| The hypoxically inducible barley alanine aminotransferase (AlaAT-2; EC 2.6.1.2) was purified to homogeneity from barley root tissue by ammonium sulphate precipitation and a series of chromatographic columns. The purified protein had a specific activity of 2231 IU/mg protein, and was very specific in the substrates it used. AlaAT-2 was shown to be a homodimer composed of 50 kD subunits. A full-length barley AlaAT-2 cDNA clone was isolated, and had an open reading frame of 1446 base pairs, giving a predicted protein molecular mass of 52,885 Da. The coding region of the cDNA was 88% identical at the nucleic acid level and 90% identical at the amino acid level to that of the light inducible AlaAT cDNA from Panicum miliaceum leaves. A maize AlaAT genomic DNA clone was isolated and a partial sequence analysis of the coding region showed 95.1% and 87.9% identity to the the P. miliaceum and barley cDNAs, respectively.;The 6-fold increase in AlaAT activity after 96 h of hypoxia was similar to that of AlaAT-2 protein concentration, as indicated by protein gel immunoblots. AlaAT-2 mRNA concentration increased almost 4-fold during 24 h of hypoxic stress, followed by a gradual decrease in mRNA levels in the following 72 h. This pattern of AlaAT-2 protein and mRNA production indicated that the AlaAT-2 protein is either very stable in hypoxic cells, or there is an increase in translation rate upon prolonged hypoxia. The hypoxically inducible AlaAT-2 in the C;In barley and wild rice, hypoxia resulted in the accumulation of substantial amounts of ethanol. Alanine also accumulated in tissues of both of these species. Malate concentrations increased in wild rice roots and leaves, however it did not accumulate over the long-term in hypoxic barley roots. Lactate, aspartate and succinate were not significant end-products of hypoxic metabolism in either of these two species. The universal synthesis of alanine and ethanol in plants exposed to hypoxic stress suggests that these two compounds have important roles in a plant's ability to tolerate short-term flood conditions. Cytoplasmic acidosis and the availability of adequate carbohydrate reserves maybe important in long-term flood-tolerance. |
