Planta
Planta (1983)159:151-158
9 Springer-Verlag 1983
Thylakoid protein kinase activity and associated control of excitation energy distribution during chloroplast biogenesis in wheat Nell R. Baker* 1, John P. Markwell 2, Michael Bradbury 1, Maxine G. Baker 1 and J. Philip Thornber 3 1 Department of Biology, University of Essex, Colchester CO4 3SQ, Essex, UK, 2 Department of Agricultural Biochemistry, University of Nebraska, Lincoln, Nebraska 68583, USA, and 3 Department of Biology and Molecular Biology Institute, University of California, Los Angeles, CA 90024, USA
Abstract. The activity of thylakoid protein kinase and the regulation of excitation energy distribution between photosystems I and II was examined during chloroplast biogenesis in light-grown Triticure a e s t i v u m (wheat) leaves. The specific activity of the thylakoid protein kinase decreased some sixfold during development from the young plastids at the base of the 7-d-old leaf to the mature chloroplasts at the leaf tip. Appreciable activity was also detected in plastids isolated from etiolated leaves. In mature chloroplasts the majority of phosphate was incorporated into the Mr=26,000 apo-proteins of the light-harvesting chlorophyll a/b-protein complex (LHCP). However, at early stages of chloroplast development and in the etioplast, the phosphate was predominantly incorporated into a polypeptide of Mr=9,000 dalton. Immature thylakoids, isolated from the base of the leaf, had relatively low concentrations of LHCP and could perform a State 1-State 2 transition, as demonstrated by ATP-induced quenching of photosystern II fluorescence. Analyses of photosystem I and photosystem II fluorescence-induction curves from intact leaf tissue demonstrated that this transition occurs in vivo at early stages of leaf development and, therefore, may play an important role in regulating energy transduction during chloroplast biogenesis. Key words: Chlorophyll fluorescence - Chloroplast development - Thylakoid protein kinase - T r i t i c u m (chloroplast biogenesis).
* To whom correspondence should be addressed Abbreviations. LHCP=light-harvesting chlorophyll a/b-protein complex; PSI, PSII =photosystem I, II
Introduction
In mature chloroplasts of higher plants the quantum yield of non-cyclic electron flow is constant over most of the visible light spectrum, even in spectral regions where photosystem II (PSII) i s preferentially excited (Myers 1971). This constancy has been explained by the existence of a physiological control to balance quantal distribution between photosystemI (PSI) and PSII which is often termed the State 1-State 2 transition (Barber 1976). In State 1, quanta are preferentially directed to PSII, whereas in State 2 an increased proportion of harvested quanta are directed to PSI; 10% of the quanta absorbed by PSII are thought to be diverted to PSI on transition from State 1 to State 2 (Bonaventura and Myers 1969). Recently, phosphorylation of the light-harvesting chlorophyll a/b-protein complex (LHCP) has been proposed for a major role in the control of the State l-State2 transition (Bennett etal. 1980; Allen et al. 1981; Chow et al. 1981; Horton and Black 1981 ; Horton et al. 1981 ; Telfer and Barber 1981). Phosphorylation of LHCP increases the density of negative surface charges on the thylakoids, which is proposed to increase interaction between LHCPPSII and PSI particles and increase excitation-energy spillover from LHCP-PSII to PSI (Barber 1982, 1983). The ability to undergo State 1-State 2 transitions could enable the photosynthetic apparatus to modify the rate of non-cyclic relative to cyclic photosynthetic electron transport and thereby regulate the ratio of A T P : N A D P H produced by the thylakoids. At early stages of leaf development, thylakoids become capable of light-induced, coupled electron transport and can produce ATP and N A D P H (Howes and Stern 1973; Plesnicar and Bendall
~52
N.R. Baker et al. : Protein-kinase activity in wheat chloroplasts
1973; Weistrop and Stern 1971; Duysen etal. 1980) despite having a qualitatively and quantitatively incomplete complement of chlorophyll-protein complexes (Argyroudi-Akoyunoglou and Akoyunoglou 1979; Kalosakas etal. 1981). The metabolic requirements of the leaf must change rapidly during expansion and greening and clearly it would be advantageous for the developing leaf to be able to regulate the ratio of ATP:reducing power generated by photosynthetic electron transport as early as possible. If current dogma is correct, to achieve this via a State 1-State 2 control, both the thylakoid protein kinase and LHCP would have to be present in the thylakoid membranes at early stages of development. During plasrid biogenesis in the majority of plants the chlorophyll a/b ratio rapidly decreases (e.g. De Greef et al. 1970; Thorne and Boardman 1971; Plesnicar and Bendall 1973; Weistrop and Stern 1977; Egneus et al. 1976) indicating that LHCP constitutes an increasing proportion of the total chlorophyllprotein complexes in the thylakoid membrane as development proceeds. The developing leaf thus offers a useful system with which to evaluate the specific roles of the thylakoid protein kinase and LHCP in the regulation of excitation energy distribution between PSI and PSII. Leaves of Gramineae, such as wheat, grown under a diurnal light regime are especially applicable to such a study since cell division occurs in a basal meristem with the result that a gradient of cellular and plastid development exists from the base to the tip of the leaves (Boffey et al. 1979). In this paper, we examine the activity of thylakoid protein kinase and associated control of excitation energy distribution between the two photosystems during chloroplast biogenesis in the 7-d-old, light-grown wheat leaf.
