RESUMEN
One of the key regulatory enzymes of the chlorophyll biosynthesis pathway is magnesium (Mg) chelatase, consisting of three different subunits CHLI, CHLD and CHLH. While CHLH and CHLD are encoded by a single gene each in Arabidopsis, CHLI is encoded by two homologous genes, ChlI 1 and ChlI 2. Analysis of the acifluorfen herbicide resistant mutant aci5 revealed an alteration of the ChlI 1 gene. This mutant as well as wild type plants contained similar transcript levels of the ChlI 1 and ChlI 2 genes. Moreover, the transcripts of both alleles of the ChlI 1 gene were present in the cs (ch42-2)/aci5 hybrid which showed an albina phenotype. Comparison of the amino acid sequence of CHLI 1 and CHLI 2 encoded in the genome of aci5 and wild type revealed in particular alterations of the C-terminal end which are suggested to be responsible for the decreased ability of CHLI 2 to participate in the formation of the CHLI ring-like structure of the Mg chelatase complex.
Asunto(s)
Arabidopsis/genética , Cloroplastos/ultraestructura , Herbicidas , Liasas/genética , Nitrobenzoatos , Secuencia de Aminoácidos , Arabidopsis/ultraestructura , Resistencia a los Herbicidas , Heterocigoto , Homocigoto , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Mutación , Fenotipo , Estructura Cuaternaria de Proteína , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
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.