Studies on the Respiratory Properties of Mitochondria Isolated from Developing Winter Wheat Seedlings

Abstract

The studies of Sarkissian and Srivastava (20,22) have demonstrated that excellent respiratory control indices and ADP:O ratios greater than the "theoretical" discussed by Lehninger (13) can be obtained from mitochondria of wheat seedlings. They have reported further (25) that respiratory control and ADP:O ratios decline with decreasing age of seedlings, and that these parameters are significantly greater in mitochondria from shoots than from roots or leaves. These investigations have demonstrated that the shoots of young wheat seedlings provide an excellent system for the examination of the respiratory properties of plant mitochondria.Current studies (18,23,24) in our laboratory are focused on structural and biochemical changes associated with the development of cold hardiness in plants. Developing winter wheat seedlings attain a high degree of freezing tolerance when grown at 2 to 4 C (4, 17), and this resistance has been correlated with changes in cell membrane composition during growth at low temperature (3, 4). It was decided, therefore, to use the developing winter wheat seedling system to examine both functional responses of isolated wheat mitochondria and structural changes in mitochondrial membranes to growth at low temperature. This paper describes the respiratory properties of mitochondria isolated from developing winter wheat seedlings grown in light and dark at 21 C, from which the system for subsequent low temperature studies was adapted. MATERIALS AND METHODSSeedlings of winter wheat (Triticum aestivum L. cv. Rideau) were germinated and grown at 21 C on moist filter paper in dark and in light (8000 lux) for 2, 3, 5, and 7 days. Two to five g of shoots were harvested in a cold room at 2 to 3 C using precooled glassware and media, and all subsequent operations were carried out at this temperature, unless otherwise noted. The shoots were homogenized for 10 to 20 sec in a mortar and pestle, with grinding medium delivered at a uniform rate from a burette by a fine tube. When the first grinding was completed, the mortar contained approximately 8 ml of grinding medium/g of tissue. The homogenate was filtered through two layers of nylon cloth (mesh about 50 ,um) into centrifuge tubes. The residue in the nylon mesh was transferred to the mortar and pestle, reground with grinding medium, and refiltered through the nylon. Several grinding media were tested (9,19,20), and the most satisfactory was that of Sarkissian and Srivastava (20), composed of 0.5 M sucrose, 1 mM EDTA, 67 mM KH,PO,, and 0.75% (w/v) bovine serum albumin at pH 7.2.Several centrifugation procedures were used initially to isolate mitochondria, including the rapid isolation procedure of Sarkissian and Srivastava (20,21) and the more conventional procedures of Ikuma and Bonner (9) and Raison and Lyons (19). The procedure finally adopted for this study was a modification of the above methods. After filtering through nylon, the homogenate was centrifuged at 2,000g for 5 min, and the resulting supernatant fraction was centrifuged at 20,000g for 4...

Swelling and Contraction of Mitochondria from Cold-hardened and Nonhardened Wheat and Rye Seedlings

“…The observation that ca-ketoglutarate in the presence of ADP is oxidized rapidly with good respiratory control and close to theoretical ADP/O ratios (14) suggests that processes other than those normally associated with respiration are involved in mitochondrial contraction. This is supported further by the rapid decline observed in the contraction response when seedlings are grown at 2 C or transferred from 24 to 2 C, although previous studies (11) have demonstrated good respiratory properties of mitochondria isolated from 2 C grown seedlings.…”

“…Mitochondria were isolated by the procedure of Miller et al (12) using media previously described (14). Swelling and contraction of mitochondria, as indicated by light scattering (18), were followed at 24 C by measuring changes in absorbance at 520 nm on either a Beckman DK or a Gilford 2000 spectrophotometer.…”

“…Oxygen consumption was measured polarographically at 24 C using a conventional Clark oxygen electrode inserted into a 1.5-ml reaction chamber (14). The substrates used were L-malate and succinate, and state 3 oxidations were induced by additions of 100 ,UM ADP.…”

The Effect of Nucleotides and Inhibitors on Respiration in Isolated Wheat Mitochondria

“…Oxygen uptake was measured polarographically at 24 C with a conventional Clark electrode using media and methods previously described (22 Figure 1A and The addition of dinucleotides, in the absence of exogenous ADP did not stimulate 02 consumption (Fig. 1, B, C, and D).…”

“…Rideau) were germinated and grown in the dark at 24 C on moist filter paper for 2 days. Mitochondria were isolated from the shoots by differential centrifugation (22) and where indicated, further purified by sucrose density gradient centrifugation. Sucrose gradients were prepared by layering 3.7 ml each of 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, and 2 M sucrose solutions containing 10 mm KCl, 10 mm tris-HCl 10 mM KH2PO4, and 0.75% (w/v) BSA at pH 7.2 in 30-ml centrifuge tubes and stored 24 hr at 2 C to produce a linear gradient.…”