Biochemical and Structural Correlations in Isolated Spinach Chloroplasts Under Isotonic and Hypotonic Conditions

Abstract

Methods are described for obtaining preparations of isolated spinach chloroplasts which are predominantly either "whole", i.e. retain the outer limiting membrane, or "naked", i.e. with the outer membrane removed. The whole chloroplasts are shown to retain more of their soluble components as evidenced by higher endogenous capacities for CO2-fixation, photophosphorylation, and light-dependent reduction of triphosphopyridine nucleotide. The isolation is also described of whole chloroplasts whose peripheral jackets have retained the property of mobility seen in living cells. Chloroplast fragments, obtained by mechanically rupturing intact chloroplasts, exhibited a higher Hill reaction activity than did intact chloroplasts. Parallel microscopic and biochemical observations of isolated chloroplasts under increasingly hypotonic conditions revealed a marked decline in photophosphorylating capacity along with a progressive swelling of the chloroplast. In contrast, Hill reaction capacity is increased threefold at sucrose concentrations below 0.005[image]. Enhancement of Hill reaction and loss of photophosphorylating capacity coincided with osmotic fragmentation of the chloroplast mass and formation of many small, discrete, balloon-like structures. Low concentrations of Mg2+ suppress to a large extent both structural and biochemical effects of hypotonicity. Water-treated preparations must be restored to a higher solute concentration (0.1[image]) before the enhanced rate of Hill reaction is elicited. The Hill reaction of such water-activated chloroplasts was compared with that of non-swollen chloroplasts with respect to light intensity relationships, temperature coefficient, and action spectra. The Hill reaction of manganese-deficient plants was not activated by water treatment.