Purification, molecular cloning and ethylene-inducible expression of a soluble-type epoxide hydrolase from soybean (Glycine max [L.] Merr.)

Abstract

A soybean protein was purified from mature dry seeds. Amino-acid sequencing of the nine internal peptides derived from this N-terminally blocked protein showed that it has a significant similarity to the soluble epoxide hydrolases known to date. A degenerate series of 23-met oligonucleotides with sequences corresponding to an internal region of eight amino-acid residues was synthesized as a probe mixture for detection of a putative epoxide hydrolase cDNA in a developing cotyledon cDNA library. The 1332-bp cDNA obtained was found to have an open-reading frame encoding the seed epoxide hydrolase-like precursor consisting of 341 amino-acid residues, suggesting that 25 amino-acid residues upstream from the second methionine correspond to a transit peptide. Employing an Escherichia coli expression system, the putative mature epoxide hydrolase-like protein was overexpressed and purified to homogeneity. This recombinant protein was confirmed to exhibit its epoxide-diol converting activity using styrene oxide as substrate. The V(max) and K(m) values for styrene oxide are 1.36 μmol·min-1·mg-1 and 1500 μM, respectively. Sedimentation equilibrium experiments showed that the active form of this epoxide hydrolase is monomeric in solution. Using the above cDNA as a probe, a 12-kb genomic clone was selected and the sequence of a 1933-bp fragment from this clone was found to cover the entire coding region together with 5'- and 3'-flanking regions of the soybean epoxide hydrolase gene. The coding region of the genes interrupted by two short introns, was identical to the corresponding regions of the cDNA. Northern blot analyses showed that this epoxide hydrolase gene was expressed strongly at a very early stage (13 days after flowering) and then the level of expression gradually decreased and almost ceased at a very late stage (58 days after flowering) of seed development, whereas its expression was markedly up-regulated by ethylene treatment. In stems (hypocotyl portion), the epoxide hydrolase transcript was detected at significant levels and was also up-regulated in response to ethylene. On the other hand, it is hardly expressed in leaves, even though they were treated with the phytohormone. Overall, the results obtained may indicate that soluble-type epoxide hydrolase mRNA is expressed at the maximum level in an early stage of seed development. Later, oil bodies are formed and subsequently epoxy fatty acids, naturally occurring metabolites, accumulate within those bodies. The temporal induction of this epoxide hydrolase transcript in some tissues in response to ethylene also indicates that this epoxide hydrolase may play a crucial role in self-defense systems of plant.