Photosynthesis Research23:111-117, 1990. © 1990KluwerAcademicPublishers. Printed in the Netherlands.

Modification of excitation energy distribution to photosystem I by protein phosphorylation and cation depletion during thylakoid biogenesis in wheat Guy J. Bredenkamp ~ & Neil R. Baker 2 Department of Biology, University of Essex, Colchester, C04 3SQ, Essex, UK; 1Current address: Department of Biology, University College, Gower Street, London, WCIE 6BT, UK; 2To whom all correspondence regarding the manuscript and reprint requests should be sent Received25 October 1988;accepted in revisedform 13 March 1989

Key words: chlorophyll fluorescence, cation depletion, chloroplast development, electron transport, light-harvesting chlorophyll proteins, photosystem I, protein phosphorylation, wheat Abstract The effects of protein phosphorylation and cation depletion on the electron transport rate and fluorescence emission characteristics of photosystem I at two stages of chloroplast development in lightgrown wheat leaves are examined. The light-harvesting chlorophyll a/b protein complex associated with photosystem I (LHC I) was absent from the thylakoids at the early stage of development, but that associated with photosystem II (LHC II) was present. Protein phosphorylation produced an increase in the light-limited rate of photosystem I electron transport at the early stage of development when chlorophyll b was preferentially excited, indicating that LHC I is not required for transfer of excitation energy from phosphorylated LHC II to the core complex of photosystem I. However, no enhancement of photosystem I fluorescence at 77 K was observed at this stage of development, demonstrating that a strict relationship between excitation energy density in photosystem I pigment matrices and the longwavelength fluorescence emission from photosystem I at 77 K does not exist. Depletion of Mg 2÷ from the thylakoids produced a stimulation of photosystem I electron transport at both stages of development, but a large enhancement of the photosystem I fluorescence emission was observed only in the thylakoids containing LHC I. It is suggested that the enhancement of PS I electron transport by Mg 2+-depletion and phosphorylation of LHC II is associated with an enhancement of fluorescence at 77 K from LHC I and not from the core complex of PS I.

Introduction

Phosphorylation of the apoproteins of the lightharvesting chlorophyll a/b complex associated with photosystem II (LHC II) is widely considered to result in an increase in excitation energy distribution to photosystem I (PS I) due to phospho-LHC II migrating from the appressed, granal membranes to the non-appressed, stromal membranes where P S I complexes are located (Anderson 1986, Andersson et al. 1982, Barber 1982, 1983, Bennett 1984, Bennett et al. 1980, Haworth et al. 1982b, Horton 1983). However, an increase in P S I elec-

tron transport is not always observed on phosphorylation of LHC II (Baker and Webber 1987) and the factors determining the interaction of phospho-LHC II with P S I are not well understood. The developing chloroplast in the light-grown wheat leaf offers a useful system with which to examine factors determining excitation energy transfer from phospho-LHC II to PS I. At the early stages of chloroplast biogenesis found at the base of the developing wheat leaf LHC II and photochemically competent PSI and photosystem II (PS II) complexes are present and the apoproteins of LHC II can be phosphorylated, but the light-harvesting

!12 chlorophyll a/b protein complex associated with PS I (LHC I) is absent (Baker et al. 1984, Bredenkamp and Baker 1988, Covello et al. 1987, Percival et al. 1986). During chloroplast development LHC I accumulates in the thylakoids producing an increase in the quantum yield of P S I electron transport (Bredenkamp and Baker 1988). An increase in the lateral segregation of PSI and PS II complexes also occurs (Baker et al. 1984, Webber et al. 1984). In this study we examine the possible importance of LHC I and the lateral segregation of PSI and PS II complexes in the thylakoid membranes in mediating changes in excitation energy distribution to PS I produced by phosphorylation of LHC II. The effects of protein phosphorylation and divalent cation depletion on PS I electron transport and fluorescence emission characteristics at 77 K are determined for thylakoid membranes isolated from the base and tip of 5-day-old light-grown wheat leaves. It should be emphasized that base thylakoids contain negligible amounts of LHC I compared to tip thylakoids (Bredenkamp and Baker 1988) and also exhibit considerably less lateral segregation of PSI and PS II complexes (Baker et al. 1984, Webber et al. 1984).