and the thylakoid membranes recovered by centrifugation for 3 rain at 3,000 g. Starch was removed from preparations prior to final pelleting of the membranes by centrifugation at 1,000 g for 30 s. The membranes were resuspended in 5 m M MgC12, 5 m M KzHPO4, 25 mM Na Tricine (pH 7.8) and stored in the dark on ice.
Materials and methods Plant material. Seeds of Triticum aestivum cv. Maris Dove (wheat) (National Seed Development Organization, Cambridge, UK) were washed in running tap water for 17 h and sown in John Innes No. 2 potting compost. Plants were grown for 7 d at 20 ~ C and 70% relative humidity in a 16-h photoperiod with a photon flux density of 280 gmol m-2s -1 prior to harvesting of the first leaf. Hordeum vulgare chlorina f-2 (barley) mutant (Highkin, H.R., California State University, Northridge, Cal. 91324, USA), which lacks chlorophyll b, was grown from seed in John Innes No. 2 potting compost in a glasshouse at 20 ~ C.
Thylakoid isolation. Leaves were homogenized in ice-cold 300 mM sorbitol, 5 m M MgC12, 25 m M Na N-tris-(hydroxymethyl)-methylglycine (Tricine) (pH 7.8), filtered through five layers of cheesecloth and five layers of 25 gm nylon mesh prior to centrifugation for 90 s at 3,000 g. The pellet was thoroughly washed With ice-cold 5 m M MgC12, 25 mM Na Tricine (pH 7.8)
Thylakoid protein-kinase activity. Assays were carried out in a volume of 0.1 cm 3 containing I mM KC1, 5 m M MgSO 4, 2 5 r a m Na Tricine (pH7.5), 185 kBq of y32p ATP (1' 1 TBq g m o l - 1), 200 ~tM unlabelled ATP and a pre-illuminated aliquot of membranes containing approx. 1 mg c m - 3 of protein. Assays were performed in triplicate. The mixture was incubated at 30~ for 2 rain using photosynthetically active light (400 700 nm) at a photon fluence rate of I mmol m-Z s- 1. The reaction was halted by spotting 50 mm 3 aliquots onto 23 mm circles of Whatman 3 MM filter paper which were immediately plunged into ice-cold 7.5% (w/v) trichloroacetic acid. The filters were washed three times, for at least 20 rain each, in cold 7.5% trichloroacetie acid (15 cm 3 per filter paper) followed by 20 rain at 90 ~ C and an additional wash in cold 7.5% trichloroacetic acid. The filter papers were then bathed in absolute ethanol, absolute ethanol:petroleum ether (1:1, v/v), and finally in petroleum ether. After air drying, the amount of 3zp incorporated was determined by Cerenkov spectrometry (Bennett 1977). Addition of 5 m M sodium fluoride, an inhibitor of thylakoid phosphatase activity (Bennett 1980), had negligible effects on the results obtained from the assays of kinase activity. Electrophoretie separation and autoradiography of polypeptides. Labelling of thylakoid membranes was carried out in 0.5 cm 3 of the medium and under the conditions used for the assay of kinase activity. After 5 rain, labelling was terminated by addition of 2% (w/v) sodium dodecyl sulphate. Samples were incubated at 60 ~ C for I h and loaded onto a 7.5 to 17.5% linear polyacrylamide gel containing 4 M urea, according to the method of Laemmli (1970). Following electrophoresis, the gels were stained with Coomassie brilliant blue and vacuum dried prior to autoradiography using X-Omat A R film (Kodak Ltd., Hemel Hempstead, Herts., UK)
Kinetics of chlorophyll fluorescence. The chlorophyll fluorescence of thylakoids was monitored in stirred cuvette after dilution of the sample to 10 ~tg chlorophyll cm-3 with resuspension medium and the addition of 1 gM nigericin and 5 m M sodium fluoride. Fluorescence was excited with 100 gmol photons m-2s -1 at 435 nm (10 nm band pass) and monitored at 685 nm (2 nm band pass) and 20~ using a SPF-500 spectrofluorometer (American Instrument Co., Silver Springs, Md. 209J0, USA). Adenosine 5'-triphosphate was added to a final concentration of 200 gM to induce fluorescence quenching. For measurements on intact leaf tissue, samples were kept in the dark for 20 rain prior to excitation. Trifurcated fibreoptic light pipes (Applied Photophysics Ltd., London, UK) were used to transmit excitation to, and fluorescence from, the upper surface of the leaf. Simultaneous measurements of fluorescence emission at 685 and 740 nm were made using two of the light pipes. The third was used to transmit the 632.8 nm excitation radiation produced by a helium-neon laser (Spectra Physics Ltd., St. Albans, Herts., UK) through a 632.8 nm interference filter (Ealing Beck Ltd., Watford, Herts. UK). The photon flux density at the leaf surface was 100 gmol m - 2S -- 1, Fluorescence was measured at 685 nm through a 685 nm interference filter (Balzers High Vacuum Ltd., Berkhampsted, Herts., UK) and at 740 nm through a high-radiance monochromator (Applied Photophysics Ltd., London, UK) with entry and exit slits
