Figure 6.

Molecular and Biochemical Analysis of Transgenic Lines Expressing the Dex-Inducible AtPLC1 Sense Transgene.

(A) Dex induction of the AtPLC1 transgenic transcript in AtPLC1 sense lines and in a vector control transgenic line. Plants were collected at 3, 6, and 20 hr after treatment with Dex (30 μM), and the expression of PLC1, KIN2, RD29a, and RD22 probes and 18S rDNA was examined by RNA gel blot hybridization.

(B) Transgenic lines containing the AtPLC1 sense transgene were grown on plates for 2 weeks, and the seedlings were transferred to a hydroponic medium (Aoyama and Chua, 1997). After 2 days, the medium was replaced with fresh medium containing either Dex (30 μM) or ABA (50 μM) or both. Total RNA was isolated at various times (0, 4, and 24 hr), and the gel blots were hybridized with RD29a and KIN2 probes and 18S rDNA. Vector control transgenic lines contained the pTA211 plasmid alone. For details, see Methods. ABA-0, plants collected at 0 hr in the ABA medium; ABA-4, plants incubated with ABA (50 μM) for 4 hr; DEX-24, plants incubated with Dex (30 μM) for 24 hr; DEX-24/ABA-4, plants incubated with Dex (30 μM) for 24 hr followed by an additional 4 hr with ABA (50 μM).

(C) and (D) Seedlings of a transgenic line containing the inducible AtPLC1 sense transgene were grown and treated with Dex as described in (B), except that plants were collected at 0, 3, and 6 hr after treatment. Total protein extracts were assayed for PLC1 enzyme activity (C), and the amount of Ins(1,4,5)P₃ was determined (D). Each bar represents the average value of three independent induction experiments with standard deviations. For details, see Methods. Solid bars, transgenic line with the inducible AtPLC1 sense gene; open bars, vector control transgenic line. PIP₂, phosphatidylinositol 4, 5 bisphosphate.