The Function of Ascorbate Oxidase in Arabidopsis thaliana

Abstract

The apoplastic enzyme, ascorbate oxidase (AO), is a blue copper oxidase that catalyses oxidation of ascorbate (AsA) to monodehydroascorbate (MDHA). In Arabidopsis thaliana, AO is encoded by three genes (At4g39830, At5g21105 and At5g21100) designated AO1, AO2, and AO3 respectively. Since AsA is the most abundant antioxidant in the apoplast and AO is active in this compartment, the regulation of apoplastic AsA redox status by AO and its role in development and environmental perturbations has become a subject of interest. Phylogenetic analysis showed that AO is present in higher plants, pteridophytes, mosses and green algae. Amino acid sequence analysis showed that AO2 and AO3 shared higher sequence identity than AO1. In silico analyses found that AO1 had a distinct expression pattern and subcellular localisation compared to AO2 and AO3, suggesting AO1 might be involved in alternative functions. Consistent with previous studies, AO activity was high in actively growing tissue of wild-type (WT) A. thaliana, supporting a possible role of AO in cell expansion. ao1, ao3 and ao1ao3 T-DNA insertion mutants were characterised. ao1 had similar level of AO activity to WT, while ao3 and ao1ao3 had 10-20% of WT AO activity. Compared with WT, these T-DNA insertion mutants did not show any phenotypic differences under unstressed or stressed (high light and drought) growth conditions. An artificial microRNA construct (amiR-AO) to silence all three AO genes was developed. Also, an overexpression plasmid (35S::AO3) harbouring AO3 gene was constructed. These constructs were used to transform A. thaliana. AO activity was undetectable in the amiR-AO line, while the 35S::AO3 line had 3-fold higher AO activity than the WT. Under unstressed normal growth conditions, the amiR-AO line had bigger rosette size, whereas the 35S::AO3 line exhibited early flowering and smaller number of rosette leaves. The amiR-AO line accumulated more anthocyanin and AsA than WT when acclimated to high light, whereas the 35S::AO3 line accumulated less anthocyanin than WT. In response to drought, the amiR-AO line did not show phenotypic differences compared to WT, while the 35::AO3 line had higher rate of leaf water loss and appeared to have greater sensitivity to drought. These results suggest that AO perturbation could, to some extent, affect the growth and stress response of A. thaliana although the effect is small.