Relative Insensitivity of Mitochondria in Hardened and Nonhardened Rye Coleoptile Cells to Freezing in Situ

Abstract

Freezing studies of plant cells point to membranes at cell surfaces and in cell organelles as being the primary site of freezing injury (5,7,8,11,12). The correlation of increases in phospholipid content in tree cells with extreme hardening has also focused attention on the involvement of membranes in the adaptation of the plant cell to freezing (13,14).While evidence exists showing that injury to the plasma membrane occurs when the whole plant cell is damaged by the dehydrative stresses of extracellular freezing (12), evidence pointing to plant mitochondria as also the targets in such cell injury is based largely on observations made on the freezing of the isolated organelle (3,4,6,7). Neither the important functional examination of the level of mitochondrial impairment sustained in situ during extracellular freezing of the plant cell nor whether the mitochondria, the cell, and other cellular membranes experience similar degrees of injury at freezing temperatures which are lethal to the cell has been reported.In this communication, we present evidence for the relative insensitivity of plant mitochondria in situ. to damage under extracellular freezing regimes which are clearly lethal to the cell.MATERIALS AND METHODS Plant Material. Coleoptiles (15-20 mm in length) were isolated from winter rye (Secale cereale L. cv. Puma) seedlings grown in the dark for 65 hr at 24 C (nonhardened) and from seedlings grown in the dark for 4 weeks at 2 C (hardened).Freezing Procedure. Freezing experiments were performed on the excised coleoptiles as follows. One-g batches of coleop- tiles were placed in polyethylene bags (10 x 15 cm) with moist cotton and sprayed with a fine mist of water before the bags were sealed with a 200 w bar sealer. The bags were first equilibrated at 0 C for 1 hr in a programmed freezer, the temperature was then allowed to drop at 1 C/hr to -20 C and thereafter at 3 C/hr until the desired temperature was attained. Cell survival after freezing was estimated by vital staining of the cells with neutral red or from observation of protoplasmic streaming. Survival of the isolated coleoptiles as a function of the freezing temperature was correlated with the survival of whole seedlings which had been subjected to identical freezing conditions and replanted on vermiculite flats as described by de la Roche et al. (2). Fast freezing of the coleoptiles was performed by immersing the polyethylene bags containing the coleoptiles into a beaker containing ethylene glycol cooled to -20 C. A thermometer placed in the polyethylene bag indicated that approximately 30 sec were required to reach -20 C. Freezing of the coleoptile tissues at different freezing rates was also examined on a microscope freezing stage. At rates of freezing comparable to the fast freezing described above, complete intracellular freezing was observed whereas only extracellular freezing was noted when the slower freezing rate was employed. Cell survival after extracellular freezing was dependent on the degree of hardening and the temperature to which ...

Increase in Linolenic Acid Is Not a Prerequisite for Development of Freezing Tolerance in Wheat

“…Duplicate lots were removed every 2 hr and allowed to thaw at 2 C. The seedlings were planted in Vermiculite after 12 hr of thawing and survival was assessed after 1 week of growth in a controlled environment (24 C light, 18 C dark, 16-hr photoperiod). Freezing tests were also performed on detached shoots (15). The excised shoots, after thawing, were incubated under continuous light at 22 C in Petri plates containing 10 ml of dilute salt solution (containing 45 mm NaCl and 5 mm CaCl2).…”

“…Shoots were also examined microscopically immediately after thawing and after 48 hr of incubation. Cell vitality was assessed by the presence or absence of protoplasmic streaming, the ability to take up neutral red, and the response to salt plasmolysis (15 1 week regrowth in a controlled environment (24 C light, 18 C dark with a 16-hr photoperiod). All seedlings were killed by freezing to -24 C. Seedlings grown at 24 C were killed by freezing to -4 C. at 2 C was even less than the level observed in the untreated control grown at 24 C. These data were similar to those obtained by Willemot (16) for roots and leaves of the same cultivar grown to a more advanced stage of development.…”

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

“…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).…”

Relative Sensitivity of Photosynthesis and Respiration to Freeze-Thaw Stress in Herbaceous Species

“…Al though one can gain valuable information from such studies, regarding some aspects of freeze-induced impairment of photosynthetic functions, one can clearly not gain an understanding of the interactions among the various cellular compartments within an intact tissue during freeze-thaw stress. For example, Singh et al (27) have demonstrated that fully functional plant mitochondria can be isolated from cells which have been lethally damaged by freezing stress. Prolonged exposure of mitochondria to the altered cellular environment, however, was found to result in the loss of function.…”