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THE EFFECT of NUTRIENT DEFICIENCIES on the HILL REACTION of ISOLATED CHLOROPLASTS from TOMATO by D

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THE EFFECT of NUTRIENT DEFICIENCIES on the HILL REACTION of ISOLATED CHLOROPLASTS from TOMATO by D THE EFFECT OF NUTRIENT DEFICIENCIES ON THE HILL REACTION OF ISOLATED CHLOROPLASTS FROM TOMATO By D. SPENCER* and J. V. POSSINGHAM* [Manuscript received April 26, 1960] Summary Tomato plants were grown deficient in each known essential macro- and micronutrient (except chlorine) and the effect of each deficiency on the Hill reaction activity of chloroplasts isolated from these plants was studied. All deficiencies, except that of iron, resulted in chloroplasts with reduced Hill reaction activity per unit chlorophyll. Among the micronutrients, deficiencies of manganese, copper, zinc, boron, or molybdenum caused impairment of activity in that decreasing order of severity. In no case was the activity of chloroplasts from a deficient plant enhanced by the addition of the missing nutrient to the chloroplast suspension. A comparison of the absorption spectra of suspensions of chloroplasts from healthy and deficient plants, and of 80 per cent. acetone extracts of these suspensions, revealed no shifts in the absorption peaks, and no gross change in the relative proportions of the major components. In some deficiencies a small but significant decrease was observed in the ratio of chlorophyll a to chlorophyll b. Chloroplasts from healthy and deficient plants were assayed over a range of limiting light intensities up to saturation, and the effect of light intensity on the degree of impairment was recorded. In some cases, the degree of impairment due to deficiency was measured at 30 and 10°C with saturating light intensity. Some tentative conclusions are drawn concerning the site of the impaired component of the Hill reaction associated with a number of the deficiencies. 1. INTRODUCTION Manganese deficiency causes a reduction in the rate of photosynthesis per unit amount of chlorophyll in a number of algae. This is true whether photosynthesis is measured as CO 2 fixed, O2 evolved in presence of CO 2, or O2 evolved in the presence of an artificial oxidant such as a quinone (Pirson 1937; Pirson, Tichy, and Wilhelmi 1952; Arnon 1954; Eyster, Brown, and Tanner 1956). This evidence indicates that manganese is essential, either directly or indirectly, for the Hill reaction (the photo­ lysis of water, and the evolution of oxygen accompanied by the reduction of an artificial oxidant). Kessler (1955) demonstrated that in the green alga Ankistrodesmus manganese deficiency causes no reduction in the rate of the initial photolytic reaction, but is involved rather in the evolution of O2 from the products of photolysis. Evidence concerning the role of manganese, and other nutrients, in the Hill reaction of higher plants is meagre. Gerretsen (1949) has shown that CO 2 assimilation per unit leaf area of oat leaves was reduced in manganese deficiency. He also found that breis of manganese-deficient leaves without any added oxidant formed less peroxides upon illumination that those of normal leaves (Gerretsen 1950). Mehler * Division of Plant Industry, C.S.I.R.O., Canberra. 442 D. SPENCER AND J. V. POSSINGHAM (1951) found that manganous ions stimulated oxygen uptake by isolated chloroplasts in the presence of catalase and ethanol. Manganous ions have been shown to stimulate CO 2 fixation, but not the Hill reaction, of isolated chloroplasts from spinach (Allen et al. 1955). Little is known about the effects of other nutrient deficiencies on the Hill reaction, especially in higher plants. Kessler (1955, 1957) has shown that phosphate deficiency results in lowered photosynthesis and photoreduction in intact Ankistrodesmus, whilst iron deficiency lowers the rate of photoreduction much more than photo­ synthesis. The aim of the present work was to test whether manganese deficiency in a higher plant results in a reduction in the Hill reaction activity as measured in isolated chloroplasts. Having determined that this was the case, the effect of other nutrient deficiencies on the Hill reaction was also studied in order to test whether the effect of manganese deficiency is specific. Since most of the work in this field to date has been carried out with intact algae, it has not been possible to decide whether the effects observed were due to direct or indirect participation in the Hill reaction of the nutrients concerned. By working with chloroplasts isolated from deficient tissues, rather than with intact cells, it was possible to attempt direct restoration of the Hill reaction in vitro. II. METHODS (a) Water Oulture Methods Tomato plants (Lycopersicon esculentum Mill., cv. Bonny Best) were grown in nutrient solutions in a glass-house in which the air temperature was controlled to approximately 25°C by day and to 20°C by night. For studies of micronutrient deficiencies, seedlings were germinated over distilled water and then transferred to nutrient solution of the composition described by Tsui (1948). For macronutrient