Contribution of Malate and Amino Acid Metabolism to Cytoplasmic pH Regulation in Hypoxic Maize Root Tips Studied Using Nuclear Magnetic Resonance Spectroscopy

Abstract

ABSTRACT31P., 13C-, and "IN-nuclear magnetic resonance spectroscopy were used to determine the roles of malate, succinate, Ala, Asp, Glu, Gin, and y-aminobutyrate (GABA) in the energy metabolism and regulation of cytoplasmic pH in hypoxic maize (Zea mays L.) root tips. Nitogen status was manipulated by perfusing root tips with ammonium sulfate prior to hypoxia; this pretreatment led to enhanced synthesis of Ala early in hypoxia, and of GABA at later times. We show that: (a) the ability to regulate cytoplasmic pH during hypoxia is not significantly affected by enhanced Ala synthesis. (b) Independent of nitrogen status, decarboxylation of Glu to GABA is greatest after several hours of hypoxia, as metabolism collapses. (c) Early in hypoxia, cytoplasmic malate is in part decarboxylated to pyruvate (leading to Ala, lactate, and ethanol), and in part converted to succinate. It appears that activation of malic enzyme serves to limit cytoplasmic acidosis early in hypoxia. (d) Ala synthesis in hypoxic root tips under these conditions is due to transfer of nitrogen ultimately derived from Asp and Gin, present in oxygenated tissue. We describe the relative contributions of glycolysis and malate decarboxylation in providing Ala carbons. (e) Succinate accumulation during hypoxia can be attributed to metabolism of Asp and malate; this flux to succinate is energetically negligible. There is no detectable net flux from GIc to succinate during hypoxia. The significance of the above metabolic reactions relative to ethanol and lactate production, and to flooding tolerance, is discussed. The regulation of the pattems of metabolism during hypoxia is considered with respect to cytoplasmic pH and redox state.

Ammonium Assimilation and the Role of [gamma]-Aminobutyric Acid in pH Homeostasis in Carrot Cell Suspensions

“…In vivo 14N NMR experiments showed that the cells contained a substantial ammonium pool at the start of the labeling period and that the size of this pool varied throughout the 18-h time course (data not shown). Increases in GABA, ranging from 5-fold in 24 h in rice shoots (Reggiani et al, 1988) to 100-fold (with a 7-fold increase in Ala) in 4 h in radish leaves (Streeter and Thompson, 1972a) have been observed previously in a wide range of plants under hypoxia (Streeter and Thompson, 1972a;Tsushida and Murai, 1987;Reggiani et al, 1988;Menegus et al, 1989;Fan et al, 1992;Roberts et al, 1992). However, in these studies oxygen-free nitrogen was supplied to ensure complete anaerobiosis, whereas in the experiments reported here the oxygen supply was only mildly restricted.…”

“…However, in these studies oxygen-free nitrogen was supplied to ensure complete anaerobiosis, whereas in the experiments reported here the oxygen supply was only mildly restricted. Increases in Ala (Streeter and Thompson, 1972a;Roberts et al, 1992) and glutamate (Roberts et al, 1992) have also been observed following the onset of hypoxia, and in the cas12 of hypoxic maize root tips the accumulation of Ala, glutamate, and GABA was enhanced by pretreatment with ammonium (Roberts et al, 1992). The accumulation of these amino acids is consistent with p&, being regulated, at least in part, by changes in primary metabolism because the synthesis of Ala, arising from the decarboxylation of malate as a rlrsult of malic enzyme activation by cytoplasmic acidosis, the conversion of Gln to glutamate, and the decarboxylation of glutamate to GABA are all proton-consuming reactions.…”

“…It is mainly produced by the action of GDC on glutamate and it can be further metabolized to succinic semialdehyde and succinate via GAB-T and SSADH (Streeter and Thompson, 1972b;Satya Narayan and Nair, 1986). It has been suggested that the accumulation of GABA can be used as a marker for dead or dying hypoxic cells (Roberts et al, 1992). However, recent reports have…”

“…GABA is present in virtually all plant tissues and often accumulates under conditions of environmental stress, including hypoxia (Streeter and Thompson, 1972a;Tsushida and Murai, 1987;Reggiani et al, 1988;Menegus et al, 1989;Fan et al, 1992;Roberts et al, 1992), low temperature (Wallace et al, 1984), heat shock (Mayer et al, 1990), and low pH (Lane and Stiller, 1970). It is mainly produced by the action of GDC on glutamate and it can be further metabolized to succinic semialdehyde and succinate via GAB-T and SSADH (Streeter and Thompson, 1972b;Satya Narayan and Nair, 1986).…”

“…It has also been suggested that the high accumulation of GABA in many tissues could be the result of a change in the relative activities of GDC and GAB-T following a decrease in pH,, (Streeter and Thompson, 1972b;Wallace et al, 1984). Because GDC activity increases as pH decreases and because glutamate decarboxylation is a protonconsuming reaction, it follows that GABA production could act as a sink for excess protons in the cytoplasm and thus contribute to pH regulation (Murphy et ai., 1983;Reid et al, 1985;Menegus et al, 1989;Roberts et al, 1992;Snedden et al, 1992).…”