Planta (1996)199:74 78 P l a n t ~ 9 Springer-Verlag 1996 Nitrate assimilation during the anaerobic germination of rice: expression of ferredoxin-dependent glutamate synthase Monica Mattana, Immacolata Coraggio, Ida Brambilla, Alcide Bertani, Remo Reggiani Istituto Biosintesi Vegetali, C.N.R., via Bassini 15, 1-20133 Milano, Italy Received: 15 May 1995/Accepted: 14 August 1995 Abstract. Ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) is the last enzyme involved in the pathway of nitrate assimilation in higher plants. This paper describes the synthesis and expression of the en- zyme in anaerobic coleoptiles of rice (Oryza sativa L.) and its regulation by exogenous nitrate. The activity of Fd- GOGAT was strongly inhibited by cycloheximide be- tween 4 and 9 d of anaerobic germination. The addition of nitrate slightly increased, in the first 5 h, the specific activ- ity of Fd-GOGAT as well as the amount of a 160-kDa protein specifically immunoprecipitated with anti-Fd- GOGAT serum. Northern blot analysis, performed with a specific riboprobe, showed the presence of mRNA of the expected size and the inductive effect of nitrate. The role of Fd-GOGAT is discussed in relation to the anaerobic assimilation of nitrate by rice coleoptiles. Key words: Anoxia - Coleoptile - Glutamate synthase Nitrate Oryza Introduction Rice is one of the few terrestrial plants which can germi- nate under anaerobic conditions (Opik 1973). The only organ which develops during the anaerobic germination of rice seed is a white coleoptile (Kordan 1976). In order to sustain elongation, the coleoptile predominantly accumu- lates amino acids which help in maintaining an elevated osmotic potential in the tissue (Atwell et al. 1982; Menegus et al. 1984; Reggiani et al. 1993a). It has been shown that the most represented amino acids in anaerobic tissues are alanine and 7-aminobutyric acid (Streeter and Thompson 1971; Reggiani et al. 1988), both derived from glutamate. A continuous accumulation of amino acids in Abbreviations: CHX = cycloheximide; Fd = ferredoxin; GOGAT = glutamate synthase; GS = glutamine synthetase; NiR = nitrite reductase; NR = nitrate reductase Correspondence to: R. Reggiani; FAX: 39 (2) 2362946; E-mail: regre@icm.mi.cnr.it the coleoptile is sustained by their translocation (mainly glutamine) from the caryopsis (Aurisano et al. 1995). Recent papers have shown the ability of some species, such as rice and Erythrina caffra, to reduce and assimilate nitrate ions translocated from the caryopsis (Kemp and Small 1993; Reggiani et al. 1993b). In particular, it was reported that rice coleoptiles are able to synthesize nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS1 and GS2) enzymes during anaerobic germination, suggesting that the pathways of nitrate re- duction and ammonia assimilation into glutamine are maintained in an active state during anoxia (Mattana et al. 1994a, b). Complete ammonia assimilation in higher plants is obtained by the action of GS and glutamate synthase (GOGAT, Lea et al. 1990a). Two forms of this last enzyme have been described in plant tissue, an NADH- and a ferredoxin (Fd)-dependent form (Suzuki and Gadal 1984). Both these forms are present in roots and leaves, but their relative amounts vary between tissues and developmental stages (Matoh and Takahashi 1982; Wallsgrove et al. 1982). In maize roots, the presence of external nitrate has been shown to increase the accumula- tion of a transcript encoding Fd-GOGAT protein (Redin-i baugh and Campbell 1993). As far as the anaerobic rice coleoptile is concerned, Fd-GOGAT activity has been observed, whereas NADH-GOGAT activity was unde- tectable (Mattana et al. 1993). The aim of the present work was to study the ability of the anaerobic rice coleopfile to synthesize Fd-GOGAT, using the antibodies raised against this protein and analyzing the mRNA production. Moreover, the effect of exogenous nitrate on the synthesis of Fd-GOGAT was examined. Materials and methods Plant material and growth conditions. Seeds of rice (Oryza sativa L. cv. Arborio; Ente Nazionale Risi, Castel d'Agogna, Italy) were steril- ized and germinated anaerobically as previously reported (Reggiani et al. 1993a). The seeds were germinated for 7 d in the absence~or presence of 5 mM KNO3. To test the effect of time of NO3 treat- ment on the accumulation of protein and RNA, after 7 d of anaer- obic germination, 5 mM KNO3 was added for 0.5, 2, 5, and 8 h and