Cryoprotection by Glucose, Sucrose, and Raffinose to Chloroplast Thylakoids

Abstract

cerning the differential cryoprotection by glucose, sucrose, and raffimose afforded to thylakoids during freezing and thawing (13) or heat stress (12) were interpreted as evidence supportive of a noncolligative-type mechanism. Here, we present data concerning the anomalous behavior of these sugars during freezing which have necessitated a reinterpretation of this earlier hypothesis concerning the mechanism of cryoprotection by neutral sugars.MATERIALS AND METHODS Chloroplast Isolation. Washed, deveined spinach leaves (Spinacia oleracea L. "Winter Bloomsdale") were ground in a Waring Blendor in a medium containing 20 mm Tricine, 250 mm NaCi, 20 mm sodium ascorbate, and 0.1% BSA (pH 7.8). Leaves were sprayed with Foamkill antifoam spray (Nutritional Biochemicals) prior to grinding. Following filtration through two layers of Miracloth, the homogenate was centrifuged for 1 min at 3,000g. The pellet was resuspended in 10 ml of 10 mm NaCl and homogenized by sucessive passages through a 10-ml volumetric pipet. The suspension was diluted to 40 ml with 10 mm NaCl and centrifuged for 7 min at 20,000g. Thylakoids were washed a second time in 10 mM NaCl and diluted to a Chl concentration of 1.0 mg/ml with 10 mM NaCl. Chl was determined by a modification of the Arnon method (18). Initial isolation was carried out at 4 C, and all washing operations were performed at 0 C.Freezing and Thawing of Thylakoid Suspensions. Thylakoid suspensions containing 500 jig Chl/ml, 5 mm NaCl, and 0-100 mm glucose, sucrose, or raffinose were frozen to -18 C in a stirred methanol bath freezing unit programmed to cool at 2.8 C/h. Samples were held at -18 C for 3 h and thawed in the same bath at 10 C/h. Biochemical assays were performed immediately after thawing.Cyclic Photophosphorylation. The reaction mixture (0.5 ml) for cyclic photophosphorylation consisted of the following compounds at the stated final concentrations: 5 ,ug Chl equivalents; 3 mM KH2PO4, 1 mm sodium ascorbate, 50 pm phenazine methosulfate, 3 mm ADP (pH 8.2). Carrier-free 32Pi was added at a dilution of 1 ,uCi/ml. Illumination was provided by two 300-w flood lamps at a distance of 4 cm from the reaction vessels, and the temperature was maintained at 16 C. The reaction was stopped by adding 0.5 ml of a solution containing 1.5 g ammonium molybdate, 2.75 ml concentrated HCI, 0.5 ml triethylamine, and 0.5 ml saturated bromine water/50 ml of solution (14). The suspensions were allowed to settle 10 min at room temperature and then were centrifuged at 12,000g for 10 min. A 0.5-ml aliquot of the supernatant was transferred to a planchet, dried, and counted on a Nuclear-Chicago gas flow counter operated at 1,300 v with a quenching gas flow of 50 ml/min.

Alternative Cold Response Modes in Chlorella (Chlorophyta, Trebouxiophyceae) from Antarctica

“…In the cold, C. vulgaris ATCC 11468 accumulates raffinose in addition to sucrose (Salerno & Pontis 1989). Sucrose, raffinose, and trehalose could either provide cryoprotection on photosynthetic membranes (Lineberger & Steponkus 1980) or generally enhance the freeze tolerance of microorganisms (Welsh 2000). We detected these di-or trisaccharides in the two Antarctic species (Fig.…”

Responses to Extreme Temperatures. Cellular and Sub-Cellular Bases

“…The differential cryoprotection appears, however, to be due to nonideal activity at the extremely high concentrations experienced during freezing (LINEBERGER and STEPONKUS 1980). When cryoprotection is analyzed as a function of the mole fraction of NaCI to which the thylakoids are exposed during freezing, protection of cyclic photophosphorylation and its component reactions is not dependent upon the chemical identity of the protective sugar.…”

Chloroplast Membrane Remodeling during Freezing Stress Is Accompanied by Cytoplasmic Acidification Activating SENSITIVE TO FREEZING2

“…This enhanced proximity of membranes, lack of fluidity, and low hydration allow nonlamellar structures to form between membranes, fusing subcellular compartments and ultimately resulting in cell death after rehydration (Webb et al, 1994;Uemura et al, 1995). Multiple mechanisms have evolved in plants to avoid both dehydration and membrane fusion, including solute accumulation, cell wall modification, lipid desaturation, and lipid composition changes (Lineberger and Steponkus, 1980;Browse and Xin, 2001;Degenkolbe et al, 2012;Chen and Thelen, 2013;Ji et al, 2015). These changes typically occur during a period of cold acclimation or cold hardening in which plants are exposed to low, nonfreezing temperatures prior to freezing and transcriptional changes accompany increased freezing tolerance.…”