Photosynthesis Research 12:243-254 (1987) © Martinus Nijhoff Publishers, Dordrecht--Printed in the Netherlands
243
Regular paper
Phosphorylation of thylakoid proteins during chloroplast biogenesis in greening efiolated and light-grown wheat leaves P A T R I C K S. C O V E L L O , 1 A N D R E W N. WEBBER, 1'2'3 S T E P H E N J. D A N K O , 2 J O H N P. M A R K W E L L 2 & N E I L R. BAKER1, * ~Department of Biology, University of Essex, Colchester C04 3SQ, Essex, UK; ~Department of Agricultural Biochemistry, University of Nebraska, Lincoln, .Nebraska 68583, USA; 3Current address: Botany School., University of Cambridge, Downing Street, Cambridge CB2 3EA, UK (*author for correspondence and offprints) Received 19 November 1986; accepted in revised form 6 January t987
Abstract.Phosphorylation of polypeptidesin isolated thylakoids was examined during chloroplast biogenesisin greeningetiolated wheat leavesand 4 day-old wheat leavesgrown under a diurnal light regime. At early stages of plastid development standard thylakoid preparations were heavily contaminated with nuclear proteins, which distorted the pob~peptide phosphorylation profiles, Removal of contamination from membranes by sucrose density centrifugation demonstrated that the major membrane phosphoprotein in etioplasts was at 35kDa. During etioplast greening a number of phosphoproteins appeared, of which the 25-27 kDa apoproteins of the light-harvesting chlorophyll a/b protein complex associated with photosystem II (LHCII) became the most dominant. At the early stages of thylakoid development found at the base of the 4-day-old light grown leaf the LHCII apoproteins were evident as phosphoproteins; however the major phosphoprotein was polypeptide at ca. 9 kDA. Phosphorylation of both the LHCII apoproteins and the 9 kDa polypeptide in these thylakoids was not light-dependent. In the older thylakoids isolated from the leaf tip the LHCII apoproteins were the major phosphoproteins and their phosphorylation had 'become light-regulated; howeverphosphorylation of the 9 kDa polypeptide remained insensitive to light.
Introduction M a n y endogenous proteins o f the thylakoid membrane are phosphorylated by m e m b r a n e - b o u n d protein kinases [11]. During short incubations (2-10min) of mature thylakoids with A T P the major polypeptides phosphorylated are the apoproteins of the light-harvesting chlorophyll a/b complex associated with photosystem I I ( L H C I I ) , which are in the 23-29 k D a molecular weight range, and a smaller polypeptide with a molecular weight of less than l0 k D a [9, t0-12, 19, 20, 22]. The phosphorylation of the L H C I I apoproteins is light regulated and has been implicated as a mechanism for regulating the distribution o f excitation energy within the photosynthetic apparatus [7-9, 11, 12, 20, 22]; reduction o f plastoquinone by photosynthetic electron transport is thought to be responsible for the activation of the L H C I I protein kinase [1, 2, 3]. However, the physiological role of phosphorylation of the large number of other thylakoid polypeptides is unclear. The observation o f greater thylakoid protein kinase activities, on a unit membrane protein basis, in etioplasts and developing plastids compared to in mature chloroplasts [5, 6] has offered support to the suggestion that thylakoid
244 polypeptide phosphorylation may be important in the biosynthesis and structural organization of the photosynthetic apparatus [32]. Unfortunately, few studies have been made of thylakoid phosphoproteins and protein kinases during chloroplast development in higher plants. In this paper the changes in thylakoid polypeptide phosphorylation that occur during chloroplast biogenesis in greening etiolated and light-grown wheat leaves are examined. The greening etiolated leaf offers a developmental system in which the LHCII apoproteins, the major substrates for phosphorylation in mature thylakoids, accumulate after the appearance of photosystern II electron transport activities [4, 25] and consequently after many other functional thylakoid polypeptides. The pattern of LHCII accumulation in light-grown leaves is considerably different with LHCII being present and functional at early stages of development and then increasing in concert with photosystem II complexes [4-6, 13, 34].
Materials and methods
Seeds of wheat (Triticum aestivum var Broom) were soaked in running water for 17 h before sowing. For etiolated plants seeds were sown on Levington compost, germinated in the dark under moist paper towels and grown in darkness at 25 °C for 6 days. Etiolated plants were greened at 23 °C by exposing the plants to white light with a photosynthetically active (400-700nm) photon flux density of 200/~mol m -2 s -i, measured at the top of the canopy. After harvesting, 1-cm segments were removed from the leaf tips and the next 4-cm segment of tissue used for thylakoid isolations. For light-grown plants, seeds were sown in a 50% soil-vermiculite mixture and plants were grown at 23 °C in a 16-h photoperiod using white light with a photosynthetically active photon flux density of 250/~molm -2 s -~ at the top of the canopy. A gradient of cellular and plastid development exists from the base to the tip of the leaf [14]. Primary leaves from light-grown plants were harvested after 4 days and cut into segments, which were stored on ice during the time required to harvest sufficient leaf material for experiments. Thylakoid membranes were routinely isolated as previously described [35] except that the resulting pellet was resuspended and washed in a medium containing 50 mM HEPES, 10 mM NaC1, 5 mM MgC12 at pH 7.6, then repelleted by centrifugation at 3000g for 2 min and finally suspended in this medium. In order to obtain thylakoid membrane preparations with minimal contamination from organelles and microorganisms an isolation procedure based upon that described by Hoyer-Hansen and Simpson [21] was used. Leaf material was homogenized and filtered as in the routine preparation of thylakoids. The homogenate was centrifuged at 1400g for 5 rain. The pellet was resuspended in isolation medium and centrifuged at 1400g for 5 min. The pellet was then resuspended in 25 mM HEPES, 5 mM EDTA at pH 7.6 and centrifuged at 12,000g for 10min. This resuspension and centrifugation procedure was then repeated. The resulting pellet was resuspended in 5-15cm 3 of 1.9 M sucrose,