N.R. Baker et al. : Protein-kinase activity in wheat chloroplasts
153
of 0.5 mm and 2.0 nm, respectively. Hamamatsu R446 photomultiplier tubes (Hakuto International, Enfield UK) were used to detect fluorescence. The reduction-oxidation (redox) state of the PSII electron acceptors throughout the fluorescence-induction curve of leaf samples was estimated from the ratio of the variable fluorescence (Fv2) and the minimal level of fluorescence (Foa) generated upon addition of a second excitation of 632.8-nm radiation, produced from a helium-neon laser, which was saturating for PSII trap closure (Bradbury and Baker 1981; Baker and Bradbury 1981). The percentage of PSII traps which are oxidised at any given time, t, can be estimated from the ratio of F~2/Fo2 at t and Fv2/Fo2 at the onset of excitation, i.e. at t = 0 . The ratio, Fv2/Fo2 at t = 0, was determined by exciting the darkadapted sample simultaneously with both the initial excitation and the second saturating irradiation, and can be assumed to be indicative of maximal PSII trap opening, i.e. 100% oxidised. When the second excitation produces no variable fluorescence, Fv2/Foa = 0 and the PSII traps are 100% reduced. Experiments on fluorescence quenching in isolated thylakoid membranes (Bradbury 1982) and protoplasts personal communication by P. Horton, University of Sheffield, Sheffield, UK) have demonstrated that determinations of the redox state of PSII traps by F~2/Fo2 produce similar results to those obtained from experiments when 15 gM 3-(3',4'-dichlorophenyl)-l,1,-dimethylurea (DCMU) is used to induce trap closure.
in the basal 1 cm of the leaf, thylakoids exhibited an approx, sixfold greater rate of endogenous protein phosphorylation than the mature thylakoids isolated from tissue near the leaf tip, i.e. 14 cm from the leaf base. The chlorophyll a/b ratio decreased from 13.1 at the base to 2.6 at the tip, indicating that the ratio of LHCP to total chlorophyll is considerably reduced at early stages of thylakoid biogenesis compared with the situation in the mature membranes. Since the apoproteins of the LHCP are the major substrates of the kinase in mature thylakoids (Bennett 1977; Alfonzo et al. 1980), the possibility of the total kinase activity being substrate-limited at early stages of development was examined by also determining the enzymic activity in the presence of an excess of exogenous histones IIA, which act as substrate for the kinase (Alfonzo et al. 1980; Markwell et al. 1983). Addition of histones IIA increased the kinase activity of thylakoids isolated from the leaf base and tip by 22 and 25%, respectively (Table 1), indicating that the protein kinase was no more limited with respect to endogenous protein substrate at the early stages of development than in the mature thylakoids. However, a 72% histone IIA-induced stimulation of kinase activity of plastids isolated from 7-d-old etiolated wheat leaves demonstrated a major substrate limitation in etioplasts (Table I). Sodium dodecyl sulphate polyacrylamide gel polypeptide profiles and the associated autoradiograms of thylakoid membranes isolated from etiolated leaves and segments taken from the base and tip of light-grown leaves are shown in Fig. 1. The mature thylakoid preparation had at least 12 polypeptides labelled, the majority of the label being incorporated into the two apo-proteins of the LHCP, which migrate at approx. Mr=26,000. Samples from etiolated leaves and the light-grown leaf base showed that at least eight polypeptides were la-
Chlorophyll andprotein concentrations. Total chlorophyll (a + b) concentrations were determined in 80% (v/v) acetone by the method of Arnon (1949). Ratios of chlorophyll a/b were determined in diethylether extracts by the sensitive fluorometric method of Boardman and Thorne (1971). Protein concentration was measured by the method of Lowry et al. (1951) except that the absorbance was determined at 720 nm to avoid a contribution due to chlorophyll.
