Liniiting Factors in Photosynthesis II . IRON STRESS DIMINISHES PHOTOCHEMICAL CAPACITY BY REDUCING THE NUMBER OF

It has been proposed that Fe stress may be used in the study of limiting factors in photosynthesis as an expermental means of varying photochemical capacity in vivo (Plant Physiol 1980 65: 114-120). In this paper the effect of Fe stress on photosynthetic unit number, size, and composition was investigated by measuring P700, cytochrome (Cyt)f, chlorophyll (Chi) a, and Chi b in sugar beet leaves. The results show that when Fe stress reduced Chl per unit area by 80% (from 60 to 12 micrograms per square centimeter), it decreased the number of P700 molecules per unit area by 88% and Cytf per unit area by 86%; over the same range the ChM to P700 ratio increased by 37% but there was no significant change in the Chi to Cyt f ratio. These data suggest that Fe stress decreases photochemical capacity and Chl per unit area by diminishing the number of photosynthetic units per unit leaf area. The ratio of Chi a to Chi b did not change with Fe stress. This suggests that the proportion of light-harvesting Chi a/b-protein complex within the photosynthetic unit remained constant. Electron microscopy ofchloroplasts revealed that the decrease in the number of photosynthetic units which occurred during Fe stress was accompanied by a reduction in the number of granal and stromal lamellae per chloroplast and by a reduction in the number of thylakoids per granum. In the first paper of this series (26), it was proposed that mineral nutrient stress might be used as an experimental tool in the study of limiting factors in photosynthesis. Data were presented which showed that Fe stress in sugar beet plants reduced the photochemical capacity of leaves. These data also showed that the maximum rate of photosynthesis per unit Chl was not decreased by Fe stress, suggesting that the size of the photosynthetic unit was unchanged. The hypothesis tested here was that the reduction in photochemical capacity which accompanied Fe chlorosis resulted from fewer photosynthetic units per leaf area rather than from a change in photosynthetic unit size. According to current models of the photosynthetic unit there is 1 molecule of P700 and 1 molecule of Cytfper photosynthetic unit (5) (except in some low-light-grown plants [4, 61). On this assumption, we measured P700 and Cyt f as a means of estimating the number of photosynthetic units per unit leaf area. The ratio of Chl/P700 and Chl/Cytf provided indices of photosynthetic unit 1 This work was partly supported by the Beet Sugar Development Foundation. Taken from a thesis submitted by S. C. Spiller in partial fulfillment of requirements for the Ph.D., University of California, Berkeley. 2 This work was presented at the Annual Meeting of the American Society of Plant Physiologists at Blacksburg, Virginia, June 29, 1978 (Plant Physiol. 61: S-87). size (4, 5). In addition, we measured the Chl a/Chl b ratio. Brown et al. (9) and Genge (12) concluded that Chl b is located only within the light-harvesting complex while Chl a is found throughout the photosynthetic unit. Based on their work we assumed that a change in the amount of light-harvesting Chl a/Chl b-protein complex per photosynthetic unit would be indicated by a change in the Chl a/Chl b ratio. Since earlier work indicated that Fe stress may influence the formation of the entire light-harvesting and electron transport apparatus (25, 26), electron micrographs were prepared to show the progressive effects of Fe stress on chloroplast structure. MATERIALS AND METHODS Plant Culture and Harvest Procedure. Sugar beet plants (Beta vulgaris L. cv. F58-554H1) were cultured in growth chambers at 25 C with an illumination of 35,000 lux (7.0 mw cm-2; 45 nE cm-2 s ' [400-700 nm]) supplied over a 16-h daylength. The procedure for culturing and inducing Fe stress was the same as previously described (26). Leaves were harvested from two control plants and three Fe-stressed plants between 7:00 and 8:00 AM. After removal from the plant the harvested leaf was immediately enclosed in a plastic bag and stored at 2 C until it was used (usually on the same day). P700 Extraction and Assay. The method employed for the extraction and assay of P700 was modified from that used by Shiozawa et al. (22). After removing the midrib the leaves were chopped and ground for 5 s in a Waring Blendor with a preparative solution containing 0.5 M sucrose, 0.1 M Tris-HCl (pH 8.0) and 30 mm sodium ascorbate. The resulting suspension was filtered through six layers of cheesecloth and centrifuged at 30,000g for 5 min. The pellet was resuspended and washed in 40 ml of a wash solution containing 0.10 M Tris-HCl (pH 8.0) and 30 mm sodium ascorbate and centrifuged at 30,000g for 10 min. This procedure gave a pellet