Comparison of Temperature Dependency of Tonoplast Proton Translocation between Plants Sensitive and Insensitive to Chilling

Abstract

Proton transport activities in isolated tonoplast vesicles were measured as quenching of fluorescence of acridine orange. A marked difference in the temperature dependency of two types of tonoplast proton transports, i.e. ATP-and pyrophosphatedriven, was observed between two leguminous plants sensitive (mung bean, Vigna radiata [L.J Wilczek) and insensitive (pea, Pisum sativum L.) to chilling. In tonoplast vesicles isolated from hypcotyls of mung bean seedlings that were germinated for 3.5 days at 260C in the dark, the total amount of fluorescence quenching at the steady state in both types of proton pumps, as a measurement of the inside-acidic pH gradient across the membrane vesicles, was markedly suppressed under temperatures below 10°C. In tonoplast vesicles isolated from epicotyls of pea seedlings, which were germinated for 7 days at 180 to 230C in the dark, no suppression occurred in the formations of the pH gradient in either type of proton pump, even at 0°C. The cause of the low temperature-induced suppression of the proton pumps in mung bean tonoplasts seems to be not an increased permeability of the membrane vesicles to protons or accompanying anions and cations, but instead a marked inhibition in the catalytic activity of both enzymes under low temperatures.Mung bean (Vigna radiata [L.] Wilczek) is a subtropical plant indigenous to Southeast Asia. The etiolated seedlings are extremely sensitive to chilling and suffer irreversible injury after exposure to 0°C in the dark more than 48 h, whereas no permanent injury occurs within the first 24 h of chilling (4). This indicates that chilling injury in mung bean seedlings proceeds in two distinct processes: an early reversible process and a later irreversible process. In the early process, i.e. within the first 24 h of chilling, there are reversible declines in a variety of physiological functions including respiration (15), 1-aminocyclopropane-1-carboxylic acid-dependent ethylene formation (4), and protein synthesis (7). The time courses of enzyme activities associated with various cellular membranes in mung bean hypocotyls during in vivo chilling at 0°C showed that the tonoplast H+-ATPase was the most sensitive to chilling, being inactivated in the first 24 h, when membranebound enzymes associated with other cellular membranes 'Supported in part by Grant-in-aid remained intact (15). Given the putative role of the tonoplast H+-ATPase in the formation ofproton electrochemical potential gradient across tonoplast membranes, which may be employed for the active transport ofions and metabolites into vacuoles (12), the early decline in the H+-ATPase activity during chilling may give rise to an alteration of the cytoplasmic environment, especially the pH and ionic concentrations. As a result, a variety of physiological functions may be disordered, eventually leading to cell death.To gain insight into more immediate response of cells to low temperatures, we have examined and compared the temperature dependency of tonoplast proton transports in vitro betwe...

Low Temperature-Induced Cytoplasmic Acidosis in Cultured Mung Bean (Vigna radiata [L.] Wilczek) Cells

“…As reported earlier (Yoshida and Matsuura-Endo, 1991), in tonoplast vesicles prepared from mung bean hypocotyls that were also highly sensitive to chilling, the permeability to protons and proton equivalents decreased linearly with decreasing incubation temperature from 25 to O°C without any indication of an abrupt change. Instead, both types of proton-translocating enzymes, namely H+-ATPase and proton-translocating inorganic pyrophosphatase, were markedly suppressed at temperatures below 10°C.…”

“…Suspension-cultured cells of mung bean (Vigna radiata [L.] Wilczek), derived from young roots as described previously (Yoshida, 1991), were used in the present study. The culture medium contained Murashige and Skoog major and minor inorganic nutrients and vitamins, with the exception that the concentration of thiamine was increased 10-fold, plus 3% SUC, 0.5% Glc, 0.1 mg/L 2,4-D, and 0.05 mg/L kinetin.…”

“…In etiolated young seedlings of mung bean (Vigna radiata [L.] Wilczek), which are highly susceptible to chilling (Etani and Yoshida, 1987), vacuoles are thought to be among the main cellular components that respond to chilling stress (Yoshida et al, 1989). The activities of both types of vacuolar proton pumps are markedly depressed in vitro upon lowering of the temperature to less than 10°C as a result of the high-temperature dependency of the enzymes (Yoshida and Endo-Matsuura, 1991). Among various membrane-associated enzymes in the hypocotyls, the vacuolar H+-ATPase is the most sensitive to cold and may be preferentially inactivated during chilling in vivo at O°C (Yoshida et al, 1989;Matsuura-Endo et al, 1992).…”

Chill-Induced Changes in the Activity and Abundance of the Vacuolar Proton-Pumping Pyrophosphatase from Mung Bean Hypocotyls

“…Although these results firmly indicate that the V-PPase responds to low temperatures in vivo, its actual role in protecting cell function can only be speculated. For example, when assayed in vitro at 4°C the activity of the V-PPase is only 10 to 15% of that of control values from mung beans grown at 24°C (Yoshida and Matsuura-Endo, 1991). Therefore, a 2-fold restorative increase in the amount of the enzyme may not appear to be of great physiological significance.…”

Chilling-Induced Inactivation and Its Recovery of Tonoplast H+-ATPase in Mung Bean Cell Suspension Cultures

“…The activities of two types of proton pumps located on tonoplast membranes are markedly suppressed with temperatures below 10°C in vitro (17). The H+-ATPase activity extracted from cells subjected to chilling treatment decreased rapidly with a loss of 50% within 48 h (18).…”