Results
Total thylakoid protein kinase activities at different stages of chloroplast development in the 7-dold light-grown wheat leaves are shown in Table 1, together with changes in total chlorophyll content and chlorophyll a/b ratio. A large decline in the kinase activity was observed during development. At the early stages of chloroplast biogenesis, found
Table 1. Total thylakoid protein kinase activity during chloroplast development in the 7-d-old light grown wheat leaf. Data presented are the mean of three experiments. Total thylakoid protein kinase activity is expressed in terms of pmol phosphate incorporated per mg total thylakoid protein per rain. Activities of some samples were measured in the presence of 3 mg cm -3 Histones IIA, which was a saturating substrate concentration Tissue samples (cm from leaf base)
Total chlorophyll (pg segment- 1)
0-1
0.078
1-2
1.06
~3 4-5 7-8 14-15 Etiolated
1.47 3.8 10.2 18.2 0
Total chlorophyll (pg mg - 1 FW)
0.107 0.150 0.194 0.530 1.17 2.38 0
Chlorophyll a/b
13.1 10.4 7.9 5.0 2.7 2.6 -
Total kinase activity (pmol mg ~ rain- 1) - H i s t o n e s IIA
+Histones IIA
91.4
112
71.0
57,3 23.7 20.9 15.1 31.2
18.8 53.6
154
N.R. Baker et al. : Protein-kinase activity in wheat chloroplasts
l t
D, Time
Fig. 2. Adenosine 5'-triphosphate-induced decrease of 685 nm fluorescence from isolated, uncoupled wheat thylakoids at different developmental stages. The regions of the 7-d-old lightgrown wheat leaf from which the membranes were isolated are given as distance from the leaf base. Arrows." addition of ATP, to a final concentration of 200 gM. Dashed lines." maximum rate of fluorescence quenching in the absence of ATP Fig. i. Polyacl2clamide gel profiles of labelled thylakoid membranes, isolated from wheat leaves at different developmental stages, after staining for protein (lanes b-d) and autoradiography (lanes e-J). Lane a is stained protein standards; b and e are from 7-d-old etiolated leaves; c and f are from the basal 1 cm of 7-d-old light-grown leaves; d and g are from tissue taken 14 cm from the base of 7-d-old tight-grown leaves
Qox[%) 10
e--
Ix'e/
F6B5 6s
belled, the majority of label being incorporated into a polypeptide of M~ = approx. 9000. This polypeptide cannot be readily identified on the Coomassie-stained gel (Fig. 1) as it was present in extremely low concentrations. In the presence of nigericin and sodium fluoride, ATP-induced quenching of LHCP-PSII fluorescence, which is indicative of a State 1-State 2 transition, was observed for thylakoid membranes isolated from all regions of the 7-d-old, light-grown wheat leaf (Fig. 2). Although quenching was proportionally greater in thylakoids isolated from the more mature leaf regions, appreciable quenching was found in the relatively poorly developed thylakoids isolated from the basal 1 cm of the leaf, which did not contain a high concentration of LHCP. The response to ATP of thylakoids isolated from segments 14.5 cm from the base of leaves was similar to that observed with thylakoids isolated from mature wheat leaves. These results demonstrate that a State l-State 2 transition can occur in membranes in which the LHCP is not a major proportion of the total chlorophyll-proteins. The kinetics of fluorescence emission at 685 and 740 nm were measured simultaneously on irradiation of dark-adapted leaf segments taken I cm
60s
~j/.j~J light on
FT&O 9
~Tirne
9
8
-FT#O/F6B5
~o .~ 1.00
Fig. 3. Induction curves of 685- and 740-nm fluorescence, F685 and F740 respectively, generated on excitation of the basal 1 cm of the 7-d-old wheat leaf. The curves are normalized on the maximal signal of F685 and F740. Changes in the ratio of fluorescence at 740 nm to that at 685 nm, F685/F740, together with the percentage of PSII electron accepters in the oxidised state, Qox, are also shown throughout the fluorescence induction
(Fig. 3) and 14 cm (Fig. 4) from the leaf base. Changes in the 740/685 nm fluorescence-emission ratio (F740/F685), which is indicative of the ratio of the excitation densities in PSI and PSII-LHCP (Kyle et al. 1983), and changes in the redox state of PSII electron accepters throughout the fluorescence-induction curves of these leaf segments are also shown in Figs. 3 and 4. Although only a small amount of fluorescence quenching was observed in the basal 1-cm segment, an increase in F740/ F685 was observed during the quenching. Since the PSII electron accepters did not show any redox changes during this quenching, remaining highly reduced throughout, the enhancement of PSI over PSII-LHCP fluorescence cannot be attributed to
N.R. Baker et al. : Protein-kinase activity in wheat chloroplasts
FTOI L,F6BS P
F%0
IF66~
16
H
15
("
f/
~ \ ~ e
/
F6o~s
11 1.o
pTime Light on
Fig. 4. Legend as for Fig. 3 except that leaf tissue 14 cm from the base of a 7-d-old wheat leaf was used
G - -
Qox%l [ 30 60s
/
6s
25 20
-~/
Planta (1983)159:151-158
9 Springer-Verlag 1983
Thylakoid protein kinase activity and associated control of excitation energy distribution during chloroplast biogenesis in wheat Nell R. Baker* 1, John P. Markwell 2, Michael Bradbury 1, Maxine G. Baker 1 and J. Philip Thornber 3 1 Department of Biology, University of Essex, Colchester CO4 3SQ, Essex, UK, 2 Department of Agricultural Biochemistry, University of Nebraska, Lincoln, Nebraska 68583, USA, and 3 Department of Biology and Molecular Biology Institute, University of California, Los Angeles, CA 90024, USA
