Pergamon 0031-9422(94)00774-8 Phytochemistry, Vol. 38, No. 5, pp. t 147-1150, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/95 $9.50 + 0.00 ANAEROBIC ACCUMULATION OF 4-AMINOBUTYRATE IN RICE SEEDLINGS; CAUSES AND SIGNIFICANCE NICOLETTA AURISANO,ALCIDEBERTANI and REMO REGGIANI Istituto Biosintesi Vegetali, C.N.R., Via Bassini, 15, 20133 Milano, Italy (Received in revisedform 25 July 1994) Key Word Index--Oryza sativa; Gramineae; rice; 4-aminobutyrate; anoxia; root; shoot; glutamate; protein; amino acids. Abstract--Accumulation of 4-aminobutyrate is induced by anoxia in rice seedlings. The induction of 4-aminobutyrate accumulation in aerobic conditions by treatments with exogenous 4-aminobutyrate, Gabaculine and glutamate is well tolerated by the seedlings. The inhibition of protein synthesis in aerobic and anaerobic conditions by cycloheximide shows that this process competes with glutamate decarboxylase for glutamic acid. The sensitivity of the anaerobic 4- aminobutyrate accumulation to azaserine indicates that glutamate synthase is important in maintaining glutamate availability. The different tolerance to anoxia and protein metabolism of shoot and root of rice suggests that the causes leading to 4-aminobutyrate accumulation in these tissues are different. It is suggested that ammonia reassimilation in root plays an important role in the synthesis of 4-aminobutyrate. INTRODUCTION In the plant kingdom, the non-protein amino acid 4- aminobutyrate (Gaba) is ubiquitous and its concentra- tion in the tissues is often similar to those of the normal protein amino acids [I, 2]. The physiological role of Gaba in plants has not been clearly established. This compound is present in transport fluids and can provide, through the Gaba shunt, carbon for energy production and nitrogen for amino acid biosynthesis [2]. In the central and peripheral nervous system of vertebrates, Gaba acts as an inhibitory neurotransmitter by increas- ing the membrane conductance to CI- ions and mem- brane polarization [3]. Evidence of a similar function in plants is lacking. Many reports indicate that the level of Gaba increases rapidly in plant tissues in response to various forms of stress [4-6] among which anaerobiosis is the condition inducing the largest accumulation [7, 8]. In wheat roots, the process of Gaba accumulation has been shown to be mediated by the phytohormone ABA [9]. Gaba is synthesized in plant tissues by the irreversible ~-decarboxylation of L-glutamic acid in a reaction cata- lysed by glutamate decarboxylase (GDC). An alternative source of Gaba is the oxidation of polyamines but this is probably lower than for glutamate decarboxylation and null under anaerobiosis [7, 9]. The synthesis of Gaba has been suggested to be an adaptive response of plant tissues to stress-induced cellular acidosis [2]. The advantage of this process would be the concomitant H + consumption during decarboxylation which ameliorates the eytosolic acidification [10]. A decline in the cytosolic pH is a phenomenon extremely marked under oxygen deficit stress [11, 12]. This investigation used seedlings of rice which tolerate an anaerobic stress [12]. The coleoptile of rice is also capable of anaerobic elongation [13]. This study seeks to widen the knowledge about the role of Gaba in plants and the causes leading to its accumulation under anoxia. RESULTSAND DISCUSSION The effect of anaerobic conditions on the Gaba content in shoot and root of rice is shown in Fig. I. As can be seen, 24 hr of anoxia induced an accumulation of ca 3.5 and 6.3/zmolg-i fr. wt of Gaba in shoot and root, respect- ively. No change in Gaba concentration was observed after an additional day of growth in air. The large increase in Gaba content in anaerobiosis is in agreement with that previously described by us [7] and other authors [8, 14-16]. Moreover, the synthesis of Gaba under stress conditions, advantageous for the pH-stat of the cell [I, 2, 9], could also occur due to the low toxicity of this amino acid. To test this hypothesis, we carried out 24 hr treat- merits with exogenous Gaba (I mM), Gabaculine (I mM, an inhibitor of Gaba transaminase, Fig. 2, arrow H) or glutamate (I raM) in order to increase the level of Gaba in non-stress conditions (aerobiosis). As can be seen in Fig. I, the Gaba and glutamate treatments increased the concentration of this amino acid in both shoot and root. The exogenous supply of Gaba led to endogenous values similar to those observed in anaerobic shoot and root. The Gabaculine treatment induced Gaba accumulation 1147