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, andADH 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 mutantabi1 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 increasedRD29A-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. 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, andADH 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 mutantabi1 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 increasedRD29A-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. abscisic acid zeaxanthin epoxidase polyethylene glycol 9-cis-epoxycarotenoid dioxygenase(s) polyethylene glycol wild type Osmotic stress resulting from either high salinity or water deficit induces the expression of numerous stress-responsive genes in plants (1Hasegawa P.M. Bressan R.A. Zhu J.K. Bohnert H.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 463-499Crossref PubMed Scopus (3541) Google Scholar, 2Bray E.A. Plant Physiol. 1993; 103: 1035-1040Crossref PubMed Scopus (660) Google Scholar, 3Ingram J. Bartel D. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 377-403Crossref PubMed Scopus (1757) Google Scholar, 4Shinozaki K. Yamaguchi-Shinozaki K. Plant Physiol. 1997; 115: 327-334Crossref PubMed Scopus (889) Google Scholar, 5Zhu J.K. Hasegawa P.M. Bressan R.A. Crit. Rev. Plant Sci. 1997; 16: 253-277Crossref Scopus (328) Google Scholar). Understanding the mechanisms that regulate the expression of these genes is a fundamental issue in basic plant biology and is instrumental for future genetic improvement of plant productivity under abiotic stresses. Considerable information has been accumulated as a result of molecular studies of gene regulation under osmotic stress (1Hasegawa P.M. Bressan R.A. Zhu J.K. Bohnert H.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 463-499Crossref PubMed Scopus (3541) Google Scholar, 2Bray E.A. Plant Physiol. 1993; 103: 1035-1040Crossref PubMed Scopus (660) Google Scholar, 3Ingram J. Bartel D. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 377-403Crossref PubMed Scopus (1757) Google Scholar, 4Shinozaki K. Yamaguchi-Shinozaki K. Plant Physiol. 1997; 115: 327-334Crossref PubMed Scopus (889) Google Scholar, 5Zhu J.K. Hasegawa P.M. Bressan R.A. Crit. Rev. Plant Sci. 1997; 16: 253-277Crossref Scopus (328) Google Scholar). In contrast, genetic analysis of osmotic signal transduction has been very limited. Because the phytohormone abscisic acid (ABA)1 is known to be involved in plant responses to various environmental stresses, the availability of ABA-deficient mutants (aba) or ABA-insensitive mutants (abi) in Arabidopsis has provided invaluable opportunities to investigate the role of ABA in plant stress responses. Using these mutants, changes in transcript levels of a few stress-responsive genes were analyzed under cold, drought, or salt stress (for reviews, see Refs. 3Ingram J. Bartel D. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 377-403Crossref PubMed Scopus (1757) Google Scholar, 4Shinozaki K. Yamaguchi-Shinozaki K. Plant Physiol. 1997; 115: 327-334Crossref PubMed Scopus (889) Google Scholar, and 6Thomashow M.F. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 571-599Crossref PubMed Scopus (2608) Google Scholar). A general consensus resulting from these studies is that low temperature signaling is less influenced by ABA, whereas drought and salt stress signal transduction has both ABA-dependent and ABA-independent pathways (4Shinozaki K. Yamaguchi-Shinozaki K. Plant Physiol. 1997; 115: 327-334Crossref PubMed Scopus (889) Google Scholar, 6Thomashow M.F. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 571-599Crossref PubMed Scopus (2608) Google Scholar).We have been using a reporter gene approach to dissect osmotic stress signal transduction networks. In this approach, Arabidopsisplants expressing the firefly luciferase gene under the control of the stress- and ABA-responsive RD29A promoter were used to screen for mutants with altered gene regulation by osmotic stress, cold, and/or ABA. In this report, we describe the isolation, cloning, and characterization of a mutation isolated in this screen. This mutation was designated as los6 (for low expression of osmotic stress-responsive genes6). Compared with wild type plants, los6 mutant plants exhibit reduced luminescence induction by osmotic stress.los6 plants were found to be ABA-deficient. Genetic analysis and cloning indicated that LOS6 is allelic toABA1 and encodes a zeaxanthin epoxidase (ZEP). Analysis ofLOS6/ABA1 gene expression reveals a very intriguing phenomenon (i.e. the expression ofLOS6/ABA1 is significantly enhanced by exogenous ABA). Importantly, in los6 mutant plants, osmotic stress up-regulation of LOS6/ABA1 is reduced severalfold, whereas the expression level under ABA treatment is similar to that of the wild type. We found similar regulation of two other ABA biosynthesis genes LOS5/ABA3 andAAO3 by ABA. These observations uncover a positive feedback loop in the regulation of expression of ABA biosynthesis genes. In addition, we found that ABA regulation of ABA biosynthesis genes is partially impaired in the ABA-insensitive mutant abi1 but not in abi2. In addition, we show that overexpression ofLOS6 leads to increased gene induction by osmotic stress. Osmotic stress resulting from either high salinity or water deficit induces the expression of numerous stress-responsive genes in plants (1Hasegawa P.M. Bressan R.A. Zhu J.K. Bohnert H.