Materials and methods

Seeds of wheat (Triticum aestivum var. Maris Dove) were soaked in running water for 17 h before sowing in Levington compost. Plants were grown at 20°C in a 16h photoperiod using white light, produced by a mixture of fluorescence tubes and incandescent bulbs, with a photosyntheticallyactive photon flux density (PPFD) of 200#molm-2s -1 at the top of the canopy. A gradient of cellular and plastid development exists from the leaf base to the tip in primary wheat leaves (Boffey et al. 1979). Leaf tissue was harvested 5 days after sowing the seeds by cutting the plant immediately above the hypocotyl insertion and collecting the basal and apical I cm segments of the primary leaves. Leaf segments were homogenized, using a Polytron blender, in a medium containing 50mM HEPES, 10mM NaCI and 5mM MgCI2 (pH 7.6) and centrifuged at 3000g for 3 min before resuspending the resulting pellet in the same medium. For the Mg 2+ depletion experiments the 5mM MgCI2 was omitted from the washing and

resuspension media. Chlorophyll contents of thylakoid suspensions were determined using the method of Arnon (1949). Thylakoid membrane proteins were phosphorylated by incubating thylakoid preparations in the resuspension medium, described above, containing 400/~m ATP and 10mM NaF for 10min at 20°C in white light with a photon flux density of 50/~molm-2s -~. The polypeptide and phosphoprotein profiles of thylakoids isolated from the basal and tip segments of the leaves were essentially identical to those previously published for thylakoids isolated from the leaf base and tip; the LHC II apoproteins and a 9 kDa polypeptide were heavily phosphorylated and were the major phosphoproteins in both samples (Covello et al. 1987). It has also been previously established that phosphorylation of the apoproteins of LHC II in thylakoids at all stages of development in the light-grown wheat produce a disconnection of a similar proportion of the LHC II population from PS II complexes (Percival et al. 1986). Photosystem I-mediated electron transport by thylakoids from reduced tetramethyl-p-phenylene diamine (TMPD) to methyl viologen was determined polarographically in a Clark oxygen electrode. Oxygen uptake was monitored at 20°C in 2cm 3 of reaction medium containing 50raM HEPES, 10mM NaC1, 0.2mM TMPD, 3mM sodium ascorbate, 0.1 mM methyl viologen, 0.5mM sodium azide, 20/~M DCMU and 20/~g chlorophyll. Monochromatic radiation at 440 and 470nm (half-band-width 10nm) was produced from a 900 W xenon arc lamp filtered with a high irradiance monochromator and attenuated with neutral density filters. Fluorescence emission spectra of isolated thylakoids were measured at 77 K using a fibre-optic scanning spectrofluorometer (Dominy and Baker 1980). 0.2cm 3 aliquots of dark-adapted thylakoid preparations containing 10/~gchlorophyll cm-3 were frozen to 77 K in liquid nitrogen in a cuvette to produce sample of ca. 1 mm thick. Samples were excited from above via one branch of a bifurcated fibre optic with 100/~molm 2s t of 470nm radiation produced as described above. Fluorescence emission from the sample surface was passed via the second arm of the bifurcated fibre optic to a scanning monochromator with a half-band-width of 2nm and detected using a Hamamatsu R446

113 photomultiplier. Emission spectra were measured at the maximal level o f fluorescence. The kinetics of chlorophyll fluorescence emission at 695 and 740 nm from thylakoid samples at 77 K were monitored simultaneously using a trifurcated fibre optic spectrofluorometer (Dominy and Baker 1980). Thylakoid preparations containing 10 #g chlorophyll cm -3 were dark-adapted at 20°C for 10min prior to freezing to 7 7 K in the dark in liquid nitrogen. The frozen samples were ca. 1 m m thick and were excited from above with broad band blue light (380-580nm) having a P P F D of 1 0 0 # m o l m - 2 s -~ at the sample surface and produced from a quartz-iodide source and a 580nm short-pass filter (Ealing 30-5362). Fluorescence transients were captured by transient recorders (Datalab DL901) and the minimal (F0), maximal (Fm) and variable (Fv) fluoresence levels determined. The absolute values of these parameters varied considerably between individual replicate samples due to unpredictable changes produced in the optical properties of the samples on freezing to 77 K. This variability precluded calculation o f the yield of excitation energy transfer from PS II to P S I using the tripartite model (Butler and Strasser 1977), however ratios of fluorescence parameters, where the variability factor cancels

60

f

out, can be usefully applied to the analysis of phosphorylation effects on excitation energy transfer.

