Effects of Freezing on Spinach Leaf Mitochondria and Thylakoids in Situ and in Vitro

Abstract

The sensitivity of spinach (Spinacia oleracea L.) leaf mitochondria and chloroplast membranes to subzero temperature stress was compared after freezing of the membrane systems in situ and in vitro. Respiratory and photosynthetic activities were measured polarographically.When leaves were frozen under controlled conditions for 2 hours to various minimum temperatures and mitochondria and chloroplasts isolated after thawing, the membrane systems showed a nearly simultaneous inactivation of respiratory and photosynthetic activities between -5 to -7 C. At that temperature range in both membrane systems phosphorylation became only slightly more affected than electron transport, ie. after freezing in situ conspicuous uncoupling of phosphorylation from electron transport was not observed.In contrast, mitochondria and thylakoids isolated from the same preparation of intact leaves under comparable conditions using NaCI as osmoticum exhibited differences in sensitivity towards freezing for 2 to 4 hours at -25 C in vitro. In the absence of cryoprotectants, photophosphorylation of isolated thylakoids became completely uncoupled from electron transport which was increased several-fold compared with the unfrozen controls. Inactivation of respiratory functions of isolated mitochondria followed the same pattern as observed after freezing in situ. In the presence of sucrose for protection of thylakoids significantly higher concentrations of the cryoprotectant were necessary than for preservation ofmitochondria. Thus, under the conditions used in this study chloroplast membranes proved to be more sensitive to freezing in vitro than mitochondria.It is a common view that inactivation of cellular membranes is the primary cause of frost damage in plant cells (11,15). In previous investigations with isolated plant membrane systems, thylakoids and mitochondria turned out to be highly sensitive to freezing (4,6,7,24). Inactivation of chloroplast membranes also took place when intact leaves were killed during extracellular ice formation (7,10,12). Singh et al. (23) found that mitochondria in situ can retain their normal function even after the cell was killed by freezing. However, gas exchange measurements on partly frostdamaged spinach leaves have shown that photosynthesis and respiration decrease almost simultaneously (12).To clarify these discrepancies, we have investigated the sensitivity of spinach leaf mitochondria and chloroplast membranes to subzero temperature stress in vitro. Both membrane systems were isolated from one leaf batch in similar isolation media and subjected to freezing and thawing under identical conditions. Subsequently, respiratory and photosynthetic activities ofthe membrane systems were tested. In addition, the effect of freezing on mito-1 This research was supported by the Deutsche Forschungsgemeinschaft. chondria and thylakoids in situ was studied. Intact leaves were frozen at various temperatures; after thawing, mitochondria and chloroplasts were isolated and the biochemical activities of the membran...

Age-related differences in frost sensitivity of the photosynthetic apparatus of two Plagiomnium species

“…5). In herbaceous higherplant species freezing affects photosynthesis and respiration either similarly, or respiration is distinctly less sensitive to a freeze-thaw treatment than photosynthetic CO2 uptake (Klosson and Krause 1981a;Thebud and Santarius 1981 ;Krause and Klosson 1983 ; see also Singh et al 1977). However, it cannot be excluded that in freeze-injured moss tissue photorespiration contributes to oxygen uptake of the cells (Rastorfer 1970).…”

Effects of High-Temperature Stress on Various Biomembranes of Leaf Cells In Situ and In Vitro

“…The addition of various exogenous substrates did not stimulate 02 uptake in the dark probably due to permeability barriers of the plasmalemma. Photosynthesis was started by addition of 2 mm NaHCO3 and illumination with saturating red light (550 w/m2) provided by a 150-w halogen lamp using a filter combination as described earlier (26) completely inhibited the HC03--dependent 02 evolution of free intact chloroplasts contaminating the preparation. The 02 consumption of mitochondria was measured as outlined by Douce et al (6) using 40 mm malate plus 25 mm glutamate as substrate.…”

“…The 02 consumption of mitochondria was measured as outlined by Douce et al (6) using 40 mm malate plus 25 mm glutamate as substrate. Methylviologen-mediated photosynthetic electron transport of thylakoids was determined in the presence of KCN under conditions described earlier (26). ATP synthesis, respiratory control, photosynthetic control, and ADP:O ratios were calculated according to Estabrook (8) and Robinson and Wiskich (20).…”

“…Isolation of Mitochondria and Thylakoids. Both biomembrane systems were isolated from intact and heat-treated spinach leaves in a single procedure as described earlier (26). The washing medium always contained mannitol (300 mM) as osmotic agent.…”

Freezing damage and frost tolerance of the photosynthetic apparatus studied with isolated mesophyll protoplasts of Valerianella locusta L.

“…In contrast to freezing of thylakoid membranes in vitro in the presence of high levels of inorganic salts [see ref . 6], uncoupling of photophosphorylation was not a primary effect of freezing in vivo [7,10,11,21].…”