T-DNA tagged mutants - in a RD29A::LUC background in Arabidopsis thaliana (C24)

In the population analyzed, a total of approximately 43,000 T-DNA insertion lines had been generated by in planta transformation with Agrobacterium harboring the T-DNA vector pSKI015 for tagging (Weigel et al., 2000). We used as the genetic background an Arabidopsis (ecotype C24) line expressing the firefly luciferase reporter under control of the Arabidopsis RD29A promoter. This promoter is responsive to cold, osmotic stress, and ABA (Yamaguchi-Shinozaki and Shinozaki, 1993). To date, approximately 30,000 lines have been screened for stress-related gene regulation mutants using our luminescence imaging system (Xiong et al., 1999). Cold (1 day), ABA (3 hours) and salt (5 hours) stresses have been included into the mutant screen. A detailed procedure can be found in Ishitani et al. (1997). Several hundred putative mutants that showed the altered luminescence images were isolated and again tested in the next generation. Of these, the mutant phenotype could be confirmed for 243 putative mutants (Table 1). From among these, 142 mutants were chosen based on their phenotype and based on Basta resistance in the F2 population for TAIL-PCR amplification (Liu et al., 1995). Primer sets were as follows; for degenerate primers: DEG1, 5'-WGC NAG TNA GWA NAA G-3' and DEG2, 5'-AWG CAN GNC WGA NAT A-3' (W = A or T; N = A, C, G, or T). For nested specific primers the following were used, AtLB1, 5'-ATA CGA CGG ATC GTA ATT TGT C-3'; AtLB2, 5'-TAA TAA CGC TGC GGA CAT CTA C-3'; AtLB3, 5'-TTG ACC ATC ATA CTC ATT GCT G-3'. In the table, the mutant identification number is hyperlinked to a description of the mutant (where available). Seeds from the ~43,000 lines have been submitted to the Ohio Stock Center and to the University of Wisconsin T-DNA knockout facility.

References
Ishitani, M., Xiong, L., Stevenson, B., and Zhu, J.K. (1997). Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis: interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways. Plant Cell 9, 1935-1949.
Liu, Y.G., Mitsukawa, N., Oosumi, T., and Whittier, R.F. (1995). Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8, 457-463.
Weigel, D., Ahn, J.H., Blazquez, M.A., Borevitz, J.O., Christensen, S.K., Fankhauser, C., Ferrandiz, C., Kardailsky, I., Malancharuvil, E.J., Neff, M.M., Nguyen, J.T., Sato, S., Wang, Z.Y., Xia, Y., Dixon, R.A., Harrison, M.J., Lamb, C.J., Yanofsky, M.F., and Chory, J. (2000). Activation tagging in Arabidopsis. Plant Physiol 122, 1003-10013.
Xiong, L.M., David, L., Stevenson, B., and Zhu, J.K. (1999). High throughput screening of signal transduction mutants with luciferase imaging. Plant Mol Biol Rep 17, 159-170.
Yamaguchi-Shinozaki, K., and Shinozaki, K. (1993). Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol Gen Genet 236, 331-340.