The metabolism of carbohydrates was investigated in rice (Oryza sativa 1.) seedlings grown under anoxia. Two phases can be recognized in the utilization of carbohydrates: during the first days of germination under anoxia, the metabolism of sugars is mainly degradative, whereas after the induction of a-amylase (EC 3.2.1.1) has taken place, the increased presence of glucose and sucrose indicates that both starch degradation and sucrose synthesis operate. The analysis of the enzymes involved in carbohydrate metabolism indicates that anoxic rice seedlings possess a set of enzymes that allow the efficient metabolism of starch and sucrose to fructose-6-phosphate. We propose that cytosolic sucrose metabolism in anoxic rice seedlings takes place mainly through a sucrose synthase (EC 2.4.1.1 3) pathway with nucleoside diphosphate kinase (EC 2.7.4.6), allowing the cycling of urydilates needed for the operation of this pathway.

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An adequate carbohydrate supply contributes to the survival of seeds under conditions of limited oxygen availability. The amount of soluble, readily fermentable carbohydrates in dry cereal seeds is usually very limited, with starch representing the main storage compound. Starch breakdown during the germination of cereal seeds is the result of the action of hydrolytic enzymes and only through the concerted action of a-amylase (EC 3.2.1 .l), P-amylase (EC 3.2.1.2), debranching enzyme (EC 3.2.1.41 1, and a-glucosidase (EC 3.2.1.20) can starch be hydrolyzed completely. We present here data concerning the complete set of starch-degrading enzymes in three cereals, rice (Oryza sativa l.), which is tolerant to anaerobiosis, and wheat (Triticum aestivum 1.) and barley (Hordeum vulgare l.), which are unable to germinate under anoxia. Among the cereal seeds tested under anoxia, only rice is able to degrade nonboiled, soluble starch, reflecting the ability to degrade the starch granules in vivo. This is explained by the presence of the complete set of enzymes needed to degrade starch completely either as the result of de novo synthesis (a-amylase, P-amylase) or activation of preexisting, inactive forms of the enzyme (debranching enzyme, a-glucosidase). These enzymes are either absent or inactive in wheat and barley seeds kept under anaerobic conditions. Cereal seeds other than rice (Oryza sativa L.) fail to germinate in anoxic environments (Alpi and Beevers, 1983;Perata and Alpi, 1993). The metabolic basis explaining the different degree of tolerance among plant species is still unknown (Drew, 1990;Kennedy et al., 1992;Perata and Alpi, 1993). Starchy seeds are usually more tolerant to anoxia than fatty seeds, which is a possible consequence of the higher fermentative metabolism observed in these seeds (AI-Ani et al., 1985;Raymond et al., 1985) leading to an adequate ATP production. It is therefore inferred that an adequate supply of carbohydrates contributes to the surviva1 of seeds under conditions of limited oxygen availability. The amount of soluble, readily fermentable carbohydrates in dry cereal seeds is usually very limited, because starch is the main storage compound in these seeds.Starch breakdown during the germination of cereal seeds is the result of the action of hydrolytic enzymes, and it is '

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Carbon (C) and nitrogen (N) are essential elements for metabolism, and their availability, called the C/N balance, must be tightly coordinated for optimal growth in plants. Previously, we have identified the ubiquitin ligase CNI1/ATL31 as a novel C/N regulator by screening plants grown on C/N stress medium containing excess sugar and limited N. To elucidate further the effect of C/N balance on plant growth and to determine the physiological function of ATL31, we performed C/N response analysis using an atmospheric CO2 manipulation system. Under conditions of elevated CO2 and sufficient N, plant biomass and total sugar and starch dramatically increased. In contrast, elevated CO2 with limited N did not increase plant biomass but promoted leaf chlorosis, with anthocyanin accumulation and increased senescence-associated gene expression. Similar results were obtained with plants grown in medium containing excess sugar and limited N, suggesting that disruption of the C/N balance affects senescence progression. In ATL31-overexpressing plants, promotion of senescence under disrupted CO2/N conditions was repressed, whereas in the loss-of-function mutant it was enhanced. The ATL31 gene was transcriptionally up-regulated under N deficiency and in senescent leaves, and ATL31 expression was highly correlated with WRKY53 expression, a key regulator of senescence. Furthermore, transient protoplast analysis implicated the direct activation of ATL31 expression by WRKY53, which was in accordance with the results of WRKY53 overexpression experiments. Together, these results demonstrate the importance of C/N balance in leaf senescence and the involvement of ubiquitin ligase ATL31 in the process of senescence in Arabidopsis.