Characterization of Laccase-like Multicopper Oxidases (LMCOs) in Arabidopsis thaliana

Abstract

Significant Findings:1. The At2g30210 LMCO harbors strong phenoloxidase activity, but no detectable ferroxidase or other metal oxidase activity. At the time the original proposal was submitted we had demonstrated expression of the At2g30210 protein to high levels in E. coli. However, we were not able to recover active enzyme, nor were we able to reconstitute activity in the heterologously expressed protein using protocols developed for metal-replacement studies of Rhus vernicifera laccase, the archetype LMCO. We subsequently prepared appropriate constructs for expression of the gene in plant cells, and isolated several lines of transgenic tobacco (BY2) cells that produced measurable amounts of active phenoloxidase. None of the cell lines produced large quantities of the enzyme or secreted freely soluble enzyme to the medium. After considerable effort, we were able to recover cellular extracts several tens of micrograms of partially purified enzyme that were tested using multiple assays for phenoloxidase, as well as ferroxidase and metal oxidase, activities. Although the enzyme proved highly active as a phenoloxidase ( Fig. 1), no ferroxidase, copper oxidase or manganese oxidase activity was detected, even when the enzyme was added to the assays in great excess over the positive controls. Although the remote possibility remains that the At2g30210 LMCO might be able to oxidize metal ion substrates if properly assisted by unknown ancillary proteins, the simplest explanation of our results is that the enzyme is primarily a phenoloxidase and carries out its physiological function(s) in Arabidopsis via this activity. coli LMCO, CueO, harboring an equivalent level of phenoloxidase activity (Lane cueO) provided a positive control for ferroxidase activity. Cleared zones representing ferroxidase activity were visualized by applying the ferrous ion chelator, 3-(2-pyridyl)-5,6-bis (4-phenylsulfonic acid)-1,2,4-triazine (ferrozine). At LMCOcueO At LMCO 2. T-DNA knockouts of At2g30210 show reduced growth, altered root morphology, and a response to sucrose. Among the most useful resources available for the study of Arabidopsis genes are the collections of T-DNA insert lines developed and cataloged at the Salk Institute and the gene-trap lines developed at Cold Spring Harbor Laboratory. A line was identified in the Salk collection as having an insert in the last intron of the At2g30210 gene. Upon obtaining a homozygous line for the insert, we found the mutation to be leaky, ostensibly because the T-DNA was spliced out of mature transcripts at a low, but sufficient, frequency for phenotypic escape. However, levels of mature transcript were always less than 30% of levels seen in wild-type Arabidopsis. In the course of working with the Salk T-DNA line, a second insertion mutant was identified among the gene-trap lines produced at CSHL (GT7855). This insertion was in the second exon of the gene, where it resulted in a "strong" allele from which no mature transcripts for At2g30210 could be detected in homozygous lines. Both knockout...