Abstract. The activity of thylakoid protein kinase and the regulation of excitation energy distribution between photosystems I and II was examined during chloroplast biogenesis in light-grown Triticure a e s t i v u m (wheat) leaves. The specific activity of the thylakoid protein kinase decreased some sixfold during development from the young plastids at the base of the 7-d-old leaf to the mature chloroplasts at the leaf tip. Appreciable activity was also detected in plastids isolated from etiolated leaves. In mature chloroplasts the majority of phosphate was incorporated into the Mr=26,000 apo-proteins of the light-harvesting chlorophyll a/b-protein complex (LHCP). However, at early stages of chloroplast development and in the etioplast, the phosphate was predominantly incorporated into a polypeptide of Mr=9,000 dalton. Immature thylakoids, isolated from the base of the leaf, had relatively low concentrations of LHCP and could perform a State 1-State 2 transition, as demonstrated by ATP-induced quenching of photosystern II fluorescence. Analyses of photosystem I and photosystem II fluorescence-induction curves from intact leaf tissue demonstrated that this transition occurs in vivo at early stages of leaf development and, therefore, may play an important role in regulating energy transduction during chloroplast biogenesis. Key words: Chlorophyll fluorescence - Chloroplast development - Thylakoid protein kinase - T r i t i c u m (chloroplast biogenesis).
* To whom correspondence should be addressed Abbreviations. LHCP=light-harvesting chlorophyll a/b-protein complex; PSI, PSII =photosystem I, II
Introduction
In mature chloroplasts of higher plants the quantum yield of non-cyclic electron flow is constant over most of the visible light spectrum, even in spectral regions where photosystem II (PSII) i s preferentially excited (Myers 1971). This constancy has been explained by the existence of a physiological control to balance quantal distribution between photosystemI (PSI) and PSII which is often termed the State 1-State 2 transition (Barber 1976). In State 1, quanta are preferentially directed to PSII, whereas in State 2 an increased proportion of harvested quanta are directed to PSI; 10% of the quanta absorbed by PSII are thought to be diverted to PSI on transition from State 1 to State 2 (Bonaventura and Myers 1969). Recently, phosphorylation of the light-harvesting chlorophyll a/b-protein complex (LHCP) has been proposed for a major role in the control of the State l-State2 transition (Bennett etal. 1980; Allen et al. 1981; Chow et al. 1981; Horton and Black 1981 ; Horton et al. 1981 ; Telfer and Barber 1981). Phosphorylation of LHCP increases the density of negative surface charges on the thylakoids, which is proposed to increase interaction between LHCPPSII and PSI particles and increase excitation-energy spillover from LHCP-PSII to PSI (Barber 1982, 1983). The ability to undergo State 1-State 2 transitions could enable the photosynthetic apparatus to modify the rate of non-cyclic relative to cyclic photosynthetic electron transport and thereby regulate the ratio of A T P : N A D P H produced by the thylakoids. At early stages of leaf development, thylakoids become capable of light-induced, coupled electron transport and can produce ATP and N A D P H (Howes and Stern 1973; Plesnicar and Bendall
~52
N.R. Baker et al. : Protein-kinase activity in wheat chloroplasts
1973; Weistrop and Stern 1971; Duysen etal. 1980) despite having a qualitatively and quantitatively incomplete complement of chlorophyll-protein complexes (Argyroudi-Akoyunoglou and Akoyunoglou 1979; Kalosakas etal. 1981). The metabolic requirements of the leaf must change rapidly during expansion and greening and clearly it would be advantageous for the developing leaf to be able to regulate the ratio of ATP:reducing power generated by photosynthetic electron transport as early as possible. If current dogma is correct, to achieve this via a State 1-State 2 control, both the thylakoid protein kinase and LHCP would have to be present in the thylakoid membranes at early stages of development. During plasrid biogenesis in the majority of plants the chlorophyll a/b ratio rapidly decreases (e.g. De Greef et al. 1970; Thorne and Boardman 1971; Plesnicar and Bendall 1973; Weistrop and Stern 1977; Egneus et al. 1976) indicating that LHCP constitutes an increasing proportion of the total chlorophyllprotein complexes in the thylakoid membrane as development proceeds. The developing leaf thus offers a useful system with which to evaluate the specific roles of the thylakoid protein kinase and LHCP in the regulation of excitation energy distribution between PSI and PSII. Leaves of Gramineae, such as wheat, grown under a diurnal light regime are especially applicable to such a study since cell division occurs in a basal meristem with the result that a gradient of cellular and plastid development exists from the base to the tip of the leaves (Boffey et al. 1979). In this paper, we examine the activity of thylakoid protein kinase and associated control of excitation energy distribution between the two photosystems during chloroplast biogenesis in the 7-d-old, light-grown wheat leaf.