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 463-499Crossref PubMed Scopus (3541) Google Scholar, 2Bray E.A. Plant Physiol. 1993; 103: 1035-1040Crossref PubMed Scopus (660) Google Scholar, 3Ingram J. Bartel D. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 377-403Crossref PubMed Scopus (1757) Google Scholar, 4Shinozaki K. Yamaguchi-Shinozaki K. Plant Physiol. 1997; 115: 327-334Crossref PubMed Scopus (889) Google Scholar, 5Zhu J.K. Hasegawa P.M. Bressan R.A. Crit. Rev. Plant Sci. 1997; 16: 253-277Crossref Scopus (328) Google Scholar). Understanding the mechanisms that regulate the expression of these genes is a fundamental issue in basic plant biology and is instrumental for future genetic improvement of plant productivity under abiotic stresses. Considerable information has been accumulated as a result of molecular studies of gene regulation under osmotic stress (1Hasegawa P.M. Bressan R.A. Zhu J.K. Bohnert H.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 463-499Crossref PubMed Scopus (3541) Google Scholar, 2Bray E.A. Plant Physiol. 1993; 103: 1035-1040Crossref PubMed Scopus (660) Google Scholar, 3Ingram J. Bartel D. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 377-403Crossref PubMed Scopus (1757) Google Scholar, 4Shinozaki K. Yamaguchi-Shinozaki K. Plant Physiol. 1997; 115: 327-334Crossref PubMed Scopus (889) Google Scholar, 5Zhu J.K. Hasegawa P.M. Bressan R.A. Crit. Rev. Plant Sci. 1997; 16: 253-277Crossref Scopus (328) Google Scholar). In contrast, genetic analysis of osmotic signal transduction has been very limited. Because the phytohormone abscisic acid (ABA)1 is known to be involved in plant responses to various environmental stresses, the availability of ABA-deficient mutants (aba) or ABA-insensitive mutants (abi) in Arabidopsis has provided invaluable opportunities to investigate the role of ABA in plant stress responses. Using these mutants, changes in transcript levels of a few stress-responsive genes were analyzed under cold, drought, or salt stress (for reviews, see Refs. 3Ingram J. Bartel D. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 377-403Crossref PubMed Scopus (1757) Google Scholar, 4Shinozaki K. Yamaguchi-Shinozaki K. Plant Physiol. 1997; 115: 327-334Crossref PubMed Scopus (889) Google Scholar, and 6Thomashow M.F. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 571-599Crossref PubMed Scopus (2608) Google Scholar). A general consensus resulting from these studies is that low temperature signaling is less influenced by ABA, whereas drought and salt stress signal transduction has both ABA-dependent and ABA-independent pathways (4Shinozaki K. Yamaguchi-Shinozaki K. Plant Physiol. 1997; 115: 327-334Crossref PubMed Scopus (889) Google Scholar, 6Thomashow M.F. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 571-599Crossref PubMed Scopus (2608) Google Scholar). We have been using a reporter gene approach to dissect osmotic stress signal transduction networks. In this approach, Arabidopsisplants expressing the firefly luciferase gene under the control of the stress- and ABA-responsive RD29A promoter were used to screen for mutants with altered gene regulation by osmotic stress, cold, and/or ABA. In this report, we describe the isolation, cloning, and characterization of a mutation isolated in this screen. This mutation was designated as los6 (for low expression of osmotic stress-responsive genes6). Compared with wild type plants, los6 mutant plants exhibit reduced luminescence induction by osmotic stress.los6 plants were found to be ABA-deficient. Genetic analysis and cloning indicated that LOS6 is allelic toABA1 and encodes a zeaxanthin epoxidase (ZEP). Analysis ofLOS6/ABA1 gene expression reveals a very intriguing phenomenon (i.e. the expression ofLOS6/ABA1 is significantly enhanced by exogenous ABA). Importantly, in los6 mutant plants, osmotic stress up-regulation of LOS6/ABA1 is reduced severalfold, whereas the expression level under ABA treatment is similar to that of the wild type. We found similar regulation of two other ABA biosynthesis genes LOS5/ABA3 andAAO3 by ABA. These observations uncover a positive feedback loop in the regulation of expression of ABA biosynthesis genes. In addition, we found that ABA regulation of ABA biosynthesis genes is partially impaired in the ABA-insensitive mutant abi1 but not in abi2. In addition, we show that overexpression ofLOS6 leads to increased gene induction by osmotic stress.

Regulation of Osmotic Stress-responsive Gene Expression by theLOS6/ABA1 Locus inArabidopsis