An Arabidopsis mutant that is resistant to the protoporphyrinogen oxidase inhibitor acifluorfen shows regulatory changes in tetrapyrrole biosynthesis

Abstract

Several Arabidopsis mutants of the ecotype Dijon were isolated that show resistance to the herbicide acifluorfen, which inactivates protoporphyrinogen oxidase (PPOX), an enzyme of tetrapyrrole biosynthesis. This enzyme provides protoporphyrin for both Mg chelatase and ferrochelatase at the branchpoint, which leads to chlorophyll and heme, respectively. One of the mutations, aci5-3, displays semidominant inheritance. Heterozygous progeny showed yellow-green leaves, while the homozygous seedlings were white and inviable, but could be rescued by supplementing the medium with sugar. Interestingly, the expression of neither of the two forms of PPOX was altered in the mutant, but the rate of synthesis of 5-aminolevulinate, the precursor of all tetrapyrroles, was drastically reduced. Genetic mapping revealed the mutant locus is closely linked to the ch42 marker, which is itself located in the CHLI-1 gene which codes for one of the three subunits of Mg chelatase. The cs mutant also shows a defect in this gene, and test for allelism with aci5-3 confirmed that the two mutations are allelic. Sequencing of the wild type and aci5-3 alleles of CHLI-1 revealed a single base change (G718A), which results in a D240N substitution in the CHLI-1 protein. In the homozygous aci5-3 mutant no CHLI-1 RNA or protein could be detected. Strikingly, CHLH and CHLI-2 transcripts were also absent. This indicates the existence of a feedback-regulatory mechanism that inactivates the genes encoding certain Mg chelatase subunits. The basis for the semidominant inheritance pattern and the relationship between herbicide resistance and modified gene expression is discussed.

The analysis of the ChlI 1 and ChlI 2 genes using acifluorfen-resistant mutant of Arabidopsis thaliana

“…We recently investigated the aci5 mutant of A. thaliana (Soldatova et al 2005), which was obtained by chemical mutagenesis and further selection for resistance to aciXuorfen, an inhibitor of protoporphyrinogen oxidase, the enzyme preceding Mg chelatase in the chlorophyll biosynthetic pathway (Ezhova et al 2001). The mutant gene identiWed by genetic mapping and sequence analysis encodes CHLI 1.…”

Arabidopsis CHLI2 Can Substitute for CHLI1

“…Data from three recombinant plants delimited the cs215 locus to the region encompassed by bacterial artificial chromosomes F28J12 and F28A21. One of the genes in this region, At4g18480 encoding CHLI1, has been shown to have mutant alleles with semidominant pale-green phenotypes (Kjemtrup et al, 1998;Fitzmaurice et al, 1999;Hansson et al, 2002;Soldatova et al, 2005). Therefore, we sequenced the region of At4g18480 from cs215 and found that cs215 indeed had a C-to-T mutation at nucleotide 584 that converted a Thr at residue 195 to an Ile (Fig.…”

“…In the cs215 mutant, the high amount of cs215 mutant protein may out-compete the low amount of CHLI2 in assembly with CHLD. It is also possible that the expression levels of CHLI1 and CHLI2 were similar (Rissler et al, 2002;Apchelimov et al, 2007) and that the presence of the cs215 mutant protein inhibited the expression of CHLI2 as suggested previously (Soldatova et al, 2005). To clarify the role of CHLI2, we compared the expression levels of CHLI1 and CHLI2 using realtime quantitative RT-PCR.…”

“…It forms a ring-shaped homohexamer. Many semidominant and recessive alleles of chli mutants have been isolated from various species (Kjemtrup et al, 1998;Fitzmaurice et al, 1999;Hansson et al, 2002;Soldatova et al, 2005). The semidominant alleles are all missense mutations that still produce full-length proteins.…”

Suppression of the Barleyuroporphyrinogen III synthaseGene by aDsActivation Tagging Element Generates Developmental Photosensitivity

“…Unlike the four preceding enzymatic steps ( Figure 1A), reduced Mg-chelatase activity does not cause photosensitivity, but rather reduced chlorophyll levels due to a negative feedback regulatory mechanism (Hansson et al, 1999(Hansson et al, , 2002Papenbrock et al, 2000aPapenbrock et al, , 2000bSoldatova et al, 2005;Sawers et al, 2006). The substrate of Mg-chelatase, protoporphyrin IX, is the branch point of chlorophyll and heme synthesis ( Figure 1A); by contrast, antisense suppression of the first committed heme synthetic enzyme, plastidic ferrochelatase, does lead to protoporphyrin IX accumulation and light-dependent necrosis (Papenbrock et al, 2001).…”