Photosynthesis Research 10." 37 49 (1986) © Martinus NijhoffPublishers, Dordrecht Printed in the Netherlands

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Regularpaper Chlorophyll-protein complex composition and photochemical activity in developing chloroplasts from greening barley seedlings ~ KENT O. BURKEY United States Department of Agriculture, Agricultural Research Service, and Departments of Crop Science and Botany, North Carolina State University, Raleigh, NC 27695-7631, USA

(Received 16 October 1985: in revised Jorm 19 December 1985: accepted 20 December ] 985) Key words: barley, chlorophyll-protein complexes, chloroplast development, mild SDSPAGE, thylakoid membranes Abstract. The time course for the observation of intact chlorophyll-protein (CP) complexes during barley chloroplast development was measured by mild sodium dodecyl sulfate polyacryhnnide gel electrophoresis. The procedure required extraction of thylakoid membranes with sodium bromide to remove extrinsic proteins. During the early stages of greening, the proteins extracted with sodium bromide included polypeptides from the cell nucleus that associate with developing thylakoid membranes during isolation and interfere with the separation of CP complexes by electrophoresis. Photosystem 1 CP complexes were observed before the photosystem I1 and lightharvesting CP complexes during the initial stages of barley chloroplast development. l'hotosystem 1 activity was observed before the photosystem 1 CP complex was detected whereas photosystem lI activity coincided with the appearance of the CP complex associated with photosystem II. Throughout chloroplast development, the percentage of the total chlorophyll associated with photosystem 1 remained constant whereas the amount of chlorophyll associated with photosystem 11 and the light-harvesting complex increased. The CP composition of thylakoid membranes from the early stages of greening was difficult to quantitate because a large amount of chlorophyll was released from the CP complexes during detergent extraction. As chloroplast development proceeded, a decrease was observed in the amount of chlorophyll released from the CP complexes by detergent action. The decrease suggested that the CP complexes were stabilized during the later stages of development. Abbreviations. Chl, chlorophyll; CP, chlorophyll-protein; CPI, P700 chlorophyll-a protein complex of photosystem 1; CPa, electrophoretic band that contains the photosystem II reaction center complexes and a w~riable amount of the photosystem I lightharvesting coinplex; CP A/B, the major light-harvesting complex associated with photosystenr 1I: DCIP, 2.6-dichlorophenolindophenol; DCMU, 3-(3,4-dichlorophenyl)1, l-dimethylurea; DPC, diphenyl carbazide; MV, methyl viologen; PAR, photosynthetically active radiation; PSI, photosystem 1; PSll, photosystem I1; SDS, sodium dodccyl sulfate: SDS-PAGE, sodium dodccyl sulfate polyacrylamide gel electrophoresis; TEMED, N,N.N',N'-tetramethylethylenediamine; TMPD, N,N,N',N'-tetramethyl-pphenylencdiam inc.

'Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and tile North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. Paper No. 9949 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601.

38 Introduction

The appearance of photochemical activity during chloroplast development has been studied extensively through the use of etiolated seedlings greened under continuous light. During the first hours of greening, PSI activity is observed before PSII activity and 02 evolution in isolated plastids [6, 7, 11, 12, 18, 22, 23]. Although the Chl content of the tissue is very low during the initial stages of development, measurable photochemical activity indicates the presence of small amounts of the CP complexes associated with PSI and PSII. The mRNA and apoproteins of the CP complexes have been detected during the first hours of chloroplast development [4, 10, 25]. The properties of the intact CP complexes and the distribution of Chl between the CP complexes during the first hours of chloroplast development have not been characterized. The CP composition of greening barley [14] and bean [1] seedlings have been observed by SDS-PAGE. However, these previous studies used detergent solubilization and electrophoresis procedures that produce large amounts of free Chl and allow only two CP complexes to be observed. More recently mild SDS-PAGE procedures have been used to analyze the CP complexes in greening bean seedlings [16]. This more recent study [16] was concerned with the CP composition at the later stages of chloroplast development and did not concern the initial appearance of the CP complexes. The present study reports the initial appearance of the CP complexes during barley chloroplast development by mild SDS-PAGE methods. The present study also shows a correlation between the development of photochemical activity and the CP complex composition in thylakoid membranes from greening barley. Materials and methods

Plant material Barley seedlings (Hordeum vulgare L. cv. Boone) were germinated in vermiculite moistened with tap water at 22 °C for 8 d in the dark. Control seedlings were grown in the same medium under continuous light at 22 °C in a growth chamber. The light was provided by a combination of fluorescent and incandescent lamps that provided an intensity of 200 to 300/aE m -2 s- 1 PAR at the top of the seedlings. Chloroplast development was monitored after transfer of the etiolated seedlings into the continuous light chamber. Thylakoid membrane isolation Thylakoid membrane preparations were isolated from plants after various periods of illumination. Leaves were removed from the coleoptile by pulling at the extreme ends of the harvested leaf. Leaves were cut into 3 to 5 mm sections and homogenized for 10s in cold buffer with a Brinkman Polytron

39 PT 10-352 equipped with probe PTA 205. Approximately 10ml of buffer were used for each g o f tissue. The buffer consisted of 0.4 M sorbitol, 10 mM NaC1, 5 mM MgC12, 0.2% (w/v) bovine serum albumin, and 50 mM TricineNaOH (pH 7.8). The homogenate was filtered through eight layers of cheesecloth. The filtrate was accelerated to 7,700 × g (4°C) and returned to rest within a total time of 2 rain. The pellet was washed once in cold grind buffer, collected at 4,000 x g for 10 rain at 4 °C, and then resuspended in grind buffer. The final suspension was kept on ice during electron transport activity measurements and then stored in liquid N 2.

Assays of pho tochemical activiO, PSI activity was measured as 02 uptake with a Clark-type electrode. The 1.8 ml assay contained 0.1 M sorbitol, 2#M DCMU, 2 mM sodium ascorbate, 0 . 2 r a m TMPD, 0.1raM MV, 4 0 r a m Tricine-NaOH (pH 8.0), and plastid membranes (~< 15~g Chl). The reaction chamber was maintained at 25 °C. Light was provided by two 75 W flood lamps and filtered through 4 cm of water. Each lamp provided an intensity of 900~E m -2 s -1 PAR. 02 uptake in the light was corrected for dark rates. The correction was most significant during the first 3 h of greening when the dark rate accounted for 50 to 60% of the overall rate. Oxygen evolution with ferricyanide as the electron acceptor was measured with the electrode described above. The 1.8 ml assay contained 0.1 M sorbitol, 40 mM Tricine-NaOH (pH 8.0), and 1.5 mM K3Fe(CN)6 and was deoxygenated with a slow stream of argon. The plastid membranes (~< 15/ag Chl) were added and the electrode chamber illuminated for 30 s. Ammonium chloride was added to a final concentration of 2 mM to obtain the uncoupled rate. DCIP reduction was measured spectrophotometrically with either water or DPC as the electron donor. The 3.0ml assay contained 0.1 M sorbitol, 40raM Tricine-NaOH (pH 8.0), 30/IM DCIP, and plastid membranes (~

Chlorophyll-protein complex composition and photochemical activity in developing chloroplasts from greening barley seedlings.

The time course for the observation of intact chlorophyll-protein (CP) complexes during barley chloroplast development was measured by mild sodium dod...
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