Results The role of L H C II in determining changes in P S I electron transport induced by protein phosphorylation can be examined by comparing the lightlimited rates of P S I electron transport in nonphosphorylated and phosphorylated membranes on excitation with 440 and 4 7 0 n m radiation; 440 nm preferentially excites chlorophyll a relative to chlorophyll b, while 470 nm will preferentially excite chlorophyll b relative to chlorophyll a (Bredenkamp and Baker 1988). Light dosage response curves for excitation with 440 and 470 nm radiation of P S I electron transport from reduced T M P D to methyl viologen for non-phosphorylated and phosphorylated thylakoids isolated from the base and tip of 5-day-old wheat leaves are shown in Figs. 1 and 2. Although phosphorylation produced negligible effects on P S I electron transport in both samples when measured with 4 4 0 n m excitation (Fig. 1), a significant enhancement of P S I activity was observed for base thylakoids excited with 470 nm radiation (Fig. 2). On phosphorylation thylakoids isolated from the leaf tip exhibited a small,

/

o - ' - -

20

,

,

,

,

40

60

SO

I00

photonflux density( ~ o ~ ) Fig. 1. Light intensity response curve for PSI electron transport

from reduced TMPD to methyl viologen in thylakoids isolated from the base (o, e) and tip (D, an) of 5 day-old wheat leaves. Measurements were made for non-phosphorylated (O, D) and phosphorylated (e, II) thylakoids using 440 nm actinic light. Each point represents the mean of four independent measurements and standard errors are given. The maximum photon flux density (100%) was 165pmol m-1 s - l

° . . . . .

O

20

40

60

80

I00

photon flux density (% max) Fig. 2. Light intensity responsecurves for PS I electron transport

from reduced TMPD to methyl viologen in thylakoids isolated from the base (o, e) and tip (D, B) of 5 day-old wheat leaves. Measurements were made for non-phosphoryolated (o, D) and phosphorylated (e, i ) thylakoids using 470nm actinic light. Each point represents the mean of four independent measurements and standard errors are given. The maximum photon flux density (100%) was 165/zmolm-Z s-l.

114

B

6° I 650

° ........ 0

20

40

photon f l u x

675

700

725

750

775

800

emissionwavelength(nm) 60

80

100

density(%max)

Fig. 3. Light intensity response curves for PSI electron transport

from reduced TMPD to methyl viologen in non-phosphorylated thylakoids isolated from the base (o, e) and tip (D, II) of 5 day-old wheat leaves. Measurements were made on thylakoids incubated in the presence (O, D) and absence (e, II) of 5 mM Mg2+ using 470 nm radiation. Each point represents the mean of four independent measurements and standard errors are given. The maximum photon flux density (100%) was 165/~mol m-2s t. r e p r o d u c i b l e , b u t non-significant, increase in P S I electron t r a n s p o r t when excited with 4 7 0 n m p h o t o n flux densities b e l o w 7 0 y m o l m - 2 s -~. T h e e n h a n c e m e n t o f P S I electron t r a n s p o r t by p r o t e i n p h o s p h o r y l a t i o n in leaf base t h y l a k o i d s when m e a s u r e d with 470 nm, b u t n o t with 440 nm, r a d i a tion indicates t h a t the effect is m e d i a t e d by an increased e x c i t a t i o n o f PS I b y a c h l o r o p h y l l b-containing a n t e n n a complex. Clearly, since L H C I is a b s e n t in t h y l a k o i d s f r o m the leaf base, an increase in e x c i t a t i o n energy transfer f r o m L H C II to P S I can be c o n s i d e r e d r e s p o n s i b l e for the e n h a n c e m e n t o f P S I activity. D e p l e t i o n o f d i v a l e n t c a t i o n s f r o m the m e d i u m in which t h y l a k o i d s are s u s p e n d e d p r o d u c e s changes in m e m b r a n e o r g a n i z a t i o n a n d excitation energy d i s t r i b u t i o n which can be used to e v a l u a t e c h a n g e s i n d u c e d in the m e m b r a n e s by p r o t e i n p h o s p h o r y l a t i o n . R e m o v a l o f M g 2÷ is c o n s i d e r e d to result in a d e t a c h m e n t o f L H C II f r o m PS II a n d a r a n d o m i z a t i o n o f L H C II, PS II a n d P S I c o m plexes within the p l a n e o f the m e m b r a n e , with a c o n s e q u e n t increase in excitation energy transfer f r o m L H C II a n d PS I I to P S I (Butler a n d K i t a j i m a 1975, Staehelin 1976, Telfer et al. 1983, Jennings 1984). T h e light intensity response curves for P S I