and the thylakoid membranes recovered by centrifugation for 3 rain at 3,000 g. Starch was removed from preparations prior to final pelleting of the membranes by centrifugation at 1,000 g for 30 s. The membranes were resuspended in 5 m M MgC12, 5 m M KzHPO4, 25 mM Na Tricine (pH 7.8) and stored in the dark on ice.
Materials and methods Plant material. Seeds of Triticum aestivum cv. Maris Dove (wheat) (National Seed Development Organization, Cambridge, UK) were washed in running tap water for 17 h and sown in John Innes No. 2 potting compost. Plants were grown for 7 d at 20 ~ C and 70% relative humidity in a 16-h photoperiod with a photon flux density of 280 gmol m-2s -1 prior to harvesting of the first leaf. Hordeum vulgare chlorina f-2 (barley) mutant (Highkin, H.R., California State University, Northridge, Cal. 91324, USA), which lacks chlorophyll b, was grown from seed in John Innes No. 2 potting compost in a glasshouse at 20 ~ C.
Thylakoid isolation. Leaves were homogenized in ice-cold 300 mM sorbitol, 5 m M MgC12, 25 m M Na N-tris-(hydroxymethyl)-methylglycine (Tricine) (pH 7.8), filtered through five layers of cheesecloth and five layers of 25 gm nylon mesh prior to centrifugation for 90 s at 3,000 g. The pellet was thoroughly washed With ice-cold 5 m M MgC12, 25 mM Na Tricine (pH 7.8)
Thylakoid protein-kinase activity. Assays were carried out in a volume of 0.1 cm 3 containing I mM KC1, 5 m M MgSO 4, 2 5 r a m Na Tricine (pH7.5), 185 kBq of y32p ATP (1' 1 TBq g m o l - 1), 200 ~tM unlabelled ATP and a pre-illuminated aliquot of membranes containing approx. 1 mg c m - 3 of protein. Assays were performed in triplicate. The mixture was incubated at 30~ for 2 rain using photosynthetically active light (400 700 nm) at a photon fluence rate of I mmol m-Z s- 1. The reaction was halted by spotting 50 mm 3 aliquots onto 23 mm circles of Whatman 3 MM filter paper which were immediately plunged into ice-cold 7.5% (w/v) trichloroacetic acid. The filters were washed three times, for at least 20 rain each, in cold 7.5% trichloroacetie acid (15 cm 3 per filter paper) followed by 20 rain at 90 ~ C and an additional wash in cold 7.5% trichloroacetic acid. The filter papers were then bathed in absolute ethanol, absolute ethanol:petroleum ether (1:1, v/v), and finally in petroleum ether. After air drying, the amount of 3zp incorporated was determined by Cerenkov spectrometry (Bennett 1977). Addition of 5 m M sodium fluoride, an inhibitor of thylakoid phosphatase activity (Bennett 1980), had negligible effects on the results obtained from the assays of kinase activity. Electrophoretie separation and autoradiography of polypeptides. Labelling of thylakoid membranes was carried out in 0.5 cm 3 of the medium and under the conditions used for the assay of kinase activity. After 5 rain, labelling was terminated by addition of 2% (w/v) sodium dodecyl sulphate. Samples were incubated at 60 ~ C for I h and loaded onto a 7.5 to 17.5% linear polyacrylamide gel containing 4 M urea, according to the method of Laemmli (1970). Following electrophoresis, the gels were stained with Coomassie brilliant blue and vacuum dried prior to autoradiography using X-Omat A R film (Kodak Ltd., Hemel Hempstead, Herts., UK)
Kinetics of chlorophyll fluorescence. The chlorophyll fluorescence of thylakoids was monitored in stirred cuvette after dilution of the sample to 10 ~tg chlorophyll cm-3 with resuspension medium and the addition of 1 gM nigericin and 5 m M sodium fluoride. Fluorescence was excited with 100 gmol photons m-2s -1 at 435 nm (10 nm band pass) and monitored at 685 nm (2 nm band pass) and 20~ using a SPF-500 spectrofluorometer (American Instrument Co., Silver Springs, Md. 209J0, USA). Adenosine 5'-triphosphate was added to a final concentration of 200 gM to induce fluorescence quenching. For measurements on intact leaf tissue, samples were kept in the dark for 20 rain prior to excitation. Trifurcated fibreoptic light pipes (Applied Photophysics Ltd., London, UK) were used to transmit excitation to, and fluorescence from, the upper surface of the leaf. Simultaneous measurements of fluorescence emission at 685 and 740 nm were made using two of the light pipes. The third was used to transmit the 632.8 nm excitation radiation produced by a helium-neon laser (Spectra Physics Ltd., St. Albans, Herts., UK) through a 632.8 nm interference filter (Ealing Beck Ltd., Watford, Herts. UK). The photon flux density at the leaf surface was 100 gmol m - 2S -- 1, Fluorescence was measured at 685 nm through a 685 nm interference filter (Balzers High Vacuum Ltd., Berkhampsted, Herts., UK) and at 740 nm through a high-radiance monochromator (Applied Photophysics Ltd., London, UK) with entry and exit slits
