Inactivation of the Hill Reaction in Spinach Chloroplasts by Pre-treatment of Tris Buffer in the Light

Abstract

By pre-treatment of spinach chloroplasts with a high concentration of Tris buffer, 0.8 M, pH 8. 0, oxygen evolution coupled with DPIP or ferricyanide photoreduction was inactivated only when the chloroplasts were incubated in the light , but not in the dark. This inactivation of the chloroplast reaction was not due to chemical decomposition of Tris, but was probably due to the photoinhibition as reported by others. The photoinhibition was protected by the addition of DPIP in either oxidized or reduced form.It has been shown by many authors that oxygen evolution which is closely connected to the photosystem 2 of photosynthetic electron transport in higher plants is unstable than the photosystem 1 for various treatments, such as prolonged storage of chloroplastsl', heating, or treatments with detergents and lipase2-4'. Recently, Yamashita and Horio5' demonstrated that treatment of chloroplasts with high concentrations of Tris buffer resulted in the inhibition of the Hill reaction.We have studied the effect of Tris buffer in more detail in order to elucidate the oxygen evolving system of chloroplasts. On pre-incubating the chloroplasts in a high concentration of Tris buffer (0.8 M, pH 8.0) under weak light, oxygen evolution coupled with reduction of DPIP or f erricyanide (Hill oxidant) was inhibited, although no inhibition occurred in the dark. Material and MethodsChloroplasts were isolated from spinach leaves (Spinacia oleracea L.), using a conventional methods' and washed once with homogenizing medium (0.01 M KCI-0.05 M Tris-HC1 buffer, pH 7.5, containing 0.4 M sucrose). Washed chloroplasts were suspended in the same medium and each portion of the chloroplast suspension was treated with a high concentration of Tris buffer (0.8 M, pH 8.0) according to the following procedures. To 8 ml of 1.0 M Tris buffer, pH 8.0, 2 ml of the chloroplast suspension containing 0.5 mg chlorophyll per ml was added and mixed in an open Petri dish (8 cm in diameter). Final concentrations of Tris and chlorophyll in the mixture were 0.8 M and 0.1 mg per ml, respectively. Tris solution was freshly prepared before use. The mixture was then incubated for an appropriate time in the

Mechanism of irreversible inhibition of oxygen evolution in photosystem II by tris(hydroxymethyl)aminomethane

“…Irreversible inhibition of 0, evolution upon incubation of PSII in 0.8 M Tris in the dark was reported earlier by Ikehara and Sugahara (1969) and Cheniae and Martin (1978), but the mechanism was not studied in detail. More work has been focused on the light-induced inactivation of 0, evolution by Tris (Yamashita & Horio, 1968).…”

Modification of oxygen evolving center by Tris-washing

“…Aged Tris-buffer yielded formaldehyde and inhibited chloroplasts [2]. Tris has a primary amino group which become non-ionic form (R-NH2) at higher pHs and inhibit chloroplasts [7][8]II]. Inactivation of chloroplasts by Tris-washing was stimulated by illumination (i000 lux), especially at pH lower than 8.0 [7].…”

“…Tris has a primary amino group which become non-ionic form (R-NH2) at higher pHs and inhibit chloroplasts [7][8]II]. Inactivation of chloroplasts by Tris-washing was stimulated by illumination (i000 lux), especially at pH lower than 8.0 [7]. Single and double flashes were more effective than triple flashes in stimulating the inactivation.…”

“…Tris-washing inactivates the 02-evolving center specifically and reversibly [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. The inactivated chloroplasts and particles could be reactivated by Mn2+-addition [20][21], DCPIP.H2-treatment [12,17,[22][23][24][25][26][27][28][29][30][31] and photo-reactivation [26-27, 29, 31-36].…”

Light-Induced Effects of Tris on the Ultrastructure of Leaf Tissue

“…It has been shown to affect many biological processes including ion transport (Van Steveninck 1961, 1966a, 1966bKholdebarin and Oertli 1970), the Hill reaction (Jacobi 1961), uncoupling of photosynthetic phosphorylation (Good 1962), isolation of mitochondria (Stinson and Spencer 1968), sucrose storage (Humphreys and Garrard 1969), and amino acid incorporation (Bewley and Marcus 1970). Ikehara and Sugahara (1969) suggested that Tris at high pH may alter the structure of chloroplast lamellae. A possible effect of Tris on membrane structure was suggested also by Humphreys and Garrard (1969) and by Yamashita, Tsuji, and Tomita (1971).…”