245 25 mM HEPES, 5 mm EDTA, pH 7.6. Ultracentrifuge tubes were loaded with 5 cm 3 of suspension which was then overlayed with 0.9 M sucrose, 25 mM HEPES, 5raM EDTA, pH 7.6 and centrifuged at 145,000g for I h. The membrane material at the 0.9-1.9 M sucrose interface was collected and diluted with about 40cm 3 of 50mM HEPES, 5mm MgC12, pH 7.6 and centrifuged at 12,000g for 10rain. The pellet was resuspended in 50-500mm 3 of the same medium. All thylakoid isolation and purification procedures were carried out at 4°C. To phosphorylate ptastid membrane proteins samples containing 375 pg of protein were incubated at 30 °C in a reaction medium similar to the resuspension medium but containing 100 #M ATP, including 8-40/~Ci of y-32p-ATP mol- 1at pH 7.6 in a 125-mm 3 volume. The incubations were carried out either under a white light of photon fluxdensity of 30/~motm-Zs -1 or in the dark. The phosphorylation reaction was stopped by adding 25ram 3 of concentrated PAGE sample buffer (250raM Tris-HCL, 250raM DTT, 10% SDS, 60% glycerol, 0.2% bromophenol blue, pH 7.6). Membrane proteins from etioplasts and plastids of greening etiolated leaves were fractionated by electrophoresis on polyacrylamide gels as described by Chua [16] using the buffer system of Neville [30]. Polypeptides were stained with Coomassie brilliant blue R-250. Thylakoid proteins from light-grown leaves were fractionated by PAGE using a modification of the method of Laemmli [24] which utilized a linear gradient of 8-15 % acrylamide stabilized by a 4-8-M urea gradient in the resolving gel. Polypeptides were examined by silver staining [29]. Phosphoproteins on polyacrylamide gels were visualized by autoradiography using Kodak X-Omat S X-ray film with an intensifying screen at - 7 0 °C. Quantitation of the amount of 32p incorporated into specific polypeptides was determined by excising the polypeptides from the gels and counting as previously described [28]. Assays of thylakoid protein kinase activity were performed in triplicate as previously described [27, 28]. Incorporation of 32p was linear for the 5-rain incubation period. Phosphorylation of etioplast and thylakoid membrane proteins was also examined in vivo. Two-day-old seedlings, supported by polystyrene beads, were floated on 7 cm 3 of distilled water containing 50 #Ci of 32PO]- and kept in the dark or exposed to 16-h photoperiod for 5 days. Leaves were then homogenized at 4 °C with a mortar and pestle in 5 cm 3 of 50 mM HEPES, 400 mM sucrose, 10 mM NaF, 10 mM EDTA, 0.2% bovine serum albumin and 2% polyvinylpyrollidone at pH 7.6. The homogenage was filtered through a 5-cm 3 syringe containing cotton wool and the filtrate centrifuged at 3350 g for 2 min. The pellet was resuspended, washed and recentrifuged twice using 50raM HEPES, 10 mm NaF, 10 mM EDTA at pH 7.6. The resulting membrane suspension was made up to 3.0 mg protein cm -3 and then diluted to 2.4rag with concentrated PAGE sample buffer (see above) prior to electrophoretic analysis. Total chlorophyll concentrations were determined by the method of Arnon [3]. Protein concentrations were measured by a modification [18] of the method
246 of Lowry et al. [26] except that absorbance following colour development was determined at 720 nm to avoid interference by chlorophyll.
Results
Polypeptide profiles and associated autoradiograms of thylakoids, which had been isolated from etiolated leaves after 0, 4 and 8 h exposure to white light and incubated with ATP in the light and dark, are shown in Fig. 1. Large changes in both the polypeptide and phosphoprotein profiles are observed with the transition from etioplast to chloroplast. In the membranes isolated from etioplasts the major phosphoproteins of c a . 17 and 22 kDa were also present. Light had little effect on the phosphorylation of these proteins. After 4 h of greening polypeptides of c a . 25-27 kDa, almost certainly the apoproteins of LHCII, became the major phosphoproteins and their phosphorylation was clearly lightdependent (Fig. 1B). Four other major phosphoproteins, having molecular masses of c a . 17, 22, 45 and 54 kDa and some degree of light regulation on their phosphorylation, but considerably less than observed for the LHCII apoproteins, were also evident. After 8 h of greening the LHCII apoproteins were by far the most dominant phosphoproteins and exhibited a marked light-dependent phosphorylation (Fig. 1C). A number of other phosphoproteins were observed; of note is a polypeptide of c a . 17kDa. It is of concern that at the early stages of thylakoid development found in etioplasts and in etiolated leaves greened for 4 and 8 h, the major stained
Fig. 1. Pol)q~eptide profiles (tracks labelled 1) of-thylakoids isolated from etiolated leaves after 0 (A), 4 (B) and 8 (C) hours exposure to white light. Autoradiograms (tracks labelled 2 and 3) of the polypeptide profiles are shown for thylakoids incubated with Y-32P-ATP in the light (tracks labelled 2) and the dark (tracks labelled 3). Numbers given at the left of the gels indicate the migration of marker proteins of known molecular weights (given in kDa).
