Sucrose synthase activity increased in 2-day-old rice (Oryza sativa) seedlings submitted to anaerobic stress. Likewise The unusual behavior of maize sucrose synthase isozymes under anaerobiosis led us to study the anaerobic induction of the enzyme in rice. Rice shares the metabolic strategy of maize roots in response to anaerobic stress. However, in contrast to maize roots, rice is extremely resistant to anoxia. Rice seedlings that have germinated for 48 h aerobically survive more than 21 d and are capable of coleoptile elongation under complete oxygen deprivation. Their metabolism is high enough to permit DNA, RNA, as well as protein synthesis (1,12,13). We have already shown that many of the glycolytic and fermentative enzymes increase in activity (19). ADH (16), cytosolic glyceraldehyde 3-phosphate dehydrogenase (18), and PDC (our unpublished data) are all induced at both the mRNA and protein levels. The rice seedlings referred to above are still attached to the seed that contains starchy reserves. It seemed likely that the existence of these reserves played a role in the active metabolism seen in such seedlings under anoxia. Because starch mobilization is mediated by sucrose synthase, we hypothesized that in rice, sucrose synthase activity and protein levels should be induced by anaerobiosis coordinately with the other enzymes of sugar phosphate metabolism. In maize roots, however, lack ofstarch reserves means that sucrose synthase accumulation would be unnecessary.Our results were obtained using several independent techniques, including enzyme activity measurements, immunodetection on Western blots, SDS, and two-dimensional PAGE of in vivo-and in vitro-labeled total proteins and immunoprecipitates, as well as hybridization analyses of Northern blots and run-on transcripts. Sucrose synthase activity and protein both increased upon anaerobic stress. This was accompanied by an increase in the steady-state level of sucrose synthase mRNA and in the rate of transcription ofthe sucrose synthase gene. We conclude that sucrose synthase is a typical anaerobic polypeptide in rice and is induced at both the protein and mRNA levels. MATERIALS AND METHODS Plant MaterialInbred rice (Oryza sativa L., var Cigalon) seeds were surface-sterilized with a commercial preparation of bleach, then germinated for 48 h in the dark at 25°C under distilled water with vigorous agitation ( 16). Under such conditions, seedlings are under normoxia and have coleoptiles and primary roots about 0.5 cm in length. Anaerobic treatment consisted of www.plantphysiol.org on April 3, 2019 -Published by Downloaded from

The role of mitochondria in the phosphorylation of ADP to ATP in the early steps of seed germination has been studied. Mitochondria were extracted from dry sunflower (Helianthus annuus) seeds. Adenylate kinase-dependent ATP synthesis was inhibited by p ',p5-di(adenosine-5') results were obtained in similar experiments with mung bean and cucumber seeds (14). It was concluded from all these results that, as soon as water enters into the cells, preexisting mitochondria synthesize ATP by oxidative phosphorylation. More recently, active ATPase and Cyt oxidase could be extracted from quiescent maize embryos (6). Thus protective mechanisms must occur in the dry seed which allow the membranes to withstand dryness or rehydration without disorganization (2, 9, 25). In the present paper, we show that mitochondria extracted from dry sunflower seeds (Helianthus annuus) are able to produce ATP by the oxidation of various organic acids or NADH, and this production is abolished by inhibitors of the partial reactions involved in the process of oxidative phosphorylation. The morphology of these mitochondria is also described and discussed in relation to their function. MATERIALS AND METHODS Plant MaterialThe mechanisms by which ATP is produced upon imbibition of seeds and during the early phases of their germination has been a matter of debate for about three decades (for reviews see refs. 1, 17, 19). Although seed respiration has been established for a long time, its link with the phosphorylation of ADP to ATP is in doubt mainly for two reasons. Many authors observed no inhibition of germination by cyanide (19). Also, in spite of many attempts in various laboratories, phosphorylating mitochondria ofgerminating seeds have been obtained in only two cases, from peanut cotyledons imbibed for 40 min (27), and from cucumber cotyledons imbibed for 6 h (14). Moreover, the leakage of solutes during seed imbibition suggested some degree of membrane disorganization either in the dry state or following rehydration (5,23,26). It was believed, in particular, that mitochondria were damaged and had to be repaired during the germination period to become functional at the time of radicle emergence (13). Because fermentation has a low activity in most germinating seeds under aerated conditions (20), new pathways for ATP synthesis have been sought ( 17).However, in vivo studies on lettuce seeds showed that the ATP synthesis which occurs upon imbibition of the seeds is 02-dependent and KCN-sensitive, suggesting that the mechanism involved is oxidative phosphorylation (8). The same Sunflower seeds (Helianthus annuus cv Rodeo) obtained from CETIOM (France) were stored at 4°C in closed vessels containing silica gel. The seeds were dehusked by hand 1 or 2 d before utilization and stored at 4°C in flasks containing silica gel. Their water content was 4.5% (w/w) as determined after drying 24 h at 95°C. ChemicalsAll organic compounds were from Sigma and Merck, except Percoll (Pharmacia), Triton X-100 (Packard), f3-mercaptoethanol (Serva), and glyce...

