Cyanide-Resistant Respiration in Suspension Cultured Cells of Nicotiana glutinosa L

Abstract

The respiration of dark-grown Nicotiava glutinosa L. cells in liquid suspension culture was found to be highly cyanide resistant and salicylhydroxamic acid (SHAM) sensitive, indicative of an active alternative respiratory pathway. This was especially true during the lag and logarithmic phases of the 14-day growth cycle. Mitochondria isolated from logarithmically growing cells exhibited active oxidation of malate, succinate, and exogenous NADH. Oxidation of all three substrates had an optimum pH of 6.5 and all were highly resistant to inhibition by cyanide and sensitive to SHAM. Respiratory control was exhibited by all three substrates but only if SHAM was present to block the alternative pathway and divert electrons to the phosphorylating cytochrome pathway. The cyanide-resistant oxidation of exogenous NADH has previously only been associated with Arum spadix mitochondria. Coemergence during evolution of the alternative respiratory pathway and the exogenous NADH dehydrogenase in plant mitochondria as a possible mechanism for removal of cytoplasmic NADH is proposed. Evidence is presented which suggests that mitochondrial assays should be performed at pH 6.5.Cyanide-resistant respiration has been described in a wide assortment of plant species (10,24). Most of the work has been performed with mitochondria isolated from intact plant tissues (mung bean shoots, skunk cabbage spadices, etc.). With the current interest in plant tissue culture as an experimental tool it was thought to be of importance to characterize the respiration of cultured plant cells. Wilson (28-30) has examined respiration of isolated mitochondria from cultured sycamore cells and found it to be partially resistant to inhibition by cyanide. RC2 was rather low with a ratio of 2.0 or less. With the discovery of the hydroxamates, inhibitors of the alternate respiratory pathway (22), it has been reported that they are effective in inhibiting respiration resistant to cyanide or antimycin A in cultured cells of tobacco (21) and soybeans (16). In the present investigation, using suspension cultured cells of Nicotiana glutinosa L. and isolated mitochondria from these cells, it has been established that respiration is sensitive to the hydroxamate SHAM and the sensitivity varies over the course of the growth cycle. The high sensitivity of respiration to SHAM and the low RC found in mitochondria from these cultures suggests that mitochondrial ATP production is limited. Preliminary studies showed that a final concentration of 1.6% alcohol caused no significant change in either the qualitative or quantitative activity of the mitochondria within a 10-min period.The reaction mixture for the respiratory measurements of whole cells consisted of 0.09 M sucrose, 50 mM Mes (pH 6.5). The buffer and pH were determined to be optimum for these cells (11).Mitochondrial Isolation. The mitochondrial isolation procedure was similar to that described by Charbonnier and Berville (4). The tissue was placed in a chilled mortar containing cold grinding medium (1:1, w/v) a...

Lower Growth Temperature Increases Alternative Pathway Capacity and Alternative Oxidase Protein in Tobacco

“…Suspension and callus cultures of tobacco have a CNresistant, SHAM-sensitive component of respiration (12,17). Here we show that a monoclonal antibody to the AO from S. guttatum recognizes an AO protein in suspension cells of tobacco.…”

Salicylic Acid Induces Cyanide-Resistant Respiration in Tobacco Cell-Suspension Cultures

“…An aging-related increase in the capacity of altemative pathway has been observed in cell-suspension cultures and mitochondria of Nicotiana glutinosa L. (8), soybean (Glycine max) (13), sycamore (Acer pseudoplatanus) (21), and petunia (Petunia hybrida) (1). In N. glutinosa, this phenomenon was thought to result from an abundance of carbohydrates relative to demand (8), supporting the 'overflow' hypothesis (9).…”

“…In N. glutinosa, this phenomenon was thought to result from an abundance of carbohydrates relative to demand (8), supporting the 'overflow' hypothesis (9). The overflow hypothesis stipulates that altemative pathway becomes engaged only when the Cyt pathway is saturated.…”

Mechanisms of Citrate Transport and Exchange in Corn Mitochondria

“…Table IV gives data on the response to pH. As reported for oxidation of NADH (11), malate (11,19), and to some extent succinate (11), the pH optima for oxidation of both endogenous substrates and citrate is between pH 6 and 7. Even here, however, the citrate oxidation rates and coupling are poor-compare the results with malate (Table IV).…”