247 polypeptides were found in the 14-20 kDa molecular mass range (Fig. 1). The presence of large amounts of such proteins in etioplast membrane preparations has been shown to be indicative of nuclear contamination [17, 21]. If nuclear or other contamination of thylakoid isolates was occurring at early stages of chloroplast biogenesis, the possibilities arise that some of the observed phosphoproteins may be non-chloroplastic in origin, i.e. not thylakoid proteins, and that phosphorylation of some thylakoid proteins may be produced by the activities of nuclear or other contaminating protein kinases. Purification of membrane preparations from etioptasts and developing chloroplasts using sucrose density gradient centrifugation, in a method modified from Hoyer-Hansen and Simpson [21], was found to remove a large proportion of polypeptides in the 14-20 kDa range (Fig. 2A). Also a number of higher molecular mass polypeptides were lost (Fig. 2A). In purified membrane preparations from etioplasts, whilst there is an increased proportion of a doublet at 35-36kDa and also at 58-59kDa, the 35 kDa polypeptide was still found to be the major phosphoprotein (Fig. 2A). Analyses of purified thylakoids isolated from greening etiolated leaves demonstrated the presence of a number of major phosphoproteins (Figs 2B,C) i.e. molecular mass c a . 9, 27, 32, 34, 40, 42 and 56 kDa, many of which had not been found in experiments with the 'crude' thylakoid preparations (see Fig. 1). It should also be noted that a c a . 60% decrease in total thylakoid kinase activity was observed in purified compared to 'crude' membrane preparations; it is not
Fig, 2. Polypeptide profiles of 'crude' (track Ala) and 'purified' (track Alb) membranes isolated from etiolated leaves; the autoradiogram of the polypeptide profile of 'purified' membranes (track A2) incubated with Y-nP-ATP in the light is also shown, Polypeptide profiles (tracks labelled 1) and the associated autoradiograms (tracks labelled 2) of light-incubated thylakoids isolated from etiolated leaves greened for 4 (B) and 8 (C) hours under white light. Numbers given at the left of the gels indicate the migration of marker proteins of known molecular weights (given in kDA).
248 known whether this decrease in enzyme activity is due to inactivation of thylakoid kinases, prevention of the kinases from reaching their substrates or removal of some kinases during the purification procedure, which takes ca. 2 hours longer than the preparation of 'crude' membranes. The very low yields of membranes obtained using the purification procedure unfortunately make this method unsuitable for routine, reproducible studies on small quantities of developing tissues. Phosphorylation of thylakoids isolated from the basal 0.5-cm and tip 2-cm segments of 4-day-old wheat leaves grown under a diurnal light regime demonstrated that the LHCII apoproteins and a 9 kDa polypeptide were the major phosphoproteins throughout chloroplast development. Since the wheat leaf has a basal meristem the basal 0.5 cm of the leaf represents a very early stage of leaf and chloroplast development. The leaf tip provides a much later stage of clevelopment and although photosynthetically active this tissue is not fully mature, as indicated by a chlorophyll content of 48#gcm -2 compared to c a . 60#gcm -2 in mature tissue, it does exhibit similar thylakoid polypeptide and phosphoprotein profiles to mature leaf tissue. It is of note that in thytakoids from the leaf base the 9 kDa polypeptide was the predominant phosphoprotein, whilst the LHCII apoproteins are dominant at the leaf tip (Fig. 3). Although
Fig. 3. Polypeptide profiles of thylakoids isolated from the basal 0.5cm (track A1) and tip 2cm (track B1) segments of 4-day-old leaves grown under a diurnal light regime. Autoradiograms of these polypeptide profiles are shown for thylakoids incubated with Y-32p-ATP in the light (tracks A2 and B2) and the dark (tracks A3 and B3).
249 phosphorylation of the LHCII apoproteins was found to be light regulated for thylakoids isolated from the leaf tip, this did not appear to be the case for base thylakoids (Fig. 3). Excision and counting of the LHCII apoprotein and 9 kDa polypeptide bands from the gels showed quantitatively that in thylakoids isolated from the leaf base phosphorylation of the LHCII apoproteins and the 9 kDa polypeptide was not light regulated, whereas in thylakoids isolated from the leaf tip only phosphorylation of the 9 kDa polypeptide remained insensitive to light (Table I). The absence of fight-regulation of LHCII apoprotein phosphorylation in thylakoids from the leaf base may be due to contamination of the thylakoid preparation with non-chloroplastic protein kinases which were capable of using the LHCII apoproteins as substrates but would not exhibit a light regulation. Unfortunately, it proved difficult to repeat these experiments on basal thylakoids isolated by the purification procedure described above; the membrane yields were extremely low from the basal 0.5-cm segments and precluded gel electrophoretic analyses of the polypeptides. However, determinations of total thylakoid protein kinase activities could be made from the purified thylakoid preparations. Total thylakoid kinase activity per unit of membrane protein was considerably less in thylakoids isolated from the leaf tip compared to the leaf base (Table 2). However, a marked light regulation was observed for the kinase activity in tip thylakoids which was not found for base thylakoids (Table 2), supporting the observation from the autoradiographs shown in Fig. 3 and data in Table 1 that LHCII phosphorylation in thylakoids at the leaf base does not exhibit a marked control by light. The possibility that inactivation of kinases may occur during the lengthy thylakoid purification procedure and give rise to these results should not be ignored. Consequently, a second, more rapid procedure was used to strip exogenous proteins from the surfaces of 'crude' thylakoids by washing the thylakoids with a solution containing 150 Mm NaCI and 5 mM EDTA. This procedure removes proteins loosely bound to the membranes as evidenced by the loss of ribulose 1,5 bisphosphate carboxylase. After this treatment thylakoids from the leaf base still exhibited negligible light-regulation of protein kinase activity. However, thylakoids prepared from the leaf tip by this procedure showed an increased light-regulation of kinase Table 1. Incorporation of 32p into the 9 kDa and LHCII apoproteins in thylakoids from 0.5-cm segments at the base and 2-cm segments at the tip of 4-day old wheat leaves grown under a diurnal light regime. Thylakoids were incubated with "f-32P-ATP in the light and in the dark as described in the Materials and Methods. Standard errors of the means are given for triplicate determinations.
Protein
Treatment
32p incorporated (dpm) base 0.5cm
tip 2cm
LHCII apoprotein
Light Dark
600 _+ 40 590 _ 50
2490 + 220 440 _+ 50
9kDa protein
Light Dark
2880 __+ 210 3310 _+ 230
1490 _ I00 1640 _+ 150
250 Table 2. Light regulation of protein kinase activity of thylakoids isolated from the base and tip of 4-day-old wheat leaves grown under a diurnal light regime. Thylakoids were either purified using a sucrose density gradient technique or salt-washed to remove extrinsic proteins as described in the text. Assays were performed in the light and the dark. Standard errors of the mean are given for triplicate determinations.
