citation L'llerg/.l, was also abundant (Tab le I). The photosynthet ic appara tu s appeared fully cqui pped for photoprotection in conti nuous I ight as soon as LHCIl pip-ment-protein comple \es were synt hesized. and as ,>oon as an adequate L1pH \\ as acilieH::d.

Concluding remarks Creening represents a process \\ hereby :J.diustment~

towards opt i mum photos) nthetic pcrfOrll1,lnCC ta J..e place; these adjustments include enl1:.1I1cemcnt of quantum effici ency of photosy nthesi s dIld the de"elopment of a latent c:lpacity for h,lll'lk~s

d i sipation of excess excitation energy .

Admowledgement We are very grateful to Adam Gilmore fo r

measuremen ts of NPQ and pi gments (chlorophyll. and xanthophy ll s) . C'. Funk acknowledges the recei pt of an International Researcher Exchange Fellowship under an agreement between Australia and Germany. Govi ndj ee and A. B. Hope held Vi siting Fellowships from The Photobioenergetics Group , Researc h School of Biological Sciences, Australian National University, We also thank Jan Anderson and Barry Osmond fo r he lpful comments on the manuscript.

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