A barley (Hordeum vulgare L.) full-length clone coding for long chain acyl-CoA oxidase (ACX), key enzyme of beta-oxidation, was isolated by cDNA library screening and 5'-rapid amplification of cDNA ends. The cDNA encodes for a polypeptide of 667 amino acids, with a molecular mass of 74.5 kDa. The amino acid sequence, beside an extensive similarity with other plant and mammalian ACXs, showed a PTS1 peroxisomal targeting signal at the C terminus and a conserved FAD-binding domain. The gene was over-expressed in E. coli and the fusion protein was shown to possess long chain acyl-CoA oxidase activity. Polyclonal antibodies were raised against a large fragment of the protein encoded by the barley putative ACX gene. Northern and Western analysis demonstrated that a basal level of long chain ACX is always present along the barley life cycle, while a higher level of expression is typical of actively growing tissues such as germinating embryos, ovary before anthesis, developing embryos, shoots and roots apexes. In vitro germination experiments with glucose and glucose analogues provided evidence about the involvement of a glucose-deriving signal in the positive modulation of ACX expression. This result highlights the role of ACX, not only during oil reserve mobilization, but also in plant growth and metabolism.
Cloning and characterization of barley long chain acyl-CoA oxidase and its possible regulation by glucose
PISTELLI, LAURA;ALPI, AMEDEO;
2003-01-01
Abstract
A barley (Hordeum vulgare L.) full-length clone coding for long chain acyl-CoA oxidase (ACX), key enzyme of beta-oxidation, was isolated by cDNA library screening and 5'-rapid amplification of cDNA ends. The cDNA encodes for a polypeptide of 667 amino acids, with a molecular mass of 74.5 kDa. The amino acid sequence, beside an extensive similarity with other plant and mammalian ACXs, showed a PTS1 peroxisomal targeting signal at the C terminus and a conserved FAD-binding domain. The gene was over-expressed in E. coli and the fusion protein was shown to possess long chain acyl-CoA oxidase activity. Polyclonal antibodies were raised against a large fragment of the protein encoded by the barley putative ACX gene. Northern and Western analysis demonstrated that a basal level of long chain ACX is always present along the barley life cycle, while a higher level of expression is typical of actively growing tissues such as germinating embryos, ovary before anthesis, developing embryos, shoots and roots apexes. In vitro germination experiments with glucose and glucose analogues provided evidence about the involvement of a glucose-deriving signal in the positive modulation of ACX expression. This result highlights the role of ACX, not only during oil reserve mobilization, but also in plant growth and metabolism.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