Procedure
Leaf segment
Protein kinase activity (pmol ATP min- t mg-i protein) Light
Dark
Sucrose density gradient
Base lcm Tip 2cm
19.3 -I- 1.8 5.6 ± 0.2
17.9 ___ 1.1 2.3 ± 0.2
Salt wash
Base 1cm Tip 2cm
68.4 __+2.3 19.5 ± 0.3
62.9 ± 1.6 2.7 ± 0.4
activity c o m p a r e d to thylakoids purified by the lengthy sucrose density gradient m e t h o d (Table 2). These data support the hypothesis that light-regulation o f L H C I I p h o s p h o r y l a t i o n is absent in the leaf base. In order to determine whether the 35 k D a m e m b r a n e polypeptide in etioplasts and the L H C I I apoproteins and the 9 k D a polypeptide in chloroplasts were phosphorylated in vivo, the roots o f etiolated and light-grown plants were fed with 3 2 p o 3 for 5 days prior to thylakoid isolation and polypeptide fractionation. Figure 4A demonstrates that in etiolated leaves the 35 k D a polypeptide is the m a j o r thylakoid p h o s p h o p r o t e i n and that its p h o s p h o r y l a t i o n in vitro is
Fig. 4. Polypeptide profiles (A1 and B1) and their respective autoradiograms (A2 and B2) of
thylakoids isolated from the leaves ofetiolated (A) and light-grown (B) plants whose roots bad been supplied with 32po]- for 5 days prior to the isolation.
251 unlikely to be due to a non-chloroplastic kinase. It would appear that two other etioplast membrane polypeptides of c a . 30 and 22 kDa molecular mass are also phosphorylated in vivo. In the light-grown green leaf the LHCII apoproteins and the 9 kDa polypeptide were found to be the major phosphoproteins as expected (Fig 4B.) Polypeptides at 32 kDa and 34 kDa were also found to be phosphorylated in vivo in wheat thylakoids.
Discussion
The results presented demonstrate some of the difficulties encountered when attempting to determine the role of thylakoid protein phosphorylation during chloroplast biogenesis. It cannot be overemphasized that~it is essential to remove contaminants from thylakoids isolated from developing tissues before phosphorylating the membrane proteins in vitro. If this is not done then phosphorylation of contaminating non-thylakoid proteins, especially histones, by thylakoid kinases and possibly phosphorylation of endogenous thylakoid proteins by contaminating non-thylakoid kinases can produce misleading results. Unfortunately, the procedure found to be most effective for purifying the developing wheat thylakoids is time consuming and consequently results in a decrease in thylakoid kinase activity. Also the procedure produces only low yields of thylakoids. Although these difficulties severely limited the experiments that could be performed with developing thylakoids, a number of important points have emerged from these studies. In etioplasts the major membrane phosphoprotein both in vivo and in vitro was a 35 kDa polypeptide, which appears to be the most abundant polypeptide in the membranes. This polypeptide is probably NADPH:protochlorophyllide oxidoreductase, an enzyme known to be present in large amounts in etioplasts [2, 17, 31]. During greening of the etioplast the LHCII apoproteins become the major phosphoproteins, presumably due to the increasing accumulation of these proteins in the thylakoids. Also a number of other changes in the phosphoprotein profile of the etioplast membranes, as seen in Fig. 2, occur during greening. Such changes may be important in the context of membrane component synthesis and organization; further parallel structure-function studies are required to examine such a possibility. In wheat leaves grown under a natural diurnal light regime the LHCII apoproteins and the 9 kDa polypeptide were found to be the major thylakoid phosphoproteins both in vivo and in vitro. However, these developing thytakoids did exhibit some important differences with respect to protein phosphorylation compared to those in greening etioplasts. At early stages of chloroplast development in the light-grown leaf the major thylakoid phosphoprotein was the 9kDa polypeptide and not the LHCII apoproteins. Although it is possible that this observation can be explained by an increasing stoichiometlV between the LHCII and PSII complexes during development, this would seem unlikely since previous studies have demonstrated that the interactions between
252 LHCiI and PSII complexes in terms of excitation energy transfer remain constant throughout chloroplast development in the light-grown wheat leaf [6, 13, 33, 34], implying a relatively similar ratio of LHCII to PSII. Since the LHCII apoproteins and the 9 kDa polypeptide are thought to be phosphorylated by different kinases [19, 28], a more plausible explanation of the data is that there is a greater amount of the kinase which phosphorylates the 9 kDa polypeptide relative to that responsible for LHCII apoprotein phosphorylation at early stages of development. A third possibility is that in developing thylakoids the 9 kDa polypeptide may be more accessible or the LHCII apoproteins less accessible to their respective kinases. Another interesting feature of thylakoids at early stages of development in the light-grown leaf is the absence of light regulation of the phosphorylation of the LHCII apoproteins. Light control of the LHCII kinase activity is thought to be mediated by the redox state of plastoquinone [1, 23]. Plastoquinone is present in thylakoids at the base of the wheat leaf and is at least partially oxidized in the dark and it can be reduced by excitation of PSII on transfer from dark to light, as demonstrated by the kinetics of variable fluorescence induction and the ability of these thylakoids to transfer electrons from PSII to PSI [35]. Thus, the lack of light-regulation of the kinase cannot be attributed to an inability to generate reduced plastoquinone on transfer from dark to light. In the absence of any uladerstanding of the molecular mechanisms by which the redox state of plastoquinone can activate the LHCII kinase, it is difficult to produce a satisfactory explanation of why there is no light regulation of the kinase in the leaf base. Another feature of this developing leaf system is the absence of any light-regulation of phosphorylation of the 9 kDa polypeptide throughout chloroplast development. The lack of light control on phosphorylation of this protein at later stages of chloroplast development when phosphorylation of the LHCII apoproteins is clearly light regulated supports the hypothesis that two different kinases are responsible for the phosphorylation of LHCII apoproteins and the 9 kDa polypeptide [19].
Aenowledgements This research was supported by grants from the National Science Foundation (DMB-84-04218) to JPM and from the UK Science and Engineering Research Council (GR/B/66738) to NRB. ANW and PSC were the recipients of a Wain Fellowship from the UK Agricultural Research Council and a post-graduate scholarship from the Natural Sciences and Engineering Research Council of Canada respectively.
