Arabidopsis mutants by activation tagging in which photosynthesis genes are expressed in dedifferentiated calli

Yasuo Niwa, Shingo Goto, Tatsuo Nakano, Mao Sakaiya, Takanori Hirano, Hirokazu Tsukaya, Yoshibumi Komeda, Hirokazu Kobayashi
Plant & Cell Physiology 2006, 47 (3): 319-31
In an effort to delineate the precise mechanisms underlying the organ-specific expression of photosynthesis genes, Arabidopsis lines homozygous for each transgene construct made with the gene for hygromycin B phosphotransferase or beta-glucuronidase (GUS) placed under control of the promoter of the nuclear gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBCS-3B) were constructed. Furthermore, activation tagging with T-DNA possessing quadruply repeated enhancers derived from the cauliflower mosaic virus 35S promoter was applied to a transgenic line of Arabidopsis. Mutants resistant to hygromycin B during the growth of calli generated from non-green roots on callus-inducing medium resulted from the expression of hygromycin B phosphotransferase driven by the RBCS-3B promoter. Three mutant lines, ces101 to ces103 (callus expression of RBCS), were obtained from approximately 4,000 calli resistant to a selectable marker for transformation. The active transcription driven by the RBCS-3B promoter in all the calli of ces mutants was confirmed by expression of both the GUS reporter gene and endogenous RBCS-3B. Chlorophyll and carotenoids, as well as light-dependent O(2) evolution, have been detected in the calli of all ces mutants. The loci where T-DNA was integrated in the ces101 line were determined by thermal asymmetric interlaced (TAIL)-PCR. The introduction of a DNA fragment harboring the gene for receptor-like kinase placed under the influence of enhancers into the parental line reproduced the phenotype of ces mutants. We have thus concluded that CES101 is a receptor-like kinase. The strategy presented in this investigation may promise to select a greater number of ces mutants.

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