Photosynthetic Activities of Spinach Leaf Protoplasts

Abstract

Photosynthetic activities of protoplasts isolated fronm spinach leaf (Spinacia oleracea L.) were investigated. The proto-plasts were stable up to 9 hr, without loss of the original activity of C02 fixation (33-75 ,umoles C02/mg Chl hr) and light-dependent 02 evolution (33-40 ,umoles 02/mg Chl * hr), when stored in 0.8 M mannitol-0.05 M N-tris(hydroxymethyl)-methylglvcine-NaOH buffer, pH 7, at 4 C in dark. The optimum pH of 8.5 for C02 fixation reaction carried out in the present experimental condition employed is about the same as that reported for intact spinach chloroplasts. The C02 concentration for half-maximal rate of C02 fixation by proto-plasts. "Km (CO2)," were determined to be 19.8 gM (pH 7) and 42 gM (pH 8.5) and are similar to those observed for intact spinach chloroplasts. Protoplasts showed postillumination CO2 fixation. Overall results indicate that spinach protoplasts are as active as the intact plant leaf tissues in their photo-svnthetic activities. Since the initial report by Cocking (10) on the isolation of protoplasts from tomato root tip cells, many investigations have been conducted on various types of plant cells (2, 9, 11, 14, 18-20, 26-28, 32, 33). Protoplasts with normal metabolic activities are potentially useful for physiological research, and we were interested in their application pertaining to structure-function relationship of spinach leaf RuDP2 carboxylase. We are attempting to elucidate the functional role of each individual subunit of the enzyme protein and the molecular interaction in vivo (1). Immunochemical techniques are being employed in this work (24, 25), and protoplasts appear to be an attractive system for such studies (2, 27, 33). As a basis for such research, spinach leaf protoplasts have been isolated, and their biochemical activities have been measured. Results reported below demonstrate that spinach protoplasts are potentially useful for research in photosynthesis. MATERIALS AND METHODS Photoplasts. Young leaves of freshly harvested spinach (Spinacia oleracea L. var Kyoho) were used for preparing 'This research was supported in part by grants from the Ministry of Education of Japan (811108), the Toray Science Foundation (Tokyo), and the Naito Science Foundation (Tokyo). This is paper XXIX in the series "Structure and Function of Chloroplast Proteins ." C Abbreviation: RuDP: ribulose 1, 5-diphosphate. protoplasts. Methods of the two-step enzymic maceration of spinach leaves using Macerozyme R-10 and Cellulase Onozuka R-10 (Kinki Yakult Co. Ltd, Osaka) were basically the same as those for preparing tobacco leaf (Nicotiana tabacumn) proto-plasts as reported by Takebe et al. (32). Two modifications employed were that the concentration of mannitol was raised to 0.8 M as recommended by Otsuki and Takebe (26) and that the washing medium did not contain 0.1 mm CaClh. Two g of spinach leaf segments (about 0.5 cm wide) devoid of lower epidermis were placed in a 50-ml Erlenmeyer flask containing 20 ml of maceration medium (0.5% Macerozyme R-10-1% K-dextran sulfate [18% S, Meito Sangyo Co. Ltd. Nagoya]-0.8 M mannitol [pH 5.8]). After vacuum infiltration of leaf samples for 2 min, the enzymic disintegration was carried out in a rotary shaker at 20 C. The spongy mesophyll cell suspension was collected by decantation of the greenish supernatant every 30 min, by substituting 10 ml of fresh maceration medium each time. At the same time, measurements of the absorbance increment at 680 nm as well as the phase contrast microscope inspection of the collected samples were carried out. This first step of the enzymic treatment usually goes to completion in 5 hr, and the whole supernatant fractions were filtered through a Nylon bolting cloth (35 mesh). The residues collected by centrifugation at lOOg for 3 min were washed twice with 50 ml each of 0.8 M mannitol solution (pH 5.4). The spongy cell fractions obtained (Fig. la) were then subjected to the second enzymic treatment using a medium containing 2% Cellulase Onozuka R-10 and 0.8 M mannitol (pH 5.2). The enzymic reaction was carried out at 37 C with occasional stirring. The complete conversion to protoplasts occurs within 1 to 1.5 hr incubation, and the residues collected by centrifugation (100g, 3 min) were washed twice with 50 ml each of 0.8 M mannitol solution. Fig. lb is a typical photomicrograph of the protoplast preparations. The final preparation was stored in 0.8 M mannitol at 4 C in dark until the time of analytical experiments. Photosynthetic CO2 Fixation. The reaction mixture for determining photosynthetic CO2 fixation activity contained the following components (lmoles): Tricine-NaOH at various pH values, 20: NaH"CO,, 1.6 (2,Ci); mannitol, 160; and appropriate amounts of protoplasts (15-25 ,g Chl) in a total volume of 0.2 ml. The reaction mixture except NaHCO, was incubated in the dark for 3 min at 25 C. After 5 min preillumination (3 x 104 lux of white light), NaH"4CO, was added and incubation continued at 25 C for 5 min. The reaction was then stopped by adding 0.1 ml of glacial acetic acid and radioactivity was measured by a Packard liquid scintillation spectrometer. The effect of various compounds on photosyn-thetic CO2 fixation was examined by adding them to the standard assay mixture in the preincubation period. Postillumination CO2 Fixation. Postillumination CO2 fixation activity was determined according to the method of Avron and Gibbs (5). After preillumination of the protoplast preparations for 10 min (3 x I04 lux of white light) at 25 C, test tubes con-712 www.plantphysiol.org on December 9, 2019-Published by Downloaded from