deficiency studies seeds were germinated in vermiculite and transferred to nutrient solution of the composition described by Hoagland and Arnon (1950). Each water­ culture vessel (3-1. "Pyrex" beakers) held 12 plants, and the nutrient solutions, which were aerated continuously, were not changed during the 2-3 weeks growing period. Nutrient solutions deficient in molybdenum were prepared by the method of Gentry and Sherrington (1950). Solutions deficient in copper, zinc, and manganese were prepared by the method of Stout and Arnon (1939). Solutions deficient in boron were prepared by dissolving A.R. grade salts in water from a stainless steel still. Boron contamination was further reduced by lining the beakers with thin polythene bags. Iron-deficient solutions were prepared from A.R. grade salts dissolved in distilled water. In order to obtain plants of a convenient size it was necessary to add a supple­ ment of the missing nutrient routinely to certain deficient cultures. The iron-deficient cultures were supplemented with a final concentration of 0·143 fJ-M Fe. Similarly, 0·182 fJ-MB, 0·035 mM K, 0·01 mM Ca, 0·056 mMN, 0·0133mMMg, andO'0133mM P was added to the corresponding deficient cultures. NUTRIENT DEFICIENCIES AND THE HILL REACTION 443 (b) Preparation of Ohloroplasts For each chloroplast preparation sufficient plants were harvested to yield 2-5 g fresh weight of leaves. All the leaves of each plant (excluding cotyledons) were used. In order to minimize day-to-day variation due to diurnal fluctuations in the Hill reaction activity (Hill and Scarisbrick 1939; A. W. Galston and J. Miller, personal communication) plants were harvested at approximately the same hour (9.00 a.m.) each day. The procedure adopted for the preparation of chloroplasts followed closely that of Jagendorf and Evans (1957). The major departure from their method was the omission of polyvinylpyrrolidone from the initial grinding solution. Leaves were macerated in 60 ml of solution containing sucrose (0·30M), KCl (0·01M), potassium phosphate (0'05M, pH 8·5), and sodium ethylenediamintetra-acetate (0'01M, pH 8·5). After removal of the cell debris by filtration through four layers of fine cloth, chloroplasts were sedimented by centrifugation at 1000 g for 10 min. The chloroplast pellet was suspended in 40 ml of a solution containing sucrose (0'30M), KCl (0·01M), and potassium phosphate (0'05M, pH 7 '3) and again sedimented at 1000 g for 10 min. The pellet was resuspended in the latter solution. This chloroplast suspension was then used for the determination of Hill reaction rates and chlorophyll content. Chloroplasts prepared in this way were examined under phase-contrast at X 1600 magnification. The chloroplasts appeared predominantly intact and undam­ aged, and were accompanied by insignificant amounts of other cell constituents excepting starch. The amount of starch in each preparation varied with the starch content of the leaf samples. Chlorophyll assays carried out on chloroplast pre­ parations and on whole plants indicated that more than one-third of the total plant chlorophyll was contained in the chloroplast preparation. Our aim in these experiments was to compare the activity of chloroplasts from deficient plants at an early stage of deficiency, with those from the appropriate healthy control plants. Comparisons between grossly deficient and healthy plants were avoided because of the extreme difference in size and morphology. Plants were harvested as soon as each deficiency was evidenced either by reduced fresh weight of whole tops, or by the commencement of characteristic deficiency symptoms. In most cases subsequent harvests were made within a period of 7 days. (c) Assay of Hill Reaction Rates These assays were carried out according to the procedure of Jagendorf and Evans (1957). This involves measuring the change in optical density (O.D.) at 620 mfL upon illumination for 45 sec of a reaction mixture containing chloroplasts, approximately 0·1M tris(hydroxymethyl)aminomethane (Tris) buffer, pH 7 '3, and 0·07 fLmoles of o-chlorophenoI-2,6-dichloroindophenol (B.D.H.) in a total volume of 3·0 m!. Illumination at more than saturating intensity was provided by a 250-W "Photoflood" lamp. The amount of chloroplasts (=2-10 fLg chlorophyll) in the reaction mixture was varied so as to give an O.D. change of 0·09-0,120 upon illumination for 45 sec. O.D. measurements were made in a Unicam SP 600 spectro­ photometer and, unless otherwise noted, assays were carried out at 30°C. 444 D. SPENCER AND J. V. POSSINGHAM Where a range of light intensities was required, the distance between the reaction vessel and the light source was varied. Light intensities were determined with an E.E.L. photoelectric illuminance meter. Phosphate buffer used in the preparation of the chloroplasts and Tris buffer used in the Hill reaction assay were extracted with 8-hydroxyquinoline in chloroform in order to remove heavy metal contaminants. Total chlorophyll concentration, and the relative proportions of chlorophyll a and b were calculated from the O.D. at 663 mIL and 645 mIL of an 80 per cent.
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