Fig. 4. Fluorescence emission spectra measured at 77 K for

non-phosphorylated ( ) and phosphorylated (. . . . . ) thylakoids isolated from the base (A) and the tip (B) of 5 day-old wheat leaves with 470 nm excitation. The spectra are normalized on the 686 nm peak. electron t r a n s p o r t driven b y 470 n m r a d i a t i o n for t h y l a k o i d s f r o m the base a n d the tip o f the leaves in the presence a n d absence o f 5 m M M g 2+ are shown in Fig. 3. M g 2÷-depletion p r o d u c e d large increases in the light-limited rate o f P S I electron t r a n s p o r t o f b o t h base a n d tip t h y l a k o i d s which, as expected, were c o n s i d e r a b l y g r e a t e r t h a n the c h a n g e s observed on p h o s p h o r y l a t i o n o f t h y l a k o i d p r o t e i n s (Fig. 2). F l u o r e s c e n c e emission s p e c t r a at 77 K o f nonphosphorylated and phosphorylated thylakoids f r o m the leaf base a n d tip o n excitation with 470 n m

650

675

700

725

750

775

800

emissionwavelengtla(ran) Fig. 5. Fluorescence emission spectra measured at 77 K for

thylakoids isolated from the base (A) and tip (B) of 5 day-old wheat leaves in the presence (. . . . . ) and absence ( ) of 5 mM Mg2+ and excited with 470 nm radiation. The spectra are normalized on the 686 nm peak.

115 radiation are shown in Fig. 4. In both base and tip thylakoids phosphorylation produced no significant enhancement of fluorescence emission above 700 nm, which is primarily from chlorophylls associated with P S I (Butler and Strasser 1977, Butler 1978, Bose 1982, Nechushtai et al. 1986), relative to L H C II and PS II emissions at 685nm. These results were unexpected since phosphorylation of LHC II apoproteins in mature thylakoids has been shown to result in a large enhancement of the 740nm emission band relative to the 685 and 695 nm emission bands associated with PS II (Bennett et al. 1980, Krause and Behrend 1983, Kyle et al. 1983). However, Mg2+-depletion o f thylakoids from the leaf base and tip did produce the expected enhancement of the long wavelength PS I emissions relative to the 685 nm PS II band (Fig. 5). As would be expected this enhancement of the P S I emission in the base thylakoids was small compared to that observed in tip thylakoids; the much greater segregation of P S I and PS II complexes in the thylakoids from the leaf tip, compared to the base, would result in a much larger increase in excitation energy transfer from L H C II and PS II to PS I on removal of Mg 2+ . The spectral data shown in Fig. 4 and 5 demonstrate clearly that the effects of Mg 2+ depletion on P S I fluorescence in developing thylakoids are different to those of protein phosphorylation. Analyses of the kinetics of 77 K fluorescence emission from P S I and PS II at 740 and 695 nm respectively can be used to further examine further changes in excitation energy distribution within the thylakoids (Butler and Strasser 1977, Butler 1978). From the tripartite model of the photochemical apparatus of thylakoids, the fraction of excitation energy in P S I that results from energy transfer from PS II complexes can be calculated from the compound ratio of (F~/Fm)74o" (Fv/Fm)~9~, the value

