Chloroplast Engineering for Herbicide Resistance via Protoporphyrinogen Oxidase

Introduction

Protoporphyrinogen oxidase (PPO) is an enzyme that catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX, and plays a key role in the biosynthesis of chlorophyll and heme. Among them, chlorophyll is a light-harvesting pigment that is an essential process for all green photosynthetic organisms. Disruption of porphyrin metabolism by any disruption is lethal, a property that makes PPO an excellent genetic target for herbicide development. When PPO is inhibited by PPO herbicides, protoporphyrin IX accumulates and causes light-dependent membrane damage, resulting in plant death. More than 100,000 PPO inhibitors have been synthesized, and this type of herbicide has become an effective tool for controlling a broad spectrum of weeds, including those that are resistant to glyphosate. Scientists have successfully developed PPO herbicide-resistant maize plants, as well as the isolation of plant PPO genes and the isolation of herbicide-resistant mutants. Meanwhile, rice plants resistant to PPO inhibitors have been developed by targeting the Bacillus subtilis PPO gene into the chloroplast or cytoplasm.

Fig. 1. The mode of action of protoporphyrinogen oxidase. (Dayan FE, et al., 2003)

Solutions

Our engineers have successfully isolated PPO genes from plants. In addition, Lifeasible is commmitted to increasing plant tolerance to PPO-inhibiting herbicides by overexpressing the native PPO gene in plants and selecting resistant mutants. Our available solutions for herbicide resistant via PPO include:

• Isolation of Plant PPO Gene.
• Identification of mutants resistant to PPO inhibitors.
• Production of herbicide-tolerant plants by expressing mutant PPO.
• Development of mutant PPO as a selectable marker.
• Effects of PPO selection on transgene copy number.
• Development of Herbicide Tolerant Transgenic Plants.

We used the Arabidopsis double mutant to develop a selectable marker system for Agrobacterium-mediated transformation of maize. The PPO gene was successfully introduced into the chloroplast genome of crops to obtain resistance to PPO family herbicides, including diphenyl ethers, oxazoles, cyclic imides, phenylpyrazoles, pyridine derivatives and benzene compounds. We are very mature in developing solutions for crops that are resistant to PPO inhibitors. Here, we also tried other methods to develop PPO herbicide-resistant plants.

(1) Traditional tissue culture methods.
(2) Expression of modified cofactors of protoporphyrin IX-binding subunit proteins.
(3) Overexpression of the PPO gene in wild-type plants.
(4) Engineering of P-450 monooxygenase to degrade PPO inhibitors.

Features of Our Strategy

• PPO1 and PPO2 isoforms are important targets for the evolution of PPO-resistant herbicides.
• Not dependent on any single herbicide or any single mutant PPO gene.
• Tetrabenazine as a selection agent with mutant PPO genes.
• Complete PPO-1 cDNA sequences were isolated from corn, wheat, sugar beet, cotton and soybean.
• Increasing the chance of identifying resistance loci by performing saturation site-directed mutagenesis of the identified resistance loci and screening with other plant PPO cDNAs.
• Using the PPO selection system for A. tumefaciens transformation.

Successful engineering of resistance to a PPO-targeted herbicide by overexpressing the PPO gene in crops. Lifeasible has extensive knowledge and experience in the engineering of herbicide resistance via herbicide-insensitive enzymes. Our mission is to provide customers with comprehensive, reliable, professional solutions to accelerate your research. If you are interested in our solutions, please contact us at any time.

References

1. Li X, Volrath SL, Nicholl DB, et al. (2003) Development of protoporphyrinogen oxidase as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation of maize. Plant Physiol. 133(2): 736-747.
2. Dayan FE, Barker A, Tranel PJ. (2018) Origins and structure of chloroplastic and mitochondrial plant protoporphyrinogen oxidases: implications for the evolution of herbicide resistance. Pest Manag Sci. 74(10):2226-2234.