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243
Regular paper
Phosphorylation of thylakoid proteins during chloroplast biogenesis in greening efiolated and light-grown wheat leaves P A T R I C K S. C O V E L L O , 1 A N D R E W N. WEBBER, 1'2'3 S T E P H E N J. D A N K O , 2 J O H N P. M A R K W E L L 2 & N E I L R. BAKER1, * ~Department of Biology, University of Essex, Colchester C04 3SQ, Essex, UK; ~Department of Agricultural Biochemistry, University of Nebraska, Lincoln, .Nebraska 68583, USA; 3Current address: Botany School., University of Cambridge, Downing Street, Cambridge CB2 3EA, UK (*author for correspondence and offprints) Received 19 November 1986; accepted in revised form 6 January t987
Abstract.Phosphorylation of polypeptidesin isolated thylakoids was examined during chloroplast biogenesisin greeningetiolated wheat leavesand 4 day-old wheat leavesgrown under a diurnal light regime. At early stages of plastid development standard thylakoid preparations were heavily contaminated with nuclear proteins, which distorted the pob~peptide phosphorylation profiles, Removal of contamination from membranes by sucrose density centrifugation demonstrated that the major membrane phosphoprotein in etioplasts was at 35kDa. During etioplast greening a number of phosphoproteins appeared, of which the 25-27 kDa apoproteins of the light-harvesting chlorophyll a/b protein complex associated with photosystem II (LHCII) became the most dominant. At the early stages of thylakoid development found at the base of the 4-day-old light grown leaf the LHCII apoproteins were evident as phosphoproteins; however the major phosphoprotein was polypeptide at ca. 9 kDA. Phosphorylation of both the LHCII apoproteins and the 9 kDa polypeptide in these thylakoids was not light-dependent. In the older thylakoids isolated from the leaf tip the LHCII apoproteins were the major phosphoproteins and their phosphorylation had 'become light-regulated; howeverphosphorylation of the 9 kDa polypeptide remained insensitive to light.
Introduction M a n y endogenous proteins o f the thylakoid membrane are phosphorylated by m e m b r a n e - b o u n d protein kinases [11]. During short incubations (2-10min) of mature thylakoids with A T P the major polypeptides phosphorylated are the apoproteins of the light-harvesting chlorophyll a/b complex associated with photosystem I I ( L H C I I ) , which are in the 23-29 k D a molecular weight range, and a smaller polypeptide with a molecular weight of less than l0 k D a [9, t0-12, 19, 20, 22]. The phosphorylation of the L H C I I apoproteins is light regulated and has been implicated as a mechanism for regulating the distribution o f excitation energy within the photosynthetic apparatus [7-9, 11, 12, 20, 22]; reduction o f plastoquinone by photosynthetic electron transport is thought to be responsible for the activation of the L H C I I protein kinase [1, 2, 3]. However, the physiological role of phosphorylation of the large number of other thylakoid polypeptides is unclear. The observation o f greater thylakoid protein kinase activities, on a unit membrane protein basis, in etioplasts and developing plastids compared to in mature chloroplasts [5, 6] has offered support to the suggestion that thylakoid
244 polypeptide phosphorylation may be important in the biosynthesis and structural organization of the photosynthetic apparatus [32]. Unfortunately, few studies have been made of thylakoid phosphoproteins and protein kinases during chloroplast development in higher plants. In this paper the changes in thylakoid polypeptide phosphorylation that occur during chloroplast biogenesis in greening etiolated and light-grown wheat leaves are examined. The greening etiolated leaf offers a developmental system in which the LHCII apoproteins, the major substrates for phosphorylation in mature thylakoids, accumulate after the appearance of photosystern II electron transport activities [4, 25] and consequently after many other functional thylakoid polypeptides. The pattern of LHCII accumulation in light-grown leaves is considerably different with LHCII being present and functional at early stages of development and then increasing in concert with photosystem II complexes [4-6, 13, 34].
Materials and methods
Seeds of wheat (Triticum aestivum var Broom) were soaked in running water for 17 h before sowing. For etiolated plants seeds were sown on Levington compost, germinated in the dark under moist paper towels and grown in darkness at 25 °C for 6 days. Etiolated plants were greened at 23 °C by exposing the plants to white light with a photosynthetically active (400-700nm) photon flux density of 200/~mol m -2 s -i, measured at the top of the canopy. After harvesting, 1-cm segments were removed from the leaf tips and the next 4-cm segment of tissue used for thylakoid isolations. For light-grown plants, seeds were sown in a 50% soil-vermiculite mixture and plants were grown at 23 °C in a 16-h photoperiod using white light with a photosynthetically active photon flux density of 250/~molm -2 s -~ at the top of the canopy. A gradient of cellular and plastid development exists from the base to the tip of the leaf [14]. Primary leaves from light-grown plants were harvested after 4 days and cut into segments, which were stored on ice during the time required to harvest sufficient leaf material for experiments. Thylakoid membranes were routinely isolated as previously described [35] except that the resulting pellet was resuspended and washed in a medium containing 50 mM HEPES, 10 mM NaC1, 5 mM MgC12 at pH 7.6, then repelleted by centrifugation at 3000g for 2 min and finally suspended in this medium. In order to obtain thylakoid membrane preparations with minimal contamination from organelles and microorganisms an isolation procedure based upon that described by Hoyer-Hansen and Simpson [21] was used. Leaf material was homogenized and filtered as in the routine preparation of thylakoids. The homogenate was centrifuged at 1400g for 5 rain. The pellet was resuspended in isolation medium and centrifuged at 1400g for 5 min. The pellet was then resuspended in 25 mM HEPES, 5 mM EDTA at pH 7.6 and centrifuged at 12,000g for 10min. This resuspension and centrifugation procedure was then repeated. The resulting pellet was resuspended in 5-15cm 3 of 1.9 M sucrose,
