The influence of cations and methylamine on structure and function of thylakoid membranes from barley chloroplasts

Abstract

The effects of cations on the structure and activity of isolated barley (Hordeum vulgare cv. Sval6fs Bonus) chloroplast lamellar systems were studied. Three separate effects of cations were recognizable. (I) Cations in high concentrations (in excess of 100-200 mM NaCI) are required to maintain granal stacks once the outer chloroplast envelope is broken. In 30 mM NaCI the majority of the lamellar systems exist as well-separated thylakoids, even in the presence of high concentrations of sucrose (330 and 660 mM). The lamellar systems photoreduce ferricyanide at coupled rates whether they are in the granal or non-granal configuration. (2) Cations are required for Hill reaction activity. This requirement becomes apparent following loss of thylakoid membrane integrity at very low cation concentrations. Maximum activation of the photoreduction of ferricyanide occurs at 30 mM NaCI, and divalent ions (Mg §247 , Ca *+ , Mn §247 ) are 12 times more effective than monovalent ions (Na + , K § (3) Cations are necessary to preserve the integrity of thylakoid membranes. Below about 8 mM NaCI, the thylakoids swell and this change is accompanied by progressive loss of Hill activity and the capacity to maintain a light-dependent proton gradient. Divalent cations are more than 200 times as effective as monovalent ions in preventing these changes. Hill reaction activity, but not proton pump activity, is regained by adding cations back to the swollen thylakoids (cation effect 2, above) and the new rate of Hill reaction activity is the same as in chloroplasts uncoupled with methylamine. The re-establishment of Hill reaction activity is accompanied by appression of the swollen thylakoids. The uncoupler methylamine causes stacking of thylakoids and collapsing of intrathylakoidal spaces. R. M. SMILUE et al.: Thylakoid Membranes of Barley

Composition and Function of Thylakoid Membranes from Grana-rich and Grana-deficient Chloroplast Mutants of Barley

“…Results of studies we reported earlier showed that wild type barley chloroplasts prepared as described in this report consist of naked lamellar systems with almost no grana (18). Salt concentrations higher than those used in the isolation media, or those used in the assay mixtures, would be required if grana structure were to have been maintained, or if appression between adjacent thylakoids were to have been re-established (18 Despite the lack of grana, the chloroplast preparations catalyzed high rates of electron transport from water to both ferricyanide and DCIP, and were able to establish proton concentration gradients across membranes in the light.…”

“…Salt concentrations higher than those used in the isolation media, or those used in the assay mixtures, would be required if grana structure were to have been maintained, or if appression between adjacent thylakoids were to have been re-established (18 Despite the lack of grana, the chloroplast preparations catalyzed high rates of electron transport from water to both ferricyanide and DCIP, and were able to establish proton concentration gradients across membranes in the light. These activities had the expected sensitivities to various uncouplers, phenylenediamine, DCMU, and dibromothymoquinone (16).…”

“…Finally, the pelleted chloroplasts were suspended in 1 ml of the wash medium. Examination of such preparation by electron microscopy reveals predominantly naked lamellae systems and very few grana (18). The Chl was extracted using 80%o (v/v) acetone containing 0.2% NH40H, and the A at 645 and 663 nm were measured in order to calculate the content of Chl a and b (3).…”

Salt Tolerance in Crop Plants Monitored by Chlorophyll Fluorescence In Vivo

“…Chloroplasts were isolated as described previously (24) and photochemical activities assayed at 23°C using an Aminco DW-2 spectrophotometer (American Instrument Co.) with saturating red-light side illumination. The assay mixture (1.5 ml) contained chloroplasts (4 ,ug Chl ml-'), 50 'Abbreviations: PD, p-phenylenediamine; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone.…”

Active Oxygen Produced during Selective Excitation of Photosystem I Is Damaging Not Only to Photosystem I, But Also to Photosystem II

“…The thylakoid membranes carry a net negative charge, but the presence of cations, especially divalent cations, keeps the thylakoid membranes stacked by screening the electrostatic repulsion (Barber, 1980(Barber, , 1982Sculley et al, 1980). NaCl was included at 10 mm to avoid osmotic swelling and rupture of the thylakoid membranes, but this concentration is still sufficiently low to allow destacking (Smillie et al, 1976). To facilitate migration and randomization of the photosynthetic complexes in the destacked thylakoid membranes (Sundby et al, 1986), the membranes were further incubated for 40 min at 20°C at approximately 2 mg of chlorophyll per milliliter after the last resuspension in 10 mm Bis-Tris (pH 6.8), 100 mm Suc, and 10 mm NaCl.…”