Photosynthesis, Growth, and the Role of Chloride

Abstract

Previous studies with isolated chioroplats have indiated that a-is an essential cofactor for photosynthesis. Coiderable pport for the postulted a-requirement in photosynthesis cme from the observation that a-is essential for growth. Data are presnted which show that a 60% reduction in growth which occurred in a-deficient sua beet (Beta vulgaris L.) was not due to an effect of a-on the rate of photosynthesis in vivo (net CO2 ptake per unit area of attached leaves). The principal effect of a-deficiency was to lower cell multip tion rates in leaves, thus slowing down their growth and altiately d ing their area. The absence of an effect of a-on photosynthesis in vivo was nlikely to have been due to a-retention by the chloroplasts becaue their a-concentration (measured after nouaqueous isolation) deeased progressively with decrease in leaf a-. An effect of a-with isolated chloroplasts in vitro, however, was confirmed. Addition of a-to the reaction medium after washing chloro-plasts in EDTA increaed the rate of ferricyanide photoreduction 10-fold. This effect of a-did not appear to be related to the a-concentration of the chioroplass since chloroplast a-was not decreased further by washing in EDTA. It is concuded that a-has not yet unequivocally been shown to be an essential cofactor for photosynthesis and that the response to Cl-in vitro probaby does not have a physiological basis. demonstrated the essentiality of Cl-, this theoretical objection to the role of Cl-in photosynthesis was apparently removed. In later reports related to the Cl-effect, the requirement for Cl-in plant growth is usually noted (3, 9, 15), and the assumption implicit in making such an observation is that Cl-is required for growth because of its role in photosynthesis. However, this assumption had not been tested experimentally. The objective of the present investigation therefore was to study further the role of Cl-in photosynthesis and growth and to determine in particular whether the effect of Cl-on plant growth is mediated via an effect on the rate of photosynthesis in vivo as opposed to an effect on some other facet of growth. Data are presented below which show that the reduction in growth which occurred in response to Cl-deficiency was not due to an effect of Cl-on the rate of photosynthesis in vivo as measured by net CO2 uptake of attached leaves. The principal effect of Cl-deficiency was to reduce cell multiplication rates in leaves, which in turn decreased leaf expansion, and thereby plant growth by up to 60%. Despite the absence of a "Cl-effect" in vivo in sugar beet, the Cl-effect could be readily obtained with isolated chloroplasts in vitro using the washing procedures described by Izawa et al. (10). I concluded that Cl-has not yet been shown unequivocally to be an essential cofactor for photosynthesis and that the response of Cl-in vitro may not have a physiological basis. Since Warburg and Luttgens (25) first showed that photosyn-thetic 02 evolution from isolated chloroplast fragments depended on the presence of Cl-, there has been some question as to whether Cl-is an essential cofactor for photosynthesis. Alternatively it has been suggested that Cl-might be required in vitro to protect chloroplasts during their isolation or assay (1, 6, 16). Later work (3, 8-10) indicated that Cl-was required for photo-synthetic 02 evolution at PSII. Virtually all of the evidence supporting the Cl-requirement for photosynthesis was obtained from studies carried out in vitro; in these experiments the photosynthetic activity of isolated chlo-roplasts (or chloroplast fragments) was decreased by washing to decrease the Cl-content, and then restored by adding Cl-to the reaction medium (1, 3, 8-10, 25). The improvement in the in vitro rate of photosynthesis which occurs when Cl-is added to the reaction medium compared to the rate when no Cl-is added is referred to elsewhere (9) and subsequently in this paper as the "Cl-effect." In 1949 Arnon and Whatley (1) considered it unlikely that Cl-was an essential cofactor for photosynthesis since, up to that time, Cl-had not been shown to be required for plant growth. When in 1954 Broyer et al. (4), and later others (12, 24), ' This work was supported in part by the Beet Sugar Development Foundation. 2 A brief presentation of this work was made at the Annual Meeting of the American Society of Plant Physiologists at New Orleans, La., June 3, 1976 (Plant Physiol. 57: S-95). MATERIALS AND METHODS Plant Culture and Growth Analysis. Sugar beets (Beta vul-garis L. cv. F58-554H1) were cultured hydroponically at 25 C, and illuminated at 40,000 lux over a 16-hr day. The plants were cultured for 2 weeks following planting in vermiculite with half-Hoagland solution then transplanted to culture solutions containing five different concentrations of Cl-: 0, 0.5, 5, 50, and 500 gmol 1-1. The composition of the culture solution (exclusive of Cl-) was: in mmol/l, 2 Ca(NO3)2, 1 K-phosphate, 2.5 KNO3, and 1 MgSO4, and in mg/l, 0.25 B, 0.25 Mn, 0.025 Zn, 0.01 Cu, 0.005 Mo, and 2.5 Fe (as ferric-sodium ethylenediaminetetraac-etate complex). Chloride was added as the Na salt, the Na concentrations being maintained at 0.5 mm in all treatments by adding Na2SO4. At 2 and 4 weeks from transplanting, concentrated stock solutions of nutrients other than Cl-were supplied in amounts to equal the initial culture solution composition. Each week plants were removed for the determination of fresh and dry weights of blades, petioles, storage roots and fibrous roots, and for the determination of areas and numbers of living leaves as well as the numbers of dead leaves. One complete experiment consisted of 25 plants (sufficient for five Cl-treatments to be harvested on five successive occasions) randomized within one growth chamber. This experiment was replicated five times. Cell Numbers, Mean Cell Volume. An experiment was designed to test whether the reduction in leaf area which occurred with Cl-deficiency was due to a decrease in the number of cells/ leaf, or to a reduction in mean cell volume. Three plants were chosen which had been supplied with Cl-and three which had 69