The third subunit, CHLD, has proven to be essential for the formation of an active enzyme complex in tobacco, soybean, and yeast (Gibson et al. 1993) and by ethyl methanesulfonate mutagenesis in Arabidopsis and barley (Jensen et al. The CHLH genes were found by transposon-tagged gene inactivation in snapdragon (Hudson et al. The first plant gene homologous to a Mg-chelatase subunit was the T-DNA-tagged chlI (Koncz et al. The magnesium chelatase comprises three subunits – CHLI, CHLD, and CHLH (Zsebo and Hearst 1984, Gibson et al. 1990, Walker and Willows 1997, Rissler et al.
The ch42 mutant of Arabidopsis is deficient in chelating the Mg2+ ion into protoporphyrin IX to form Mg-protoporphyrin IX, which is then converted to the chlorophylls in a series of enzymatic steps (Koncz et al. Chlorophyll is the principal pigment that traps light energy its defect results in this chlorina phenotype in various plants including Arabidopsis (Meinke and Koornneef 1997). Here, we report our study on isolating the T-DNA flanking sequence from a yellow-green (chlorina) mutant in order to identify the knockout gene that encodes for an enzyme involved in chlorophyll biosynthesis. We have screened those insertional lines and have observed that 2% are GUS positive in the leaves (Jeon et al. T-DNA-tagged lines that carry such a gene fusion can be verified by GUS assay.
Therefore, when T-DNA is inserted into a gene in the proper orientation, gene fusion can be generated between the endogenous gene and GUS (Springer 2000). The vector used for mutagenesis carries the promoter-less β-glucuronidase (GUS) reporter gene immediately next to the right border of T-DNA. Japonica), using the scutellum-driven embryonic calli (Lee et al.
We recently reported the establishment of a large pool of T-DNA insertional lines in rice (Oryza sativa cv. However, application of the T-DNA insertional mutagenesis to other plant species has been difficult because it requires development of an efficient transformation procedure. T-DNA is believed to be randomly inserted into plant chromosomes, which allows for a greater chance of finding knockouts in a given gene (Azpiroz-Leehan and Feldmann 1997, Jeon et al. Establishing T-DNA insertional mutant pools in Arabidopsis is simple and rapid because the in plants procedures result in high transformation frequencies (Clough and Bent 1998). T-DNA generates low copy insertions that are stable through the next generations (Azpiroz-Leehan and Feldmann 1997, Jeon et al. (Received DecemAccepted February 18, 2003) Introduction T-DNA insertional mutagenesis has been successfully used to isolate a number of genes from Arabidopsis (Azpiroz-Leehan and Feldmann 1997, Takechi et al. This is the first report that T-DNA insertional mutagenesis can be used for functional analysis of rice genes. The chlorophyll content was very low in the OschlH mutants.
GUS assays and RNA blot analysis showed that expression of the OsCHLH gene is light inducible, while TEM analysis revealed that the thylakoid membrane of the mutant chloroplasts is underdeveloped. Those phenotypes were co-segregated with Tos17 in the progeny. We have identified two additional chlorina mutants that have a Tos17 insertion in the OsCHLH gene.
In the T2 and T3 generations, the chlorina mutant phenotypes are co-segregated with the T-DNA. This OsCHLH gene encodes the largest subunit of the rice Mg-chelatase, a key enzyme in the chlorophyll branch of the tetrapyrrole biosynthetic pathway. Line 9-07117 has a T-DNA insertion into the gene that is highly homologous to XANTHA-F in barley and CHLH in Arabidopsis. Among the mutants, 10 lines were β-glucuronidase (GUS)-positive in the leaves. Among the 1,995 lines examined in the T2 generation, 189 showed a chlorophyll-deficient phenotype that segregated as a single recessive locus. In this study, we screened those T-DNA pools for rice mutants that had defective chlorophylls. Jung, Ki-Hong Hur, Junghe Ryu, Choong-Hwan Choi, Youngju Chung, Yong-Yoon Miyao, Akio Hirochika, Hirohiko An, GynheungĪbstract We have previously generated a large pool of T-DNA insertional lines in rice. Characterization of a Rice Chlorophyll-Deficient Mutant Using the T-DNA Gene-Trap System Characterization of a Rice Chlorophyll-Deficient Mutant Using the T-DNA Gene-Trap System