245 25 mM HEPES, 5 mm EDTA, pH 7.6. Ultracentrifuge tubes were loaded with 5 cm 3 of suspension which was then overlayed with 0.9 M sucrose, 25 mM HEPES, 5raM EDTA, pH 7.6 and centrifuged at 145,000g for I h. The membrane material at the 0.9-1.9 M sucrose interface was collected and diluted with about 40cm 3 of 50mM HEPES, 5mm MgC12, pH 7.6 and centrifuged at 12,000g for 10rain. The pellet was resuspended in 50-500mm 3 of the same medium. All thylakoid isolation and purification procedures were carried out at 4°C. To phosphorylate ptastid membrane proteins samples containing 375 pg of protein were incubated at 30 °C in a reaction medium similar to the resuspension medium but containing 100 #M ATP, including 8-40/~Ci of y-32p-ATP mol- 1at pH 7.6 in a 125-mm 3 volume. The incubations were carried out either under a white light of photon fluxdensity of 30/~motm-Zs -1 or in the dark. The phosphorylation reaction was stopped by adding 25ram 3 of concentrated PAGE sample buffer (250raM Tris-HCL, 250raM DTT, 10% SDS, 60% glycerol, 0.2% bromophenol blue, pH 7.6). Membrane proteins from etioplasts and plastids of greening etiolated leaves were fractionated by electrophoresis on polyacrylamide gels as described by Chua [16] using the buffer system of Neville [30]. Polypeptides were stained with Coomassie brilliant blue R-250. Thylakoid proteins from light-grown leaves were fractionated by PAGE using a modification of the method of Laemmli [24] which utilized a linear gradient of 8-15 % acrylamide stabilized by a 4-8-M urea gradient in the resolving gel. Polypeptides were examined by silver staining [29]. Phosphoproteins on polyacrylamide gels were visualized by autoradiography using Kodak X-Omat S X-ray film with an intensifying screen at - 7 0 °C. Quantitation of the amount of 32p incorporated into specific polypeptides was determined by excising the polypeptides from the gels and counting as previously described [28]. Assays of thylakoid protein kinase activity were performed in triplicate as previously described [27, 28]. Incorporation of 32p was linear for the 5-rain incubation period. Phosphorylation of etioplast and thylakoid membrane proteins was also examined in vivo. Two-day-old seedlings, supported by polystyrene beads, were floated on 7 cm 3 of distilled water containing 50 #Ci of 32PO]- and kept in the dark or exposed to 16-h photoperiod for 5 days. Leaves were then homogenized at 4 °C with a mortar and pestle in 5 cm 3 of 50 mM HEPES, 400 mM sucrose, 10 mM NaF, 10 mM EDTA, 0.2% bovine serum albumin and 2% polyvinylpyrollidone at pH 7.6. The homogenage was filtered through a 5-cm 3 syringe containing cotton wool and the filtrate centrifuged at 3350 g for 2 min. The pellet was resuspended, washed and recentrifuged twice using 50raM HEPES, 10 mm NaF, 10 mM EDTA at pH 7.6. The resulting membrane suspension was made up to 3.0 mg protein cm -3 and then diluted to 2.4rag with concentrated PAGE sample buffer (see above) prior to electrophoretic analysis. Total chlorophyll concentrations were determined by the method of Arnon [3]. Protein concentrations were measured by a modification [18] of the method
246 of Lowry et al. [26] except that absorbance following colour development was determined at 720 nm to avoid interference by chlorophyll.
Results
Polypeptide profiles and associated autoradiograms of thylakoids, which had been isolated from etiolated leaves after 0, 4 and 8 h exposure to white light and incubated with ATP in the light and dark, are shown in Fig. 1. Large changes in both the polypeptide and phosphoprotein profiles are observed with the transition from etioplast to chloroplast. In the membranes isolated from etioplasts the major phosphoproteins of c a . 17 and 22 kDa were also present. Light had little effect on the phosphorylation of these proteins. After 4 h of greening polypeptides of c a . 25-27 kDa, almost certainly the apoproteins of LHCII, became the major phosphoproteins and their phosphorylation was clearly lightdependent (Fig. 1B). Four other major phosphoproteins, having molecular masses of c a . 17, 22, 45 and 54 kDa and some degree of light regulation on their phosphorylation, but considerably less than observed for the LHCII apoproteins, were also evident. After 8 h of greening the LHCII apoproteins were by far the most dominant phosphoproteins and exhibited a marked light-dependent phosphorylation (Fig. 1C). A number of other phosphoproteins were observed; of note is a polypeptide of c a . 17kDa. It is of concern that at the early stages of thylakoid development found in etioplasts and in etiolated leaves greened for 4 and 8 h, the major stained
Fig. 1. Pol)q~eptide profiles (tracks labelled 1) of-thylakoids isolated from etiolated leaves after 0 (A), 4 (B) and 8 (C) hours exposure to white light. Autoradiograms (tracks labelled 2 and 3) of the polypeptide profiles are shown for thylakoids incubated with Y-32P-ATP in the light (tracks labelled 2) and the dark (tracks labelled 3). Numbers given at the left of the gels indicate the migration of marker proteins of known molecular weights (given in kDa).
247 polypeptides were found in the 14-20 kDa molecular mass range (Fig. 1). The presence of large amounts of such proteins in etioplast membrane preparations has been shown to be indicative of nuclear contamination [17, 21]. If nuclear or other contamination of thylakoid isolates was occurring at early stages of chloroplast biogenesis, the possibilities arise that some of the observed phosphoproteins may be non-chloroplastic in origin, i.e. not thylakoid proteins, and that phosphorylation of some thylakoid proteins may be produced by the activities of nuclear or other contaminating protein kinases. Purification of membrane preparations from etioptasts and developing chloroplasts using sucrose density gradient centrifugation, in a method modified from Hoyer-Hansen and Simpson [21], was found to remove a large proportion of polypeptides in the 14-20 kDa range (Fig. 2A). Also a number of higher molecular mass polypeptides were lost (Fig. 2A). In purified membrane preparations from etioplasts, whilst there is an increased proportion of a doublet at 35-36kDa and also at 58-59kDa, the 35 kDa polypeptide was still found to be the major phosphoprotein (Fig. 2A). Analyses of purified thylakoids isolated from greening etiolated leaves demonstrated the presence of a number of major phosphoproteins (Figs 2B,C) i.e. molecular mass c a . 9, 27, 32, 34, 40, 42 and 56 kDa, many of which had not been found in experiments with the 'crude' thylakoid preparations (see Fig. 1). It should also be noted that a c a . 60% decrease in total thylakoid kinase activity was observed in purified compared to 'crude' membrane preparations; it is not
Fig, 2. Polypeptide profiles of 'crude' (track Ala) and 'purified' (track Alb) membranes isolated from etiolated leaves; the autoradiogram of the polypeptide profile of 'purified' membranes (track A2) incubated with Y-nP-ATP in the light is also shown, Polypeptide profiles (tracks labelled 1) and the associated autoradiograms (tracks labelled 2) of light-incubated thylakoids isolated from etiolated leaves greened for 4 (B) and 8 (C) hours under white light. Numbers given at the left of the gels indicate the migration of marker proteins of known molecular weights (given in kDA).