of which is termed fiN, (Strasser and Butler 1977). Changes in (Fv/Fm)695, (Fv/Fm)74o and fin for base and tip thylakoids on protein phosphorylation and Mg 2+ depletion are given in Table 1. For thylakoids from the leaf base both phosphorylation and Mg 2+ depletion had negligible effects on flr~, suggesting in the context of the tripartite model that there is no change in excitation energy transfer from PS II to PS I in these treatments. On depletion of M g ~+ from tip thylakoids a large increase in fin indicates that excitation energy transfer from PS II to PS I is markedly enhanced, as would be expected when segregation of P S I and PS II complexes is decreased. However, phosphorylation of tip thylakoid proteins did not modify fiN significantly. Unexpectedly phosphorylation of both tip and base thylakoids increased (Fv/Fm)695by a small amount (Table 1); studies with mature thylakoids have shown no change (Krause and Behrend 1983) or a decrease (Haworth et al. 1982a) in (Fv/Fm)695 on phosphorylation. These data support the suggestion that the phosphorylation-induced increases in PS I electron transport are mediated by a different mechanism than those produced by Mg z+depletion. MgE+-induced changes in fin correlate with the appearance of L H C I in the developing thylakoids. However, L H C I appears to play no role in producing P S I fluorescence changes associated with the phosphorylation of L H C II apoproteins.

Discussion At the earliest stage of chloroplast development found in the base of the 5-day-old wheat leaf, phosphorylation of thylakoid polypeptides produced an increase in the light-limited rate of P S I electron transport when driven by 470nm light, which

Table 1. Effectsof protein phosphorylation and Mg2+ depletion on 77 K fluorescenceemission parameters, (Fv/Fm)695,(Fv/Fm)74o and fiN, for thylakoids isolated from the base and tip of 5 day-old wheat leaves, fN was calculated from (Fv/Fm)74o"(Fv/Em)69~"See text for definitions of these parameters. Standard errors of the means of 6 replicatesare given in parentheses Section

ATP

Mg2+

(Fv/Fro)695

(Fv/Fm)74o

fin

Base Base Base Tip Tip Tip

+ + --

+ + + + --

0.45 (0.01) 0.53 (0.02) 0.38 (0.03) 0.65 (0.02) 0.71 (0.02) 0.35 (0.02)

0.33 (0.01) 0.38 (0.02) 0.28 (0.02) 0.36 (0.02) 0.40 (0.02) 0.25 (0.01)

0.73 0.72 0.74 0.55 0.57 0.71

116 preferentially excited chlorophyll b relative to chlorophyll a. It is evident that L H C I is not required to mediate such a phosphorylation-induced increase in P S I electron transport, since LHC I is not present in thylakoids in the leaf base (Bredenkamp and Baker 1988). Presumably excitation energy transfer can occur directly from phosphoLHC II to the core complex of P S I in thylakoids from the leaf base. These data may also suggest that an association between P S I and phospho-LHC II has a greater probability of occurrence when P S I complexes are deficient in L H C I and less spatially separated from PS II complexes. The effects of protein phosphorylation on PS I electron transport in the developing wheat thylakoids reported in this paper are unlikely to have any major physiological importance for photosynthetic activity of the tissues, since no significant enhancement of P S I activity was observed in base or tip thylakoids on excitation with 440 nm radiation, or on excitation of tip thylakoids with 470 nm radiation. Although protein phosphorylation can produce significant enhancement of P S I electron transport in thylakoids from the base of the leaf, it is of note that no parallel changes are observed in either the 77 K fluorescence emission spectra or the fluorescence energy transfer parameter, fiN. In mature thylakoids phosphorylation of the apoproteins of L H C II has been associated with an enhancement of the PS 1 740 nm emission band at 77 K relative to the PS II emission bands below 700 nm (Bennett et al. 1980, Krause and Behrend 1983, Kyle et al. 1983). Clearly the data presented here demonstrates that a strict relationship between phosphorylation-induced enhancement of PS I electron transport, which is related directly to the density of excitation energy in PS I, and 77 K P S I fluorescence emission, relative to that of PS II, does not exist. The factor(s) that determine the enhancement of P S I fluorescence at 77 K on phosphorylation of the L H C II apoproteins cannot be resolved from this study. With regard to the enhancement of P S I fluorescence, it is interesting that Mg2+-depletion induced a large increase in the light-limited rate of electron transport in the thylakoids from both the leaf base and tip (Fig. 3), but a large enhancement of the P S I fluorescence at 77 K, relative to that of PS II, was only observed for tip thylakoids (Fig. 5). The Mg2+-depletion enhancement of PS I fluorescence would appear to be associated with the

