Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana.

Abstract

A recessive pale mutation, designated as cs, was identified by transferred‐DNA (T‐DNA)‐mediated insertional mutagenesis in Arabidopsis thaliana. The pale mutation, cosegregating with the hygromycin resistance marker of the T‐DNA, was mapped to the position of the ch‐42 (chlorata) locus on chromosome 4. Lack of genetic complementation between cs and ch‐42 mutants indicated allelism. Plant boundaries of the T‐DNA insert rescued from the pale mutant were used as probes for the isolation of genomic and full‐length cDNA clones of the wild‐type cs gene. Transformation of the pale mutant with T‐DNA vectors carrying these clones resulted in a normal green phenotype, thus demonstrating positive complementation of the T‐DNA induced mutation. DNA sequence comparison of the cs mutant and its wild‐type allele revealed that the T‐DNA insertion occurred 11 bp upstream of the stop codon. A fusion protein, seven amino acids longer than its wild‐type counterpart of Mr 46,251, is therefore synthesized in the pale mutant. Transcript analysis during dark‐light transition, in vitro protein transport assay, and the absence of DNA sequence homology between cs and known genes indicates that the light regulated expression of the cs gene results in the synthesis of a novel chloroplast protein.

AtGA3ox2, a Key Gene Responsible for Bioactive Gibberellin Biosynthesis, Is Regulated during Embryogenesis by LEAFY COTYLEDON2 and FUSCA3 in Arabidopsis

“…lec2-1 and fus3-8 were initially in the Wassilewskija and Columbia backgrounds, respectively, and were backcrossed to Ler as described (Raz et al, 2001). Wild-type seeds were planted on soil or surface sterilized and grown in petri dishes on Murashige and Skoog for Arabidopsis (MSAR) medium (Koncz et al, 1990).…”

Mechanism and regulation of Mg-chelatase

“…The major impediment was the lack of an in itro assay, which was only achieved 6 years ago [2]. Since that time our knowledge of Mg-chelatase has expanded dramatically to the extent that the enzyme has been cloned and sequenced from several photosynthetic species [3][4][5][6][7][8][9][10].…”

“…The expression of the chlH and chlI genes in developing plants is induced by light. When dark-adapted Arabidopsis and soybean (Glycine max) were transferred to light for 24 h, the chlI transcript was induced up to 64-fold compared with the light-adapted plants [9,10]. When etiolated barley seedlings were transferred to light, the chlH transcript increased 25-fold after 4 h and remained at this level ; in this system, the induction of the transcription of I was not so pronounced, with only a 2-fold increase after 8 h and a return to the uninduced level after 24 h [7].…”

The Regulation of Enzymes Involved in Chlorophyll Biosynthesis

“…BchH has been proposed to bind protoporphyrin IX prior to the insertion step of MgZ+, while BchI contains an ATP-binding site (Gibson et al, 1995). Gene sequences related to bchH and bchl of R. sphaeroides (Bauer et al, 1993) occur in higher plants, such as Antirrhinum majus (Hudson et al, 1993) and Arubidopsis thaliana (Koncz et al, 1990). Functional disruption of one allele of the single nuclear gene in Antirrhinum by transposon tagging (used to isolate the gene) has been shown to lead to reduced chlorophyll levels and to an altered chloroplast ultrastructure (Hudson et a]., 1993).…”

“…Functional disruption of one allele of the single nuclear gene in Antirrhinum by transposon tagging (used to isolate the gene) has been shown to lead to reduced chlorophyll levels and to an altered chloroplast ultrastructure (Hudson et a]., 1993). In Arubidopsis, the product of the ch42 gene, a homolog of bchl, is found in the stroma (Koncz et al, 1990), which suggests that it may function as a soluble component of the Mg2+-chelatase complex (Hudson et al, 1993).…”