Membrane Lipid Integrity Relies on a Threshold of ATP Production Rate in Potato Cell Cultures Submitted to Anoxia1

Abstract

In this paper we report on our study of the changes in biomass, lipid composition, and fermentation end products, as well as in the ATP level and synthesis rate in cultivated potato (Solanum tuberosum) cells submitted to anoxia stress. During the first phase of about 12 h, cells coped with the reduced energy supply brought about by fermentation and their membrane lipids remained intact. The second phase (12-24 h), during which the energy supply dropped down to 1% to 2% of its maximal theoretical normoxic value, was characterized by an extensive hydrolysis of membrane lipids to free fatty acids. This autolytic process was ascribed to the activation of a lipolytic acyl hydrolase. Cells were also treated under normoxia with inhibitors known to interfere with energy metabolism. Carbonyl-cyanide-4-trifluoromethoxyphenylhydrazone did not induce lipid hydrolysis, which was also the case when sodium azide or salicylhydroxamic acid were fed separately. However, the simultaneous use of sodium azide plus salicylhydroxamic acid or 2-deoxy-D-glucose plus iodoacetate with normoxic cells promoted a lipid hydrolysis pattern similar to that seen in anoxic cells. Therefore, a threshold exists in the rate of ATP synthesis (approximately 10 mol g ؊1 fresh weight h ؊1 ), below which the integrity of the membranes in anoxic potato cells cannot be preserved.O 2 deprivation becomes a frequent stress for plants submitted to unpredictable heavy rainfalls and flooding. The diffusion of O 2 to their submerged underground organs is severely limited, so that plants must cope with hypoxic or even anoxic conditions. Whereas ATP is produced with a high efficiency by respiration in nongreen cells, its synthesis is much lower under anoxia, with fermentation as the sole energy provider. For most higher plants, the latter condition eventually becomes lethal. The multifarious effects of O 2 deprivation stress on plants sensitive and resistant to anoxia are fairly well understood and have been extensively reviewed in this decade (Armstrong et al., 1994; Sachs, 1994; Ratcliffe, 1995; Crawford and Braendle, 1996; Drew, 1997; Vartapetian and Jackson, 1997).Aside from the obvious interest given to the responses of energy metabolism, the role of macromolecules, and in particular, of gene expression and protein synthesis, has received considerable attention (Sachs, 1994; Drew, 1997). In contrast, the behavior of membrane lipids under anoxia has scarcely been investigated. This is surprising knowing how important it is for a living cell to maintain its membrane integrity. In potato (Solanum tuberosum) tubers, for instance, changes in membrane lipids have mostly been studied during aging, and have been related to overall lipid unsaturation, lipid degradation, and peroxidation processes (Knowles and Knowles, 1989; Spychalla and Desborough, 1990a, 1990b; Kumar and Knowles, 1993; Dipierro and De Leonardis, 1997). Although these effects are not directly relevant to anoxia (Kumar and Knowles, 1996), they are probably related to the effects likely to occur unde...

A Plant Plasma Membrane ATP Binding Cassette–Type Transporter Is Involved in Antifungal Terpenoid Secretion

“…Eight hours after induction, we divided the culture in halves. To deplete the level of ATP, we treated half with the ATP synthesis inhibitor cocktail (NaF, a glycolysis inhibitor, as well as NaN 3 , and carbonylcyamide m-chlorophenylhydrazone, mitochondrial electron chain and phosphorylation inhibitors) used by Rawyler et al (1999) and found that the cell-associated radioactivity increased rapidly ( Figure 6C), indicating that sclareolide extrusion is linked to an energetic process.…”

“…Eight hours after induction, we divided the culture in halves. To deplete the level of ATP, we treated half with the ATP synthesis inhibitor cocktail (NaF, a glycolysis inhibitor, as well as NaN 3 , and carbonylcyamide m-chlorophenylhydrazone, mitochondrial electron chain and phosphorylation inhibitors) used by Rawyler et al (1999) and found that the cell-associated radioactivity increased rapidly ( Figure 6C), indicating that sclareolide extrusion is linked to an energetic process.Using another approach, we compared the labeled sclareolide derivative associated with control cells and cells that had been incubated for 12 hr with unlabeled sclareolide. The terpenoid-pretreated cells showed less accumulation than the untreated cells, and this difference was removed by ATP synthesis inhibitors ( Figure 6D).…”

Tansley Review No. 118

“…In practice this means that cells can survive for a certain period before metabolism is seriously hampered. For example, potato cells in culture showed no membrane damage for the first 12 h after imposition of anaerobic stress (Rawyler et al, 1999). Thereafter, free fatty acids accumulated in the potato cells coincident with the calculated energy supply decreasing to 1-2% of the aerobic maximum.…”

“…From this, and studies with inhibitors, it was suggested that there is a threshold of ATP production of approx. 10 µmol g −" f. wt h −" , below which membrane structure cannot be preserved (Rawyler et al, 1999). The restricted ATP supply also has direct implications for proteolysis.…”

“…It has been suggested that ethanol is toxic to plant cells, but the evidence is equivocal. In leaves, ethanol has been observed at concentrations of 2-50 mM (Kimmerer & MacDonald, 1987 ;Kato-Noguchi, 1998 ;Rawyler et al, 1999), although in vitro exogenous application requires 100-600 mM to induce death (Perata & Alpi, 1993). Metabolic attempts to remove ethanol involve translocation in the xylem and volatilization.…”