β-Cyano-alanine synthase (CAS; EC 4.4.1.9) plays an important role in cyanide metabolism in plants. Although the enzymatic activity of β-cyano-Ala synthase has been detected in a variety of plants, no cDNA or gene has been identified so far. We hypothesized that the mitochondrial cysteine synthase (CS; EC 4.2.99.8) isoform, Bsas3, could actually be identical to CAS in spinach (Spinacia oleracea) and Arabidopsis. An Arabidopsis expressed sequence tag database was searched for putative Bsas3homologs and four new CS-like isoforms, ARAth;Bsas1;1,ARAth;Bsas3;1, ARAth;Bsas4;1, andARAth;Bsas4;2, were identified in the process. ARAth;Bsas3;1 protein was homologous to the mitochondrial SPIol;Bsas3;1 isoform from spinach, whereas ARAth;Bsas4;1 and ARAth;Bsas4;2 proteins defined a new class within the CS-like proteins family. In contrast to spinach SPIol;Bsas1;1 and SPIol;Bsas2;1 recombinant proteins, spinach SPIol;Bsas3;1 and Arabidopsis ARAth;Bsas3;1 recombinant proteins exhibited preferred substrate specificities for the CAS reaction rather than for the CS reaction, which identified these Bsas3 isoforms as CAS. Immunoblot studies supported this conclusion. This is the first report of the identification of CAS synthase-encoding cDNAs in a living organism. A new nomenclature for CS-like proteins in plants is also proposed.

Mild flooding is experienced by most land plants but as its severity increases, fewer species are able to grow and survive. At the extreme, a highly exclusive aquatic lifestyle appears to have evolved numerous times over the past 120 million years. Although only 1-2% of angiosperms are aquatics, some of their adaptive characteristics are also seen in those adopting an amphibious lifestyle where flooding is less frequent. Lowland rice, the staple cereal for much of tropical Asia falls into this category. But, even amongst dry-land dwellers, or certain of their sub-populations, modest tolerance to occasional flooding is to be found, for example in wheat. The collection of papers summarized in this article describes advances to the understanding of mechanisms that explain flooding tolerance in aquatic, amphibious and dry-land plants. Work to develop more tolerant crops or manage flood-prone environments more effectively is also included. The experimental approaches range from molecular analyses, through biochemistry and metabolomics to whole-plant physiology, plant breeding and ecology.

Up-regulation of PdSUS1 transcription under anoxia may not be attributed to sugar starvation under anoxia. A positive correlation between stem elongation and the level of PdSUS1 transcripts was observed in turions treated with anoxic conditions, 2,4-D and sorbitol. The increase in SuSy activity in the cytosol may contribute to sugar metabolism and sustain stem elongation under anoxia.

The responses of two aquatic plants, arrowhead (Sagittaria pygmaea Miq.) and pondweed (Potamogeton distinctus A. Benn), to anoxia were compared with those of rice (Oryza sativa L.). Shoot elongation of arrowhead tubers was enhanced at around 1 kPa O 2 , whereas that of pondweed turions was slight in air and reached a maximum in the absence of O 2 . Anaerobic enhancement of alcohol dehydrogenase (ADH) activity took place in rice coleoptiles but not in arrowhead and pondweed shoots. Shoots of both arrowhead and pondweed maintained a more stable energy status than did the rice coleoptile under anaerobic conditions. Total adenylate nucleotide contents of arrowhead and pondweed shoots were constant under anaerobic conditions. Adenylate energy charge in both shoots remained at a high and stable level of more than 0·8 for at least 8 d. Three forms of ADH from arrowhead shoots were separated by starch gel electrophoresis, showing that the activity of each ADH form was different under aerobic and anaerobic conditions. The incorporation of 35 S-labelled Cys and Met into soluble proteins in arrowhead shoots showed active protein biosynthesis and an involvement of a special set of polypeptides in the anaerobiosis.Key-words: Oryza sativa; Potamogeton; Sagittaria; adenylate energy charge; alcohol dehydrogenase; alcoholic fermentation; anaerobiosis; aquatic plants; growth in an anoxic state; protein synthesis in an anoxic state. INTRODUCTIONStudies on the responses of plants to anaerobic conditions are important to improve the productivity of cultivated plants in fields that are in danger of flooding. Therefore, many studies have been made on major crops such as tomato, barley, corn, soybean (Perata & Alpi 1993;Sachs, Subbaiah & Saab 1996;Vartapetian & Jackson 1997). These crops only survive a few days under anaerobic conditions because of lethal damage to their roots from exposure to anoxia. Studies of early events occurring in the cells of plants exposed to anaerobic conditions have focused on adenosine 5'-triphosphate (ATP) production, fermentation, protein synthesis, gene expression, and acidosis.It has been shown, on the other hand, that several aquatic plants exposed to anoxia have the ability not only to survive but even to elongate their shoots for a certain period (Crawford 1989;Kennedy, Rumpho & Fox 1992;Crawford & Braendle 1996;Vartapetian & Jackson 1997). This does not mean that all aquatic plants can tolerate an anaerobic environment. Instead most aquatic plants avoid anaerobiosis by developing a system to ventilate tissues under water (Armstrong 1979). Some aquatic plants, however, have been known to be tolerators of anoxia. The most extreme example of this is the overwintering shoots of Potamogeton pectinatus L., which can elongate under anaerobic conditions (Summers & Jackson 1994). In this study, we found that the shoots of arrowhead (Sagittaria pygmaea Miq.) tubers and pondweed (Potamogeton distinctus A. Benn.) turions can also grow under anaerobic conditions.Arrowheads are an emergent plant and a per...

Ethanol production by anoxic, excised, 7-10 mm tips of rice coleoptiles was manipulated using a range of exogenous glucose concentrations. Such a dose-response curve enabled good estimates at which level of ethanol production (and hence by inference ATP production), injury commenced and also allowed assessments of energy requirements for maintenance in anoxia. Rates of net uptake or loss of K+ and P by these excised coleoptile tips were related to rates of ethanol production (r2 of 0.59 and 0.68, respectively). At 72 h anoxia, ATP levels in excised tips were similar at 0, 2.5, and 50 mol m(-3) exogenous glucose, despite large differences in the inferred rates of ATP production. At 96 h anoxia, tips without exogenous glucose had low ATP concentrations; these may be the cause or the consequence of cell injury. In tips without glucose, injury was indicated by losses of K+ and Cl- between 72-96 h anoxia, and during the first hour after re-aeration, while later than 1 h after re-aeration, rates of net uptake were substantially lower than for re-aerated tips previously in anoxia with exogenous glucose. Between 96 h and 124 h anoxia, ion losses from tips without exogenous glucose increased while recovery of net uptake after re-aeration was very sluggish and incomplete. The energy requirement for maintenance of health and survival of anoxic coleoptile tips, expressed on a fresh weight basis, was lower than for three other anoxia-tolerant plant tissues/cells, studied previously. However, the energy requirement on a protein basis was assessed at 1.4 micromol ATP mg(-1) protein h(-1) and this value is 2.6-5.4-fold higher than for the other plant tissues/cells. Yet, this requirement was still only 58-88% of the published values for aerated tissues. The reason for this relatively high ATP requirement per unit protein in anoxic rice coleoptiles remains to be elucidated.