presence of LHC I. It is now accepted that at 77 K the 740 nm emission band originates from LHC I, while the P S I core complex emits a considerably smaller signal with a maximum at about 720725 nm (Argyroudi-Akoyunoglou 1984, Markwell et al. 1985, Nechushtai et al. 1986). Consequently, the data presented suggests that the enhancement of P S I electron transport by Mg2+-depletion and phosphorylation of L H C II apoproteins is not associated with an enhancement of fluorescence from the P S I core complex at 77 K. A large enhancement of fluorescence above 700 nm on Mg 2÷depletion is only apparent when L H C I is associated with the P S I core complex. It is also evident from this study that the Mg2+-depletion and LHC II phosphorylation enhancements of P S I electron transport are mediated by different mechanisms; Mg2+-depletion induces a large enhancement of 740 nm fluorescence when LHC I is present in the P S I complexes, whereas this is not the case following LHC II phosphorylation. Mg2÷-depletion not only dissociates LHC II from PS II complexes (Butler 1978, Jennings et al. 1978, Loos and Kellner 1980, Wong and Govindjee 1981, Nairn et al. 1982) but also decreases the segregation of PS II and PS I complexes (Telfer et al. 1983, 1984, Hodges and Barber 1984). Consequently the differences in the fluorescence emission characteristics of PS I induced by MgZ+-depletion and LHC II phosphorylation may be due to non-phosphorylated LHC II and PS II complexes interacting with P S I complexes when Mg 2÷ is depleted, while on phosphorylation of the thylakoids only phosphorylated LHC II would be expected to interact with PS I complexes.

Acknowledgement GJB was the recipient of a research studentship from the U.K. Science and Engineering Research Council.

References Anderson JM (1986)Photoregulationof the composition,function and structure of thylakoid membranes. Ann Rev Plant Physiol 37: 93-186. Andersson B, Akerlund H, Jergil B and Larsson C (1982). Differential phosphorylation of the light-harvesting

117 chlorophyll-protein complex in appressed and non-appressed regions of the thylakoid membrane. FEBS Lett 149:181-185 Argyroudi-Akoyunoglou J (1984) The 77 K fluorescence spectrum of the photosystem I pigment-protein complex CPla. FEBS Lett 171:47-53 Arnon DI (1949) Copper enzymes in isolated chloroplasts: polyphenol oxidase in Beta vulgaris. Plant Physiol 24:1-15 Baker NR and Webber AN (1987) Interactions between photosystems. Adv Bot Res 13:1-66 Baker NR, Webber AN, Bradbury M, Markwell JP, Baker MG and Thornber JP (1984) Development of photochemical competence during growth of the wheat leaf. In: Thornber JP, Staehelin LA and Hallick RB (eds) Biosynthesis of the photosynthetic apparatus: molecular biology, development and regulation, pp 237-255. New York: Alan R. Liss Inc. Barber J (1982) Influence of surface charges on thylakoid structure and function. Ann Rev Plant Physiol 33:261-295 Barber J (1983) Photosynthetic electron transport in relation to thylakoid membranes composition and organization. Plant Cell Environ 6:311-322 Bennett J (1984) Regulation of photosynthesis by protein phosphorylation. In: Cohen P (ed) Molecular aspects of cellular regulation, Vol 3, pp 227-246. Amsterdam: Elsevier Bennett J, Steinbeck KE and Arntzen CJ (1980) Chloroplast phosphoproteins: regulation of excitation energy transfer by phosphorylation of thylakoid membrange polypeptides. Proc Natl Acad Sci USA 77:5253-5257 Boffey SA, Ellis JR, Sellden G and Leech RM (1979) Chloroplast division and DNA synthesis in light-grown wheat leaves. Plant Physiol 64:502-504 Bose S (1982) Chlorophyll fluorescence in green plants and energy transfer pathways in photosynthesis. Photochem Photobiol 36:725-731 Bredenkamp GJ and Baker NR (1988) The changing contribution of LHC I to photosystem I activity during chloroplast biogenesis in wheat. Biochim Biophys Acta 934:14-21 Butler WL (1978) Energy distribution in the photochemical apparatus of photosynthesis. Ann Rev Plant Physiol 29: 345-378 Butler WL and Kitajima M (1975) Energy transfer between photosystem II and photosystem I in chloroplasts. Biochim Biophys Acta 396:72-85 Butler WL and Strasser RJ (1977) Tripartite model for the photochemical apparatus of green plant chloroplasts. Proc Natl Acad Sci USA 74:3382-3385 Covello PS, Webber AN, Danko SJ, Markwell JP and Baker NR (1987) Phosphorylation of thylakoid proteins during chloroplast biogenesis in greening etiolated and light-grown wheat leaves. Photosynthesis Res 12:243-254 Dominy PJ and Baker NR (1980) Salinity and in vitro aging effects on primary photosynthetic processes of thylakoids isolated from Pisum sativum and Spinacea oleraceae. J Exp Bot 31:59-74 Haworth P, Kyle DJ and Arntzen CJ (1982a) A demonstration of the physiological role of membrane phosphorylation in chloroplasts using the bipartite and tripartite models of photosynthesis. Biochim Biophys Acta 680:343-351 Haworth P, Kyle D J, Horton P and Arntzen CJ (1982b) Chloroplast membrane protein phosphorylation. Photochem Photobiol 36:743-748 Hodges M and Barber J (1984) Analysis of chlorophyll fluorescence quenching by DBMIB as a means of investigating the consequences of thylakoid membrane phosphorylation. Bio-

