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American Society of Plant Biologists
Luc Genetic Screen Illuminates Stress-Responsive Gene Regulationneckardt{at}aspb.org A typical genetic screen for identifying loci associated with plant responses to stress involves searching for mutants that are hypersensitive and/or hyperresistant to a particular stress, on the basis of visual appearance of seedlings compared with the wild-type response. This type of screen has successfully identified numerous genetic loci that are important components of plant respon-ses to biotic and abiotic stresses. However, this approach has limitations. For example, acute stress conditions often are required for a visible phenotype to be manifested, and such conditions may continually fail to generate mutants in important components of stress response pathways. In other cases, visible phenotypes may be completely absent. Because of the complexity of stress response signaling networks, new and innovative types of screens may be necessary to identify all of the components.
Reporter gene screens typically make use of a reporter gene, such as firefly luciferase (LUC) or  -glucuronidase (GUS), fused to the promoter of a specifically regulated or inducible gene. This type of genetic screen (in use since the late 1980s and early 1990s) represents an important advance in genetic technologies; not only does it create a highly visible phenotypic screen, it also specifically targets genes that encode regulatory and/or signal transduction components that may play key roles in the regulation of plant responses to stress. The Arabidopsis npr1 (nonexpressor of pathogenesis-related genes) mutant provides an example of the importance of this type of screen in plant genetics. To isolate the npr1 mutant, Cao et al. (1994) made use of transgenic Arabidopsis expressing GUS under the control of the Arabidopsis -1,3-glucanase (BGL2) promoter. BGL2 is a pathogenesis-related gene that is regulated by salicylic acid and is a marker gene for the induction of systemic acquired resistance; thus, plants expressing BLG2-GUS provided a visible phenotype for mutants impaired in the induction of systemic acquired resistance after pathogen attack. Although npr1 alleles also were identified via more conventional genetic screens, the npr1 mutation yielded important information regarding the key role of NPR1 in salicylic acid signaling and the induction of systemic acquired resistance (Cao et al., 1997; Kinkema et al., 2000).
Osmotic stress is one area that has yielded a relative paucity of mutations in traditional genetic screens. Ishitani et al. (1997)
In this issue of The Plant Cell, Xiong et al. (pages 2063–2083) characterize two allelic Arabidopsis RD29A-LUC mutants, los5-1 and los5-2, that exhibit greatly reduced RD29A-LUC gene expression in response to dehydration, high salt, or low temperature. Gene expression analysis showed that stress induction of the endogenous RD29A gene and several other known stress response genes (COR15A, KIN1, P5CS, RD22, and COR47) was inhibited completely or partially by the los5 mutation, and importantly, the mutant plants also were found to have reduced tolerance to drought, salt stress, and freezing. The los5 mutants were found to be deficient in abscisic acid (ABA), and subsequently the mutation was shown to be allelic to aba3.
The LOS5/ABA3 gene was cloned via map-based cloning and was found to encode an enzyme that functions indirectly in ABA biosynthesis. This enzyme, molybdopterin cofactor sulfurylase, catalyzes the production of sulfurylated molybdopterin cofactor required by aldehyde oxidase (which functions in the last step of ABA biosynthesis). Schwartz et al. (1997)
An important feature of the RD29A-LUC screen in this case is that it led to the identification of hitherto unrecognized characteristics of a previously identified locus. aba3 was identified by Léon-Kloosterziel et al. (1996)
ABA is involved in the regulation of many aspects of plant growth and development, including seed dormancy, embryogenesis, root and shoot development, and stomatal function. ABA also is an important regulatory component of plant responses to abiotic stresses such as drought, salinity, and, possibly, low temperature.
ABA biosynthesis in plants branches off of the xanthophyll cycle. One of the key steps is cleavage of 9-cis-neoxanthin to yield xanthoxin, which is catalyzed in maize by the VP14 gene product. VP14 homologs in various species are induced by drought and other abiotic stresses, and this gene is thought to represent a key regulatory step in ABA biosynthesis (Taylor et al., 2000
ABA is thought to have a significant role in plant responses to drought and osmotic stress. In addition to its direct effect on stomatal closure, ABA also has a role in the regulation of expression of drought and desiccation tolerance genes in seed and vegetative tissues. ABA-deficient and ABA-insensitive mutants also are marked by a characteristic dehydration-sensitive "wilty" phenotype. ABA also may play a role in cold acclimation and the development of freezing tolerance, although the importance of this role is somewhat controversial. Thomashow (1999)
Several genes that are induced by drought, salinity, or cold temperature are not induced in response to exogenous ABA treatment, suggesting that there are ABA-independent as well as ABA-dependent stress response pathways. In addition, some genes that are ABA responsive, such as RD29A, KIN1, and COR47, also are induced by certain stress conditions in ABA-deficient or ABA-insensitive mutants. Yamaguchi-Shinozaki and Shinozaki (1994)
There is growing evidence that the ABA-dependent and ABA-independent pathways are interconnected in a complex and subtle manner. Ishitani et al. (1997) presented evidence for such interconnection when they identified los and hos mutants that showed reduced or enhanced RD29A-LUC expression, respectively, in response to ABA treatment and osmotic stress as well as low temperature stress conditions. The results of Xiong et al. provide further evidence for the idea that there is communication between ABA-dependent and ABA-independent stress-responsive signal transduction pathways. For example, it is perhaps surprising that a mutation in the ABA biosynthetic pathway would completely inhibit the osmotic stress induction of RD/COR/KIN genes, because previous research has shown that osmotic stress induction of these genes is controlled by both ABA-dependent and ABA-independent pathways. In an ABA-deficient mutant such as los5/aba3, we might expect to see some degree of osmotic stress induction of these genes because the ABA-independent pathway should not be affected. This is the case for other ABA-deficient and ABA-insensitive mutants, such as various aba1 and abi1 alleles that have been examined.
