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Characterization of HemY-type protoporphyrinogen IX oxidase genes from cyanobacteria and their functioning in transgenic Arabidopsis

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We investigated the functions of two cyanobacterial HemY protoporphyrinogen IX oxidase (PPO) genes with in vitro and in vivo assays and evaluated their applicability as resistance traits to PPO-inhibiting herbicides.

Abstract

We isolated HemY-type protoporphyrinogen IX oxidase (PPO) genes from cyanobacteria, OnPPO gene from Oscillatoria nigro-viridis PCC7112 and HaPPO gene from Halothece sp. PCC7418. The alignment of amino acid sequences as well as phylogenetic analyses conducted showed that OnPPO and HaPPO are classified as HemY-type PPO and are more closely related to plastidic PPOs than to mitochondrial PPOs. The PPO-deficient Escherichia coli BT3 strain, which requires heme supplementation, could obtain normal growth in the absence of heme supplementation when complemented with OnPPO and HaPPO. The enzyme assays of OnPPO, HaPPO, and Arabidopsis thaliana PPO1 (AtPPO1) proteins each revealed different kinetic properties in terms of catalytic efficiency, substrate affinity, and the degree of inhibition by PPO inhibitors. In particular, the catalytic efficiencies (kcat/Km) of OnPPO and HaPPO were approximately twofold higher than that of AtPPO1. The elution profiles of all three PPOs, acquired by size-exclusion chromatography, showed only a single peak with a molecular weight of approximately 52–54 kDa, which corresponds to a monomeric form. Moreover, functional complementation with OnPPO and HaPPO in AtPPO1-silenced Arabidopsis resulted in restored growth, whereas AtPPO1-silenced wild type Arabidopsis suffered necrotic death. In addition, we observed that overexpression of OnPPO and HaPPO in Arabidopsis conferred resistance to the PPO-inhibiting herbicides tiafenacil and saflufenacil. These results suggest that two HemY-type PPOs of cyanobacteria can functionally substitute for plastidic PPO activity in Arabidopsis and can enhance resistance to tiafenacil and saflufenacil.

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Acknowledgements

The authors thank Dr. Gyung-Hee Yu (Korean Agency for Technology and Standards) for expert comments and critical reading of this manuscript. We are also grateful to Dr. Ryouichi Tanaka of Hokkaido University for providing us with BT3 cells. This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry of the Ministry of Agriculture, Food, and Rural Affairs under Grant No. 814004-3 and by the BioGreen 21 Program of the Rural Development Administration under Grant Nos. PJ01121901 and PJ01186401.

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S-KS designed and supervised the work. JY designed, planned and performed the physiological and molecular characterization of PPOs. YH performed cyanobacteria culture and western blotting experiment. YOA performed the analysis of PPO enzyme kinetics and IC50 of PPO-inhibitors. M-KH performed the production of recombinant PPO proteins in E. coli and the analysis of monomeric form of PPOs.

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Correspondence to Soon-Kee Sung.

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J. Y., Y. H., S.-K. S. and FarmHannong Co., Ltd. have filed an international patent application (WO2016/099153A1) covering the genes reported in this study.

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Yoon, J., Han, Y., Ahn, Y.O. et al. Characterization of HemY-type protoporphyrinogen IX oxidase genes from cyanobacteria and their functioning in transgenic Arabidopsis. Plant Mol Biol 101, 561–574 (2019). https://doi.org/10.1007/s11103-019-00925-8

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