A highly sensitive radioimmunoassay has been used to determine the levels of adenosine 3',5'-cyclic monophosphate (cAMP) in five higher plants (Lactuca sativa, Helianthus annuus, Oryza sativa, Pinus pinaster, Nicotiana tabacum). Particular attention was paid to the three main sources of errors in the charactenzation of cAMP in plants: presence of interfering substances in plant tissues; possible artefactual formation of cAMP from endogenous ATP during extraction, purification, and assay; and microbial origin of cAMP. In all the tested tissues, the cAMP level was below the detection limit of 0.5 picomole per gram fresh weight, a value much lower than those reported for similar materials of the same species in many previous studies. This result is not in favor of cAMP-dependent regulations in higher plants.et al. (7,8) to check the presence of cAMP in different plants chosen among gymnosperms, monocotyledons, and dicotyledons. Great care was taken to ensure the elimination of interfering compounds and the absence of de novo synthesis of cAMP during the assay, together with maximal recovery of endogenous cAMP. In all tissues, the cAMP level was* below the detection limit ofthe assay: the discrepancy between these results and published ones on similar materials is discussed in the light of potential sources of errors. MATERIALS AND METHODS Biological MaterialCyclic AMP is present in animal tissues and in many microorganisms such as bacteria, fungi, and green algae. Its occurrence in higher plants and its role in the regulation of their metabolism are still a matter of debate. The main evidence supporting the existence of the cAMP' system in plant tissues is based on cAMP estimations, but a large diversity exists among reported determinations of cAMP in higher plants: for example, in the case of tobacco callus, published values range from 900 (15) to less than 0.5 pmol. g-' (2). On the other hand, there is only scant evidence for enzymes involved in cAMP metabolism (9). Most data in this field can be found in reviews (9) Lettuce (Lactuca sativa L., cv Val d'Orge) was obtained from Societe Clause (France). Lettuce seeds were sterilized by soaking for 20 min in commercial sodium hypochlorite (150 g ofchlorine per L). After extensive washing with sterile water, seeds were either immediately frozen in liquid nitrogen or germinated for 2 h at 20C.Sunflower (Helianthus annuus L., cv Rodeo) was obtained from Cetiom (France). Different materials were used: immature seeds were removed from plant heads 7 and 14 d after self-pollination in a growth cabinet and nondesiccated mature seeds after 28 d; these seeds, as well as mature dry seeds, were soaked for 15 min in one-fourth diluted commercial sodium hypochlorite, dehusked, and further sterilized in one-tenth hypochlorite dilution; young seedlings were obtained 3 and 8 d after the beginning of germination under sterile conditions. Rice (Oryza sativa L., var Cigalon) was obtained from Station d'Amelioration des Plantes (INRA Montpellier, France). Dehusked dry seeds wer...