N.R. Baker et al. : Protein-kinase activity in wheat chloroplasts
153
of 0.5 mm and 2.0 nm, respectively. Hamamatsu R446 photomultiplier tubes (Hakuto International, Enfield UK) were used to detect fluorescence. The reduction-oxidation (redox) state of the PSII electron acceptors throughout the fluorescence-induction curve of leaf samples was estimated from the ratio of the variable fluorescence (Fv2) and the minimal level of fluorescence (Foa) generated upon addition of a second excitation of 632.8-nm radiation, produced from a helium-neon laser, which was saturating for PSII trap closure (Bradbury and Baker 1981; Baker and Bradbury 1981). The percentage of PSII traps which are oxidised at any given time, t, can be estimated from the ratio of F~2/Fo2 at t and Fv2/Fo2 at the onset of excitation, i.e. at t = 0 . The ratio, Fv2/Fo2 at t = 0, was determined by exciting the darkadapted sample simultaneously with both the initial excitation and the second saturating irradiation, and can be assumed to be indicative of maximal PSII trap opening, i.e. 100% oxidised. When the second excitation produces no variable fluorescence, Fv2/Foa = 0 and the PSII traps are 100% reduced. Experiments on fluorescence quenching in isolated thylakoid membranes (Bradbury 1982) and protoplasts personal communication by P. Horton, University of Sheffield, Sheffield, UK) have demonstrated that determinations of the redox state of PSII traps by F~2/Fo2 produce similar results to those obtained from experiments when 15 gM 3-(3',4'-dichlorophenyl)-l,1,-dimethylurea (DCMU) is used to induce trap closure.
in the basal 1 cm of the leaf, thylakoids exhibited an approx, sixfold greater rate of endogenous protein phosphorylation than the mature thylakoids isolated from tissue near the leaf tip, i.e. 14 cm from the leaf base. The chlorophyll a/b ratio decreased from 13.1 at the base to 2.6 at the tip, indicating that the ratio of LHCP to total chlorophyll is considerably reduced at early stages of thylakoid biogenesis compared with the situation in the mature membranes. Since the apoproteins of the LHCP are the major substrates of the kinase in mature thylakoids (Bennett 1977; Alfonzo et al. 1980), the possibility of the total kinase activity being substrate-limited at early stages of development was examined by also determining the enzymic activity in the presence of an excess of exogenous histones IIA, which act as substrate for the kinase (Alfonzo et al. 1980; Markwell et al. 1983). Addition of histones IIA increased the kinase activity of thylakoids isolated from the leaf base and tip by 22 and 25%, respectively (Table 1), indicating that the protein kinase was no more limited with respect to endogenous protein substrate at the early stages of development than in the mature thylakoids. However, a 72% histone IIA-induced stimulation of kinase activity of plastids isolated from 7-d-old etiolated wheat leaves demonstrated a major substrate limitation in etioplasts (Table I). Sodium dodecyl sulphate polyacrylamide gel polypeptide profiles and the associated autoradiograms of thylakoid membranes isolated from etiolated leaves and segments taken from the base and tip of light-grown leaves are shown in Fig. 1. The mature thylakoid preparation had at least 12 polypeptides labelled, the majority of the label being incorporated into the two apo-proteins of the LHCP, which migrate at approx. Mr=26,000. Samples from etiolated leaves and the light-grown leaf base showed that at least eight polypeptides were la-
Chlorophyll andprotein concentrations. Total chlorophyll (a + b) concentrations were determined in 80% (v/v) acetone by the method of Arnon (1949). Ratios of chlorophyll a/b were determined in diethylether extracts by the sensitive fluorometric method of Boardman and Thorne (1971). Protein concentration was measured by the method of Lowry et al. (1951) except that the absorbance was determined at 720 nm to avoid a contribution due to chlorophyll.