248 known whether this decrease in enzyme activity is due to inactivation of thylakoid kinases, prevention of the kinases from reaching their substrates or removal of some kinases during the purification procedure, which takes ca. 2 hours longer than the preparation of 'crude' membranes. The very low yields of membranes obtained using the purification procedure unfortunately make this method unsuitable for routine, reproducible studies on small quantities of developing tissues. Phosphorylation of thylakoids isolated from the basal 0.5-cm and tip 2-cm segments of 4-day-old wheat leaves grown under a diurnal light regime demonstrated that the LHCII apoproteins and a 9 kDa polypeptide were the major phosphoproteins throughout chloroplast development. Since the wheat leaf has a basal meristem the basal 0.5 cm of the leaf represents a very early stage of leaf and chloroplast development. The leaf tip provides a much later stage of clevelopment and although photosynthetically active this tissue is not fully mature, as indicated by a chlorophyll content of 48#gcm -2 compared to c a . 60#gcm -2 in mature tissue, it does exhibit similar thylakoid polypeptide and phosphoprotein profiles to mature leaf tissue. It is of note that in thytakoids from the leaf base the 9 kDa polypeptide was the predominant phosphoprotein, whilst the LHCII apoproteins are dominant at the leaf tip (Fig. 3). Although
Fig. 3. Polypeptide profiles of thylakoids isolated from the basal 0.5cm (track A1) and tip 2cm (track B1) segments of 4-day-old leaves grown under a diurnal light regime. Autoradiograms of these polypeptide profiles are shown for thylakoids incubated with Y-32p-ATP in the light (tracks A2 and B2) and the dark (tracks A3 and B3).
249 phosphorylation of the LHCII apoproteins was found to be light regulated for thylakoids isolated from the leaf tip, this did not appear to be the case for base thylakoids (Fig. 3). Excision and counting of the LHCII apoprotein and 9 kDa polypeptide bands from the gels showed quantitatively that in thylakoids isolated from the leaf base phosphorylation of the LHCII apoproteins and the 9 kDa polypeptide was not light regulated, whereas in thylakoids isolated from the leaf tip only phosphorylation of the 9 kDa polypeptide remained insensitive to light (Table I). The absence of fight-regulation of LHCII apoprotein phosphorylation in thylakoids from the leaf base may be due to contamination of the thylakoid preparation with non-chloroplastic protein kinases which were capable of using the LHCII apoproteins as substrates but would not exhibit a light regulation. Unfortunately, it proved difficult to repeat these experiments on basal thylakoids isolated by the purification procedure described above; the membrane yields were extremely low from the basal 0.5-cm segments and precluded gel electrophoretic analyses of the polypeptides. However, determinations of total thylakoid protein kinase activities could be made from the purified thylakoid preparations. Total thylakoid kinase activity per unit of membrane protein was considerably less in thylakoids isolated from the leaf tip compared to the leaf base (Table 2). However, a marked light regulation was observed for the kinase activity in tip thylakoids which was not found for base thylakoids (Table 2), supporting the observation from the autoradiographs shown in Fig. 3 and data in Table 1 that LHCII phosphorylation in thylakoids at the leaf base does not exhibit a marked control by light. The possibility that inactivation of kinases may occur during the lengthy thylakoid purification procedure and give rise to these results should not be ignored. Consequently, a second, more rapid procedure was used to strip exogenous proteins from the surfaces of 'crude' thylakoids by washing the thylakoids with a solution containing 150 Mm NaCI and 5 mM EDTA. This procedure removes proteins loosely bound to the membranes as evidenced by the loss of ribulose 1,5 bisphosphate carboxylase. After this treatment thylakoids from the leaf base still exhibited negligible light-regulation of protein kinase activity. However, thylakoids prepared from the leaf tip by this procedure showed an increased light-regulation of kinase Table 1. Incorporation of 32p into the 9 kDa and LHCII apoproteins in thylakoids from 0.5-cm segments at the base and 2-cm segments at the tip of 4-day old wheat leaves grown under a diurnal light regime. Thylakoids were incubated with "f-32P-ATP in the light and in the dark as described in the Materials and Methods. Standard errors of the means are given for triplicate determinations.
Protein
Treatment
32p incorporated (dpm) base 0.5cm
tip 2cm
LHCII apoprotein
Light Dark
600 _+ 40 590 _ 50
2490 + 220 440 _+ 50
9kDa protein
Light Dark
2880 __+ 210 3310 _+ 230
1490 _ I00 1640 _+ 150
250 Table 2. Light regulation of protein kinase activity of thylakoids isolated from the base and tip of 4-day-old wheat leaves grown under a diurnal light regime. Thylakoids were either purified using a sucrose density gradient technique or salt-washed to remove extrinsic proteins as described in the text. Assays were performed in the light and the dark. Standard errors of the mean are given for triplicate determinations.
