Pea-mutant Deficient in Chlorophyll

A chlorophyll-deficient mutant of pea (Pisum sativum) was found as a spontaneous mutation of the variety Greenfeast. Total chlorophyll of the mutant leaves was about one-half that of normal pea leaves per mg dry weight, and the ratio of chl a:chl b ranged from 10 to 18, compared with 3 for normal pea. In each generation the mutant plants gave rise to normal and mutant plants and lethal plants with yellow leaves. For a normal pea plant, CO2 uptake was saturated at about 60,000 lux, whereas with mutant leaves, the rate of CO2 uptake was still increasing at 113,000 lux. At 113,000 lux the mutant and normal leaves showed similar rates of CO2 fixation per unit area of leaf surface, but on a chlorophyll basis the mutant leaves were twice as active. Hill reaction measurements on isolated chloroplasts also showed that the mutant chloroplasts werc saturated at higher intensities than the normal, and that the activity of the mutant was at least double that of the normal on a chlorophyll basis. It is suggested that the photosynthetic units of the mutant chloroplasts contain about half the number of chlorophyll molecules as compared to the normal photosynthetic units. Electron microscopy of leaf sections of normal and mutant leaves showed that the mutant chloropla,sts oontain fewer lamellae per chloroplast and fewer lamellae per granum. The lethal chloroplasts, which were virtually devoid of chlorophyll, were characterized by an absence of grana. Previous studies with a mutant of barley (Hordeum vulgare L.) devoid of chlorophyll b indicated that chlorophyll b is not essential for photosynthetic activity in a higher plant (6,11, 13). Photosynihetic rates obtained with the mutant plants did not differ significantly from those obtained in normal plants (13). Hill reaction measurements on isolated chloroplasts showed that the mutant chloroplasts were more active per mg of total chlorophyll if assayed at saturating light intensities, but less active at low light intensities (6). The mutant chloroplasts were less fluorescent (7), and their molar ratio of total chlorophyll to cytochrome b6 plus cytochrome 559 was only about one-half of the corresponding ratio of normal chloroplasts (4). It was concluded from these studies that the mutant chloroplasts have less chlorophyll in their lightharvesting assemblies than the normal chloroplasts, but comparable amounits of the components of the photosynthetic electron transport chain (4). It was further suggested that the mutant chloroplasts have a decreased amount of chlorophyll in photo'This work was supported in part by a research grant (GB3341) from the National Science Foundation. 2 Present address: Department of Biology, San Fernando Valley State College, Northridge, California. system 2, relative to photosystem 1 (7). This would result in photosystem 2 absorbing a lower fraction of the incident quanta and would account for the lower photochemical efficiency of the mutant clhloroplasts at low light intensities. Electron microscopy of mutant leaf sections showed fewer grana in the mutant chloroplasts and fewer lamellae per gralnum, in comparison with the normal chloroplast (8). In the present studies, we have investigated the photosynthetic properties of a pea mutant deficient in both chlorophyll a and clhlorophyll b, and examined the fine structure of its chloroplasts. The level of total chlorophyll is shown to be lower in the pea than in the barley mutant, and a study of its photosynthetic properties enables a useful comparison with the barley mutant, and the clilorophyll-deficient tobacco mutants of Schmid and Gaffron (18,19) and Homann and Sclhmid (14). A study of the relationship between photosynthetic activity and structure in such chloroplhyll-deficient mutants may assist in the elucidation of the molecular organization of the chloroplast. A summary of this work was presented previously (12). Materials and Methods Plant Material. The mutant pea was derived from a comnmercial variety (Pisum sativum, var. 1310 www.plantphysiol.org on October 23, 2017 Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved. HIGHKIN ET AL.