Phylogenesis of the Functional 1-Aminocyclopropane-1-Carboxylate Oxidase of Fungi and Plants

Abstract

The 1-aminocyclopropane-1-carboxylic acid (ACC) pathway that synthesizes ethylene is shared in seed plants, fungi and probably other organisms. However, the evolutionary relationship of the key enzyme ACC oxidase (ACO) in the pathway among organisms remains unknown. Herein, we cloned, expressed and characterized five ACOs from the straw mushroom (Volvariella volvacea) and the oyster mushroom (Pleurotus ostreatus): VvACO1-4 and PoACO. The five mushroom ACOs and the previously identified AbACO of the button mushroom contained all three conserved residues that bound to Fe(II) in plant ACOs. They also had variable residues that were conserved and bound to ascorbate and bicarbonate in plant ACOs and harbored only 1-2 of the five conserved ACO motifs in plant ACOs. Particularly, VvACO2 and AbACO had only one ACO motif 2. Additionally, VvACO4 shared 44.23% sequence identity with the cyanobacterium Hapalosiphon putative functional ACO. Phylogenetic analysis showed that the functional ACOs of monocotyledonous and dicotyledonous plants co-occurred in Type I, Type II and Type III, while putative functional gymnosperm ACOs also appeared in Type III. The putative functional bacterial ACO, functional fungi and slime mold ACOs were clustered in ancestral Type IV. These results indicate that ACO motif 2, ACC and Fe(II) are essential for ACO activity. The ACOs of the other organisms may come from the horizontal transfer of fungal ACOs, which were found ordinarily in basidiomycetes. It is mostly the first case for the horizontal gene transfers from fungi to seed plants. The horizontal transfer of ACOs from fungi to plants probably facilitates the fungal-plant symbioses, plant-land colonization and further evolution to form seeds.

Keywords: 1-aminocyclopropane-1-carboxylic acid oxidase; basidiomycete; horizontal gene transfer; sequence motif analysis.

Figure 1

Expression and purification of the ACO proteins from straw mushrooms and oyster mushrooms. (A), VvACO1; (B), VvACO2; (C), VvACO3; (D), VvACO4; (E), PoACO. M, protein marker; C-E, control E. coli cell extract; CDS, cell disruption supernatant; C-Y, control yeast cell extract; PE, purified enzyme.

Figure 2

Specific activities of the heterologously expressed ACO enzymes from straw mushroom and oyster mushroom. The data shown were the mean of three independent experiments ± standard deviation (SD). They were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey–Kramer multiple-comparison post hoc test. * Indicates a significant difference at p < 0.05.

Figure 3

Selected functional ACO protein sequence alignment of mushrooms, slime molds and plants. Protein sequences were aligned using ClustalX 2.0. The important and conserved amino acid residues were marked in accordance with the legend shown.

Figure 4

Unrooted phylogenetic tree of 27 ACO proteins generated by MEGA, version 11.0, with the maximum likelihood method. Numbers near branches show percentage bootstrap support. Red clades indicate fungal ACO proteins; blue clades indicate ACO proteins of gymnosperms; green clades indicate the ACO proteins of slime mold; purple clades indicate bacterial ACO proteins; black clades indicate the ACO proteins of angiosperms. The protein sequence information is listed in Table 2.

Figure 5

The phylogenetic relationships and conserved motif structure of the ACO protein sequences from the functional ACOs of fungi, slime molds and angiosperms plants, and the putative functional ACOs of cyanobacteria and gymnosperms. The phylogenetic trees were constructed MEGA version 11.0 with the maximum likelihood method. The conserved motif structure was discovered by MEME. The protein sequence information listed in Table 2.