which consisted largely ofchloroplast lamellae. These were resuspended in a small volume and diluted to give a Chl concentration ofapproximately 800 jig/ml using the wash solution. A 0.5-ml aliquot of this suspension was diluted to 5 ml to give an assay solution with a final concentration of 0.5% (v/v) Triton X100, 0.1 M Tris-HCI (pH 8.0), 30 mim sodium ascorbate and a Chl concentration of about 80 jig/ml. After 30 min at room temperature the assay solution was centrifuged at 2,500g for 10 min and the supernatant assayed immediately, or, frozen at -8 C prior to measurement. The assay of P700 was carried out with an Aminco DW2 spectrophotometer which was modified by the insertion ofan extension tube and a matt black baffle (with holes for the measuring beam) between the sample cuvette and the photomultiplier. This arrangement was made to minimize the amount of sample fluorescence reaching the photomultiplier. The concentration of P700 was determined using the dual wavelength mode by measuring the difference in A at 698 nm (slit width 3.5 nm) between the pho121 www.plantphysiol.org on July 14, 2017 Published by Downloaded from Copyright © 1980 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 165, 1980 tooxidized and dark-reduced sample with 730 nm as the reference wavelength. An extinction coefficient of 64 mm-' cm-' (14) was used to calculate P700 concentration from the difference between AO.D. (698-730 nm) in light and in darkness. Blue actinic light was provided by means of an incandescent microscope lamp filtered through two Corning No. 4-96 filters. A was first measured in darkness and then at each of three increasing irradiances to ensure that the P700 was fully photooxidized. Actinic light was prevented from reaching the photomultiplier by two red Corning filters (No. 2-64) taped to the face of the photomultiplier. Determination of Chlorophyils. To determine Chl a and Chl b, leaf plugs of known area were ground in a glass homogenizer with powdered sodium ascorbate and a solution containing 80%o (v/v) acetone in water. The resulting suspension was made up to volume with 80%o (v/v) acetone and centrifuged. The A of the supernatant was measured at 645 nm and 663 rm and the coefficients of Mackinney (16) used to determine the amounts of Chl a and Chl b per unit leaf area. The Chl content of the P700 assay solution was determined directly from the measurement of A at the 670 nm peak using an extinction coefficient of 60 mm-' cm-' (22). Cyt f Extraction and Assay. The midrib was removed from harvested leaves and the leaf blade chopped and ground in a Waring Blendor in a preparative solution consisting of 0.4 M sucrose, 20 mi Tricine-KOH (pH 8.0), 10 mm NaCl and 30 mM sodium ascorbate. The suspension was filtered through six layers of cheesecloth and centrifuged at 30,000g for 5 min. The pellet was resuspended in 40 ml of a wash solution consisting of 20 mm Tricine-KOH (pH 8.0), 10 mm NaCl and 30 mm sodium ascorbate and centrifuged at 30,000g for 10 min. This pellet was resuspended in a second wash solution of 20 mm Tricine-KOH (pH 8.0) and 10 mm NaCl and centrifuged at 30,000g for 10 min to remove sodium ascorbate. The chloroplast pellet was resuspended in a minimal volume of a solution containing 50 mm Tricine-KOH (pH 8.0) and 5mM MgCl2. An appropriate aliquot was added to the assay solution containing 50 mM Tricine-KOH (pH 8.0), 5 mM MgCl2, and 1% (v/v) Triton X-100 to give a final concentration of 65 to 110 LM Chl. The Triton was included to eliminate interference from absorption by the high potential form of Cyt bw (3). The concentration of Cytfwas determined by measuring the AO.D. at the 554nm peak obtained by ferricyanide oxidation and hydroquinone reduction of the sample using the split beam mode of the Aminco DW2 spectrophotometer (3). An extinction coefficient of 19.7 mm-' cm-' was used (based on Forti et al. [11] and personal communication from D. S. Bendall). ElectronMicroscopy. Blocks of leaf tissue (0.5-1.0 mm3) were fixed for 2 h in a solution consisting of 4% glutaraldehyde and 50 mm S0rensen's phosphate buffer (pH 7.2). The fixed tissue was rinsed using a solution of 50 mm S0rensen's phosphate buffer (pH 7.2) three times, 15 min per rinsing. Postfixation was carried out using1%OS04 in 50 mM S0rensen's phosphate buffer (pH 7.2) for 2 h and rinsed in the buffer solution for 10 min. The tissue was dehydrated using a series of ethanol-water solutions, 30, 50, 70, 85, 90, and 95% (10 min per solution), followed by two changes in 100o ethanol (20 min each) and two changes in propylene oxide solution (20 min each). The leaf tissue was embedded in Araldite. Sections were stained with Reynold's lead citrate and saturated aqueous uranyl acetate. The electron microscopy was carried out using a Siemens IA electron microscope.