-CHLOROPHYLI.. MUTANT OF P1EA Greenfeast) as a spontaneous mutation. The mutant was observed in a population of plants growing in artificial liglht at a temperature of 21°/19° and a photoperiod of 16 hr. It was distinguislhable from the other plants by the pale-green color of its leaves. The mutant was grown to maturity, and the seed saved and growvn for a second generation. Seeds from eaclh of these plants wer-e lharvested and grown for a tlhird generation, and this procedure was repeated for a number of generations to date. After the first generation, the plants were grown in a controlled temperature glasshouse of the CSIRO pliytotron in a 16 lhr photoperiod and a temperature of 210/190. Normal pea plants were grown under identical conditions. Preparation of Chlor oplasts. Leaves (3-5 g fresh wt) eitlher from mutant or normal plants were ground in a mortar, clhilled in ice, witlh 15 ml of 0.05 M phosplhate btuffer (pH 7.2), containing 0.3 M sucrose and 0.01 alt KCl. The restulting brei was filtered througlh 2 lavers of Miracloth, and the clhloroplasts sedimented by centrifugation at 10OOg for 10 min. The clhloroplasts were resuspended in the sucrose-plhosphlate buffer (30 ml) and again centrifuged at 1OOOg for 10 min. Chloroplasts obtained from 3 g of mutant leaves were resuspended in 3 ml of sucrose-plhosplhate buffer and those from 3 g of normal leaves were resuspended in 6 ml. Chlorophyfll Determlinationis on Chioroplasts or Leaves. Total chlorophyll was determined in 80 % acetone with a Cary recording spectroplhotometer, using the eqtuations of Arnon (2). For determinations witlh wlole leaves 1.0 g fresh wt samples were grounid in 80 % acetone in a Servall Omnimixer. The residue obtained after centrifugation was extracted with 80 % acetone in the Omnimixer. and the procedure repeated. Tlle 3 supernatanits were combined and made up to 250 ml. Dry weiglhts were determined after heating samples at 800 until a constant weiglht was aclhieved, tusually 24 hr. Leaf areas were obtained by outlining leaves on mm paper. Chl a :chl b ratios of mutant leaves or mutant chloroplasts were determined in ethyl alcohol by a sensitive spectrofluorimetric metlhod, ftull details of whiclh will be published elsewhere. Brieflv, the ethanol extracts were diluted to an absorbancy of 0.2 at 436 nm, and cooled to the temperature of liquid nitrogen. Fluorescence emission spectra were recorded on a fluorescence spectrometer incorporating automatic correction for plhotomultiplier and moilochromator responses, and variation in energy output of the light source (7). The extract was excited at 474 nm, the wavelength maximum for chlorophyll b, and the chl a :chl b ratio determined from the relative amplitudes of the fluorescence emission at 680 nm (Chl a) and 656 nm (Chl b). The method was calibrated with mixtures of purified chlorophyll a and chlorophyll b. Absorption spectra of intact leaves at 77°K were recorded on a Cary Model 14R spectrophotonmeter, equipped with a scattered transmission accessory (6). Carbon Dioxide Fixationt. Carbon dioxide uptake by leaves of mutant and normal plants was determined with anl infra-red gas analyzer (Grubb Parsons). A leaf attaclhed to the plant was enclosed in a perspex airtiglht container, and an air sample containing 300 ppm of CO, was pumped across the leaf at a rate of 2000 cc/min. The leaf was illuminated with 4 Mazda FBRU 1000 watt mercury vapor high pressure lamps, and the liglht intensity was varied with neutral grey "Sarlon" shade cloth. Light intensities were measured with a undirectional Kipp thermopile and also with a selenium light meter (Eel Instruments). The temperature of the leaves was determined with a tllermocouple placed under an identical set of leaves in tlle same cabinet as the experimental leaves. The temperature of the cabinet was 17.5°. Photochemical Activilies of Chloroplasts. Hill reaction measurements were made as described previously (1), except that ferricyanide reduction wvas measured directly in a Cary spectropliotometer by the absorbance decrease at 418 nm. Wlhite-light intensities in the range of 1400 to 200,000 lux were obtained from a 250 \. att photoflood lamp operated at 200 volts. The cuvette containing the reaction mixture was positioned at varying distances from the lamp. Liglht intensities were measured with a Weston meter, Model 603, calibrated in ft-c. Electron Mllicroscopy. For electron microscopy several leaf pieces, approximately 1 mm2, were cut from the second leaf of normal, mutant or lethal pea plants and fixed in 3 % glutaraldehyde (purified by vacuum distillation) in 0.025 Ai phosplhate buffer pH 7.2 for 1.5 hr at 200. After washing in plhosplhate buffer for 1 lhr the leaf pieces were post-fixed in 2 % osmium tetroxide in 0.025 M phosphate buffer pH 7.2 for 2 lhr at 200, waslhed in buffer, delhydrated in ethanol, and embedded in an araldite epon nmixture (1/7). The resin was vacuunm infiltrated at 850 and polymerized at the same temperature for 24 hr before sectioning wvitlh an LKB ultra-microtome. Sections were stainied witlh saturated uranyl acetate in 50 % etlhanol for 1 lhr followed by Fiske's lead stain (9) before examination in a Plhilips EM200 electron microscope. Electron micrographls of the chloroplasts were exanmined for distinctness and resolution of lamellae and 10 each of the mutant and normal were selected. The numbers of single lamellae, groups of lamellae (grana) per clhloroplast, and number of lamellae per group were recorded for eaclh chloroplast and the results analyzed statistically.