Purification and characterisation of two ACC oxidases expressed differentially during leaf ontogeny in white clover

Abstract

Two isoforms of ACC oxidase (ACO) (EC 1.4.3), expressed differentially during leaf ontogeny in white clover (Trifolium repens L.), have been identified and purified to homogeneity. One isoform, designated MGI, was purified from mature green leaf tissue while the second isoform, designated SEII, was purified from senescent leaf tissue. The isolation and purification of these isoforms were achieved using a combination of hydrophobic interaction chromatography, anion exchange chromatography, chromatofocusing and gel filtration column chromatography. The Mr of both MGI and SEII was determined to be 37.5 kDa by gel filtration, and 37 kDa (MGI), 35 kDa (SEII) by SDS‐PAGE, indicating that both isoforms are active as monomers. During purification, both isoforms were recognised by a polyclonal antibody directed against a recombinant polypeptide derived from a white clover ACO gene expressed in mature green leaf tissue, TR‐ACO2. In addition to molecular mass, differences between the two isoforms were observed in terms of pH optima, isoelectric point (pI), Km for ACC, optimal requirements for the co‐substrate ascorbate, and NaHCO3 and Fe2+ as co‐factors. The identification of distinct ACC oxidases from the same tissue at different developmental stages shows that the now widely observed transcriptional regulation of the ACO gene family in higher plants is also expressed in terms of differential regulation of enzyme isoforms.