Can double PPO mutations exist in a same allele and are such mutants functional?

Abstract

Background: Resistance to PPO-inhibiting herbicides is primarily endowed by target site mutations at the PPO2 gene that compromise binding of the herbicide to the catalytic domain. In Amaranthus ssp. PPO2, the most prevalent target mutations are deletion of the G210 codon and the R128G and G339A substitutions. These mutations strongly affect the dynamic of the PPO2 binding pocket resulting in reduced affinity with the ligand. Here we investigated the likelihood of co-occurrence of the most widespread target site mutations in the same ppo2 allele. Results: Plants carrying R128G+/+ ΔG210+/-, where + indicates presence of the mutation, were crossed with each other. The ppo2 of the offspring was subjected to pyrosequencing and E. coli- based Sanger sequencing to determine mutation frequencies and allele co-occurrence. The data show that R128G ΔG210 can occur only in one allele; the second allele carries only one mutation. Double mutation in both alleles is less likely because of significant loss of enzyme activity. The segregation of offspring populations derived from a cross between heterozygous plants carrying ΔG210 G399A also showed no co-occurrence in the same allele. The offspring exhibited the expected mutation distribution patterns with few exceptions. Conclusions: Homozygous double-mutants are not physiologically viable. Double-mutant plants can only exist in a heterozygous state. Alternatively, if two mutations are detected in one plant, each mutation would occur in a separate allele

The chloroplast envelope localization of protoporphyrinogen oxidase 2 prevents full complementation of Arabidopsisppo1

“…Such sequence variations have been discovered repeatedly in Amaranthus species, in which the N-terminal region of PPO2 (and thus its subcellular distribution) differs from that found in Arabidopsis (Figure 5A). The recent description of an obviously lethal phenotype caused by severe PPO2 mutations in Amaranthus (Porri et al, 2022) also deviates from the Arabidopsis ppo2-2 phenotype described in this study and hints at fundamental differences in PPO2 function between Amaranthaceae and dicots encoding Arabidopsis-type PPO2 sequences.…”

“…Regardless of the detailed import route for the Arabidopsis-type PPO2 sequences from dicotyledonous plants, it is worth mentioning that, despite the lack of a visible phenotype for Arabidopsis ppo2 knockout mutants, the assignment of plant PPO2 to the inner envelope membrane of the chloroplast is of economic and biotechnological relevance. PPO2 has recently attracted attention for several reported cases of resistance against PPO-inhibiting herbicides which are caused by PPO2 point mutations (Porri et al, 2022). Such sequence variations have been discovered repeatedly in Amaranthus species, in which the N-terminal region of PPO2 (and thus its subcellular distribution) differs from that found in Arabidopsis (Figure 5A).…”

“…However, in Arabidopsis, proteome studies have identified PPO2 as a constituent of the plastid envelope membrane (Joyard et al, 2009). While PPO2-deficient plants have not been investigated so far, recent reports on herbicide resistance specifically conferred by mutations in PPO2 have drawn renewed attention to this specific isoform (Porri et al, 2022).…”

Inhibition profile of trifludimoxazin towards PPO2 target site mutations

“…The principal difference observed between G399 and A399 is the additional methyl group (-CH3) that creates close repulsive interaction with the central phenyl ring of PPO inhibitors generating repulsive electrostatic interactions that push the herbicide from the binding site. 25 Recently, Porri et al 27 discussed the likelihood of a double PPO2 mutation on the same allele. Although R128G and ΔG210 can occur on the same allele, the presence of these mutations on both alleles is unlikely because of the fitness cost associated with this mutant combination.…”

“…discussed the likelihood of a double PPO2 mutation on the same allele. Although R128G and ΔG210 can occur on the same allele, the presence of these mutations on both alleles is unlikely because of the fitness cost associated with this mutant combination 27 . Nevertheless, other double‐mutant combinations, R128(X) ΔG210, may appear in the future if the imposed fitness cost is less severe.…”