Procedure
Leaf segment
Protein kinase activity (pmol ATP min- t mg-i protein) Light
Dark
Sucrose density gradient
Base lcm Tip 2cm
19.3 -I- 1.8 5.6 ± 0.2
17.9 ___ 1.1 2.3 ± 0.2
Salt wash
Base 1cm Tip 2cm
68.4 __+2.3 19.5 ± 0.3
62.9 ± 1.6 2.7 ± 0.4
activity c o m p a r e d to thylakoids purified by the lengthy sucrose density gradient m e t h o d (Table 2). These data support the hypothesis that light-regulation o f L H C I I p h o s p h o r y l a t i o n is absent in the leaf base. In order to determine whether the 35 k D a m e m b r a n e polypeptide in etioplasts and the L H C I I apoproteins and the 9 k D a polypeptide in chloroplasts were phosphorylated in vivo, the roots o f etiolated and light-grown plants were fed with 3 2 p o 3 for 5 days prior to thylakoid isolation and polypeptide fractionation. Figure 4A demonstrates that in etiolated leaves the 35 k D a polypeptide is the m a j o r thylakoid p h o s p h o p r o t e i n and that its p h o s p h o r y l a t i o n in vitro is
Fig. 4. Polypeptide profiles (A1 and B1) and their respective autoradiograms (A2 and B2) of
thylakoids isolated from the leaves ofetiolated (A) and light-grown (B) plants whose roots bad been supplied with 32po]- for 5 days prior to the isolation.
251 unlikely to be due to a non-chloroplastic kinase. It would appear that two other etioplast membrane polypeptides of c a . 30 and 22 kDa molecular mass are also phosphorylated in vivo. In the light-grown green leaf the LHCII apoproteins and the 9 kDa polypeptide were found to be the major phosphoproteins as expected (Fig 4B.) Polypeptides at 32 kDa and 34 kDa were also found to be phosphorylated in vivo in wheat thylakoids.
Discussion
The results presented demonstrate some of the difficulties encountered when attempting to determine the role of thylakoid protein phosphorylation during chloroplast biogenesis. It cannot be overemphasized that~it is essential to remove contaminants from thylakoids isolated from developing tissues before phosphorylating the membrane proteins in vitro. If this is not done then phosphorylation of contaminating non-thylakoid proteins, especially histones, by thylakoid kinases and possibly phosphorylation of endogenous thylakoid proteins by contaminating non-thylakoid kinases can produce misleading results. Unfortunately, the procedure found to be most effective for purifying the developing wheat thylakoids is time consuming and consequently results in a decrease in thylakoid kinase activity. Also the procedure produces only low yields of thylakoids. Although these difficulties severely limited the experiments that could be performed with developing thylakoids, a number of important points have emerged from these studies. In etioplasts the major membrane phosphoprotein both in vivo and in vitro was a 35 kDa polypeptide, which appears to be the most abundant polypeptide in the membranes. This polypeptide is probably NADPH:protochlorophyllide oxidoreductase, an enzyme known to be present in large amounts in etioplasts [2, 17, 31]. During greening of the etioplast the LHCII apoproteins become the major phosphoproteins, presumably due to the increasing accumulation of these proteins in the thylakoids. Also a number of other changes in the phosphoprotein profile of the etioplast membranes, as seen in Fig. 2, occur during greening. Such changes may be important in the context of membrane component synthesis and organization; further parallel structure-function studies are required to examine such a possibility. In wheat leaves grown under a natural diurnal light regime the LHCII apoproteins and the 9 kDa polypeptide were found to be the major thylakoid phosphoproteins both in vivo and in vitro. However, these developing thytakoids did exhibit some important differences with respect to protein phosphorylation compared to those in greening etioplasts. At early stages of chloroplast development in the light-grown leaf the major thylakoid phosphoprotein was the 9kDa polypeptide and not the LHCII apoproteins. Although it is possible that this observation can be explained by an increasing stoichiometlV between the LHCII and PSII complexes during development, this would seem unlikely since previous studies have demonstrated that the interactions between
252 LHCiI and PSII complexes in terms of excitation energy transfer remain constant throughout chloroplast development in the light-grown wheat leaf [6, 13, 33, 34], implying a relatively similar ratio of LHCII to PSII. Since the LHCII apoproteins and the 9 kDa polypeptide are thought to be phosphorylated by different kinases [19, 28], a more plausible explanation of the data is that there is a greater amount of the kinase which phosphorylates the 9 kDa polypeptide relative to that responsible for LHCII apoprotein phosphorylation at early stages of development. A third possibility is that in developing thylakoids the 9 kDa polypeptide may be more accessible or the LHCII apoproteins less accessible to their respective kinases. Another interesting feature of thylakoids at early stages of development in the light-grown leaf is the absence of light regulation of the phosphorylation of the LHCII apoproteins. Light control of the LHCII kinase activity is thought to be mediated by the redox state of plastoquinone [1, 23]. Plastoquinone is present in thylakoids at the base of the wheat leaf and is at least partially oxidized in the dark and it can be reduced by excitation of PSII on transfer from dark to light, as demonstrated by the kinetics of variable fluorescence induction and the ability of these thylakoids to transfer electrons from PSII to PSI [35]. Thus, the lack of light-regulation of the kinase cannot be attributed to an inability to generate reduced plastoquinone on transfer from dark to light. In the absence of any uladerstanding of the molecular mechanisms by which the redox state of plastoquinone can activate the LHCII kinase, it is difficult to produce a satisfactory explanation of why there is no light regulation of the kinase in the leaf base. Another feature of this developing leaf system is the absence of any light-regulation of phosphorylation of the 9 kDa polypeptide throughout chloroplast development. The lack of light control on phosphorylation of this protein at later stages of chloroplast development when phosphorylation of the LHCII apoproteins is clearly light regulated supports the hypothesis that two different kinases are responsible for the phosphorylation of LHCII apoproteins and the 9 kDa polypeptide [19].
Aenowledgements This research was supported by grants from the National Science Foundation (DMB-84-04218) to JPM and from the UK Science and Engineering Research Council (GR/B/66738) to NRB. ANW and PSC were the recipients of a Wain Fellowship from the UK Agricultural Research Council and a post-graduate scholarship from the Natural Sciences and Engineering Research Council of Canada respectively.
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