Results
Total thylakoid protein kinase activities at different stages of chloroplast development in the 7-dold light-grown wheat leaves are shown in Table 1, together with changes in total chlorophyll content and chlorophyll a/b ratio. A large decline in the kinase activity was observed during development. At the early stages of chloroplast biogenesis, found
Table 1. Total thylakoid protein kinase activity during chloroplast development in the 7-d-old light grown wheat leaf. Data presented are the mean of three experiments. Total thylakoid protein kinase activity is expressed in terms of pmol phosphate incorporated per mg total thylakoid protein per rain. Activities of some samples were measured in the presence of 3 mg cm -3 Histones IIA, which was a saturating substrate concentration Tissue samples (cm from leaf base)
Total chlorophyll (pg segment- 1)
0-1
0.078
1-2
1.06
~3 4-5 7-8 14-15 Etiolated
1.47 3.8 10.2 18.2 0
Total chlorophyll (pg mg - 1 FW)
0.107 0.150 0.194 0.530 1.17 2.38 0
Chlorophyll a/b
13.1 10.4 7.9 5.0 2.7 2.6 -
Total kinase activity (pmol mg ~ rain- 1) - H i s t o n e s IIA
+Histones IIA
91.4
112
71.0
57,3 23.7 20.9 15.1 31.2
18.8 53.6
154
N.R. Baker et al. : Protein-kinase activity in wheat chloroplasts
l t
D, Time
Fig. 2. Adenosine 5'-triphosphate-induced decrease of 685 nm fluorescence from isolated, uncoupled wheat thylakoids at different developmental stages. The regions of the 7-d-old lightgrown wheat leaf from which the membranes were isolated are given as distance from the leaf base. Arrows." addition of ATP, to a final concentration of 200 gM. Dashed lines." maximum rate of fluorescence quenching in the absence of ATP Fig. i. Polyacl2clamide gel profiles of labelled thylakoid membranes, isolated from wheat leaves at different developmental stages, after staining for protein (lanes b-d) and autoradiography (lanes e-J). Lane a is stained protein standards; b and e are from 7-d-old etiolated leaves; c and f are from the basal 1 cm of 7-d-old light-grown leaves; d and g are from tissue taken 14 cm from the base of 7-d-old tight-grown leaves
Qox[%) 10
e--
Ix'e/
F6B5 6s
belled, the majority of label being incorporated into a polypeptide of M~ = approx. 9000. This polypeptide cannot be readily identified on the Coomassie-stained gel (Fig. 1) as it was present in extremely low concentrations. In the presence of nigericin and sodium fluoride, ATP-induced quenching of LHCP-PSII fluorescence, which is indicative of a State 1-State 2 transition, was observed for thylakoid membranes isolated from all regions of the 7-d-old, light-grown wheat leaf (Fig. 2). Although quenching was proportionally greater in thylakoids isolated from the more mature leaf regions, appreciable quenching was found in the relatively poorly developed thylakoids isolated from the basal 1 cm of the leaf, which did not contain a high concentration of LHCP. The response to ATP of thylakoids isolated from segments 14.5 cm from the base of leaves was similar to that observed with thylakoids isolated from mature wheat leaves. These results demonstrate that a State l-State 2 transition can occur in membranes in which the LHCP is not a major proportion of the total chlorophyll-proteins. The kinetics of fluorescence emission at 685 and 740 nm were measured simultaneously on irradiation of dark-adapted leaf segments taken I cm
60s
~j/.j~J light on
FT&O 9
~Tirne
9
8
-FT#O/F6B5
~o .~ 1.00
Fig. 3. Induction curves of 685- and 740-nm fluorescence, F685 and F740 respectively, generated on excitation of the basal 1 cm of the 7-d-old wheat leaf. The curves are normalized on the maximal signal of F685 and F740. Changes in the ratio of fluorescence at 740 nm to that at 685 nm, F685/F740, together with the percentage of PSII electron accepters in the oxidised state, Qox, are also shown throughout the fluorescence induction
(Fig. 3) and 14 cm (Fig. 4) from the leaf base. Changes in the 740/685 nm fluorescence-emission ratio (F740/F685), which is indicative of the ratio of the excitation densities in PSI and PSII-LHCP (Kyle et al. 1983), and changes in the redox state of PSII electron accepters throughout the fluorescence-induction curves of these leaf segments are also shown in Figs. 3 and 4. Although only a small amount of fluorescence quenching was observed in the basal 1-cm segment, an increase in F740/ F685 was observed during the quenching. Since the PSII electron accepters did not show any redox changes during this quenching, remaining highly reduced throughout, the enhancement of PSI over PSII-LHCP fluorescence cannot be attributed to
N.R. Baker et al. : Protein-kinase activity in wheat chloroplasts
FTOI L,F6BS P
F%0
IF66~
16
H
15
("
f/
~ \ ~ e
/
F6o~s
11 1.o
pTime Light on
Fig. 4. Legend as for Fig. 3 except that leaf tissue 14 cm from the base of a 7-d-old wheat leaf was used
G - -
Qox%l [ 30 60s
/
6s
25 20
-~/