Xiong et al. (1999a) An interesting characteristic of the los5/aba3 mutation is that it appears to affect low temperature gene regulation independently of ABA. In the case of other ABA-deficient mutants, such as los6/aba1, exogenous application of ABA restores a wild-type phenotype in response to cold or other stress conditions. Xiong et al. suggest that cold signaling requires a function of LOS5/ABA3 that is not related directly to ABA biosynthesis, although it is not clear what this function might be.
One of the RD29A-LUC mutants isolated in the screen devised by Ishitani et al. (1997) showed superinduction of RD29A-LUC expression and was named fiery1 (fry1) because of the high level of LUC activity induced in response to dehydration, osmotic stress, exogenous ABA, and low temperature (Xiong et al., 2001b). In contrast, the hos1 and hos2 mutations affected only the cold stress response (Ishitani et al., 1998; Lee et al., 1999, 2001), and the hos5 mutation affected only the osmotic stress response (Xiong et al., 1999b). Interestingly, although the fry1 mutants showed superinduction of ABA- and stress-inducible gene expression, they were compromised in their tolerance to freezing, drought, and salt stress. FRY1 was found to encode an inositol polyphosphate 1-phosphatase, suggesting a role for phosphoinositols in ABA and stress signaling (Xiong et al., 2001b). Because the fry1 mutation affects ABA, osmotic stress, and cold stress–responsive signaling, it raises the interesting possibility that phosphoinositol signaling provides the link between ABA-dependent and ABA-independent pathways. Plant stress responses involve a remarkable array of signal transduction pathways that must be integrated successfully to allow for the normal growth and development of an organism that cannot move to escape predators or adverse environmental conditions. Innovative genetic screens, like the RD29A-LUC reporter screen, are helping to unravel the complex interactions that make up plant stress signaling networks.
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Ishitani, M., Xiong, L., Lee, H., Stevenson, B., and Zhu, J.-K. (1998). HOS1, a genetic locus involved in cold-responsive gene expression in Arabidopsis. Plant Cell 10, 1151–1161.
Kinkema, M., Fan, W., and Dong, X. (2000). Nuclear localization of NPR1 is required for activation of PR gene expression. Plant Cell 12, 2339–2350. Lee, H., Xiong, L., Ishitani, M., Stevenson, B., and Zhu, J.K. (1999). Cold-regulated gene expression and freezing tolerance in an Arabidopsis thaliana mutant. Plant J. 17, 301–308.[CrossRef][Web of Science][Medline]
Lee, H., Xiong, L., Gong, Z., Ishitani, M., Stevenson, B., and Zhu, J.-K. (2001). The Arabidopsis HOS1 gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo-cytoplasmic partitioning. Genes Dev. 15, 912–924. Léon-Kloosterziel, K.M., Gil, M.A., Ruijs, G.J., Jacobsen, S.E., Olszewski, N.E., Schwartz, S.H., Zeevaart, J.A.D., and Koornneef, M. (1996). Isolation and characterization of abscisic acid-deficient Arabidopsis mutants at two new loci. Plant J. 10, 655–661.[CrossRef][Web of Science][Medline] Llorente, F., Oliveros, J.C., Martínez-Zapater, J.M., and Salinas, J. (2000). A freezing-sensitive mutant of Arabidopsis, frs1, is a new aba3 allele. Planta 211, 648–655.[CrossRef][Web of Science][Medline] Marin, E., Nussaume, L., Quesada, A., Gonneau, M., Sotta, B., Hugueney, P., Frey, A., and Marion-Poll, A. (1996). Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana. EMBO J. 15, 2331–2342.[Web of Science][Medline] Schwartz, S.H., Léon-Kloosterziel, K.M., Koornneef, M., and Zeevaart, J.A.D. (1997). Biochemical characterization of the aba2 and aba3 mutants in Arabidopsis thaliana. Plant Physiol. 114, 161–166.[Abstract] Seo, M., Koiwai, H., Akaba, S., Komano, T., Oritani, T., Kamiya, Y., and Koshiba, T. (2000). Abscisic aldehyde oxidase in leaves of Arabidopsis thaliana. Plant J. 23, 481–488.[CrossRef][Web of Science][Medline]
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Xiong, L., Ishitani, M., and Zhu, J.-K. (1999a). Interaction of osmotic stress, temperature, and abscisic acid in the regulation of gene expression in Arabidopsis. Plant Physiol. 119, 205–211. Xiong, L., Ishitani, M., Lee, H., and Zhu, J.-K. (1999b). HOS5: A negative regulator of osmotic stress-induced gene expression in Arabidopsis thaliana. Plant J. 19, 569–578.[CrossRef][Web of Science][Medline]
Xiong, L., Ishitani, M., Lee, H., and Zhu, J.-K. (2001a). The Arabidopsis LOS5/ABA3 locus encodes a molybdenum cofactor sulfurase and modulates cold stress– and osmotic stress–responsive gene expression. Plant Cell 13, 2063–2083. Xiong, L., Lee, B., Ishitani, M., Lee, H., Zhang, C., and Zhu, J.-K. (2001b). FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis. Genes Dev. 15, in press. Yamaguchi-Shinozaki, K., and Shinozaki, K. (1994). A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6, 251–264.[Abstract] This article has been cited by other articles:
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REPORTER GENE SCREENS
los5 IS ABSCISIC ACID...
