Abstract
Drought and high salinity induce the expression of many plant genes. To understand the signal transduction mechanisms underlying the activation of these genes, we carried out a genetic screen to isolate Arabidopsis mutants defective in osmotic stress-regulated gene induction. Here we report the isolation, characterization, and cloning of a mutation, los6, which diminished osmotic stress activation of a reporter gene. RNA blot analysis indicates that under osmotic stress the transcript levels for stress-responsive genes such as RD29A, COR15A, KIN1, COR47, RD19, and ADH are lower in los6 plants than in wild type plants. los6 plants were found to have reduced phytohormone abscisic acid (ABA) accumulation and to be allelic to the ABA-deficient mutant, aba1. LOS6/ABA1 encodes a zeaxanthin epoxidase that functions in ABA biosynthesis. Its expression is enhanced by osmotic stress. Furthermore, we found that there exists a positive feedback regulation by ABA on the expression of LOS6/ABA1, which may underscore a quick adaptation strategy for plants under osmotic stress. Similar positive regulation by ABA also exists for other ABA biosynthesis genes AAO3 and LOS5/ABA3 and in certain genetic backgrounds, NCED3. This feedback regulation by ABA is impaired in the ABA-insensitive mutant abi1 but not in abi2. Moreover, the up-regulation of LOS6/ABA1, LOS5/ABA3, AAO3, and NCED3 by osmotic stress is reduced substantially in ABA-deficient mutants. Transgenic plants overexpressing LOS6/ABA1 showed an increased RD29A-LUC expression under osmotic stress. These results suggest that the level of gene induction by osmotic stress is dependent on the dosage of the zeaxanthin epoxidase enzyme.
Highlights
We have been using a reporter gene approach to dissect osmotic stress signal transduction networks
RNA Analysis—Total RNA was isolated from 10-day-old los6 and wild type plants growing in the same MS agar plates either untreated or treated with cold (0 °C for the indicated time), abscisic acid (ABA), or NaCl/polyethylene glycol (PEG)
LOS6 Encodes an Enzyme Functioning in ABA Biosynthesis—In the present study, we characterized and cloned an Arabidopsis mutation, los6, which was identified by virtue of its reduced osmotic stress induction of the RD29A-LUC reporter gene
Summary
Plant Materials and Stress Treatments—Arabidopsis plants (ecotype C24) expressing the RD29A-LUC transgene were obtained as previously described [7]. RNA Analysis—Total RNA was isolated from 10-day-old los and wild type plants growing in the same MS agar plates either untreated (unstressed controls) or treated with cold (0 °C for the indicated time), ABA (sprayed with 100 M ABA and incubated under light for 5 h), or NaCl/polyethylene glycol (PEG) (transferred onto filter paper saturated with 300 mM NaCl or 30% polyethylene glycol with a molecular weight of 6000 and treated for 5 h). Genetic Analysis, Cloning, and Overexpression Assay—The los mutants were back-crossed to the wild type RD29A-LUC plants and the F1 seedlings were tested for luminescence expression in response to stress or ABA treatment as described above. T3 generation of transgenic lines were used to test the luminescence responsiveness
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