chim Biophys Acta 767:102-107 Horton P (1983) Control of chloroplast electron transport by phosphorylation of thylakoid proteins. FEBS Lett 152:47-52 Jennings RC (1984) Independent effects of magnesium ions on energy transfer processes in chloroplasts. Biochim Biophys Acta 766:303-309 Krause GH and Behrend U (1983) Characterisation of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77 K. II. ATP-dependent quenching. Biochim Biophys Acta 723:176-181 Kyle D J, Staehelin LA and Arntzen CJ (1983) Lateral mobility of the light-harvesting complex in chloroplast membranes controls excitation distribution in higher plants. Arch Biochem Biophys 222:527-541 Loos E and Kellner E (1980) Influence of magnesium ions on the actions of photosystems I and II at different wavelengths. Zeit Naturforsch 35C: 298-302 Markwell JP, Webber AN, Danko SJ and Baker NR (1985) Fluorescence emission spectra and thylakoid protein kinase activities of three higher plant mutants deficient in chlorophyll b. Biochim Biophys Acta 808:156-163 Nairn JA, Haehnel W, Reisberg P and Sauer K (1982) Picosecond fluorescence kinetics in spinach chloroplasts at room temperature. Biochim Biophys Acta 682:420-422 Nechushtai R, Norazideh SD and Thornber JP (1986) A reevaluation of the core chlorophylls of photosystem 1. Biochim Biophys Acta 848:193-200 Percival MP, Webber AN, Markwell JP and Baker NR (1986) Modification of the interaction between photosystem II and the light-harvesting chlorophyll a[b protein complex by protein phosphorylation in developing wheat thylakoids exhibiting different degrees of lateral heterogeneity. Biochim Biophys Acta 848:317-323 Staehelin LA (1976) Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro. J Cell Biol 71; 136-158 Strasser RJ and Butler WL (1977) Energy transfer and the distribution of excitation energy in the photochemical apparatus of spinach chloroplasts. Biochim Biophys Acta 460: 230-238 Telfer A, Hodges M and Barber J (1983) Analysis of chlorophyll fluorescence induction curves in the presence of 3-(3,4dichlorophenyl)-l,l-dimethylurea as a function of magnesium concentration and NADPH-activated light-harvesting chlorophyll a[b protein phosphorylation. Biochim Biophys Acta 724:167-175 Telfer A, Hodges M, Millner PA and Barber J (1984) The cation-dependence of the degree of phosphorylation-induced unstacking of pea thylakoids. Biochim Biophys Acta 766: 554-562 Webber AN, Baker NR, Platt-Aloia K and Thomson WW (1984) Appearance of a state 1-state 2 transition during chloroplast development in wheat leaf: energetic and structural considerations. Physiol Plant 60:171-179 Webber AN, Baker NR, Paige CD and Hipkins MF (1986) Photosynthetic electron transport and the establishment of an associated trans-thylakoid proton electro-chemical gradient during development of the wheat leaf. Plant Cell Environ 9: 203-208 Wong D and Govindjee (1981) Action spectra of cation effects on the fluorescence polarization and intensity in thylakoids at room temperature. Photochem Photobiol 33:103-108