Gene Cloning And Functional Analysis Of Two Arabidopsis Chlorophyll Deficient Mutants

Chlorophylls are essential for photosynthesis. Chlorophyll biosynthesis is catalyzed by a series of enzyme complexes, such as Mg-protoporphyrin IX methyltransferase. One Arabidopsis yellow mutant was isolated using an enthyl-methane sulfonate (EMS) mutagenesis strategy. Chlorophyll contents were dramatically reduced and stacked grana were absent in the mutant. Genetic analysis indicated that the mutant was controlled by a single recessive gene. Using map-based cloning strategy, the gene responsible for the mutant phenotype was mapped to a region of 114 kb between the molecular markers F13M23 and T30C3 on chromosome 4, in which the CHLM gene encoding Mg-protoporphyrin IX methyltransferase was included. The mutant was proved to be an allelic mutant of CHLM gene by sequencing and allelism test and then was designated as chlm-4. Gly59 of CHLM was replaced by Glu59 in chlm-4, which indicated that Gly59 was essential for the function of Mg-protoporphyrin IX methyltransferase. The chloroplast development is the prerequisite of photosynthesis and requires coordinated expression of the nuclear and the plastid genome. Due to the limited encoding capability of plastid genome, most chloroplast proteins are encoded by nuclear genes and a large portion of them are PPR proteins. PPR proteins are defined by a tandem array of PPR motifs: each is a highly degenerate unit consisting of 35 amino acids, and is predicted to be folded into a pair of antiparallel helices. They are thought to recognized target RNAs and involved in editing, splicing, stability and translation of various transcripts in chloroplast. So far, fifteen PPR proteins required for chloroplast RNA editing have been isolated from Arabidopsis. As one of them, AtECB2 is essential for accD RNA editing. In this dissertation, we isolated a new allelic mutant of AtECB2 gene, which showed delayed green phenotype and mature plants were leaf-variegation. Pigment measurement showed that the content of total chlorophylls decreased substantially in the early stage and can raise to the fair level of wildtype in cotyledons but not in leaves. Microscopy observation showed that the status of chloroplast development changed accordingly. Green leaves or cotyledons contained normal chloroplast while chloroplast of yellow leaves or cotyledons is defective and lack of organized thylakoid lamella. Genetic analysis indicated that the mutant phenotype was controlled by a single recessive locus. Using map-based cloning strategy, the gene responsible for the mutant was mapped to a region of 75 kb in the BAC clone T16N11 on the short arm of chromosome I. The mutant was proved to be an allelic mutant of AtECB2gene by sequencing, allelism test and genetic complementation, and then was designated as atecb2-2. Thr500 of AtECB2 protein was replaced by Ile500 in atecb2-2, indicating that Thr500 was essential for the function of AtECB2 protein. By detecting the accD editing efficiency in the yellow and green cotyledons and 21-d-old true leaves of the mutant, we found that the cell developmenal status in atecb2-2 mutants was correlated with the accD editing rate.