Regulation and function of hemoglobin in barley aleurone tissue

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Nie, Xianzhou
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In this study, a barley Hb cDNA was demonstrated to also hybridize to genomic DNA sequences in rice, oat and bean, suggesting that barley Hb-like DNA sequences are widely distributed in the plant kingdom. The expression of Hb gene(s) in barley is tissue specific under normal growth conditions. Transcripts were found in roots, coleoptiles and aleurone layers but not in leaves. The gene, along with alcohol dehydrogenase and lactate dehydrogenase, was induced by oxygen deprivation in barley aleurone layers. Both Hb transcripts and protein were enhanced by anaerobiosis in barley aleurone layers and coleoptiles. However, the induction of protein was not as pronounced as that of mRNA. Supplying oxygen to oxygen deficient tissue decreased both Hb mRNA and protein, indicating that the gene expression is oxygen dependent. The effect of a number of respiratory inhibitors on barley aleurone layers has been examined to determine how increased hemoglobin gene expression occurs. Carbon monoxide induced Hb expression. Cyanide and antimycin A, two mitochondrial respiratory inhibitors, reduced oxygen consumption and, at the same time, strongly enhanced Hb messenger RNA levels. Treatment with the oxidative phosphorylation uncoupler 2,4-dinitrophenol markedly increased oxygen consumption and had a similar positive effect on Hb gene expression, suggesting that the expression of hemoglobin is not directly related to oxygen usage in the tissue. Hb transcription was also stimulated by the oxidative phosphorylation inhibitor oligomycin. The results suggest that the level of ATP may be critical in the induction of Hb gene transcription. The signal transduct on pathway leading to Hb gene expression in barley aleurone layers has been investigated. Ruthenium red, an organelle calcium channel blocker, inhibited anoxia-induced Hb transcription in a dose-dependent manner. The divalent ionophore, A23187, combined with EGTA dramatically reduced anoxia-induced Hb transcripts. The normal response of Hb to anoxia was restored by adding exogenous Ca$\sp{2+}$ but not Mg$\sp{2+}$. The results indicate that cytosolic calcium is involved in Hb gene regulation. W-7, a calmodulin antagonist, appeared not to affect anaerobically induced Hb mRNA even though it could induce Hb in normoxia, suggesting that calmodulin is not required in anaerobic induction of Hb in barley aleurone layers. Okadaic acid, a protein phosphatase inhibitor, was shown to abolish anaerobic-induced Hb transcripts dramatically, whereas A3, a protein kinase inhibitor, failed to do this. The results suggest that an okadaic acid-sensitive protein phosphatase is involved in the signal transduction pathway leading to Hb induction. Interestingly, both A3 and okadaic acid stimulated Hb expression under normoxia. The protein synthesis inhibitor,cycloheximide, blocked Hb gene expression, indicating that de novo synthesis of protein is required in Hb gene expression. (Abstract shortened by UMI.)