C 2 H 2 -zinc finger proteins that contain the EAR repressor domain are thought to play a key role in modulating the defense response of plants to abiotic stress. Constitutive expression of the C 2 H 2 -EAR zinc finger protein Zat10 in Arabidopsis was found to elevate the expression of reactive oxygen-defense transcripts and to enhance the tolerance of plants to salinity, heat and osmotic stress. Surprisingly, knockout and RNAi mutants of Zat10 were also more tolerant to osmotic and salinity stress. Our results suggest that Zat10 plays a key role as both a positive and a negative regulator of plant defenses.

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 ABAinsensitive 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.Osmotic stress resulting from either high salinity or water deficit induces the expression of numerous stress-responsive genes in plants (1-5). 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 (1-5). 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. 3, 4, and 6). 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 (4, 6).We have been using a reporter gene approach to dissect osmotic stress signal transduction ne...

The Arabidopsis mutation, los2, impairs cold‐responsive gene transcription, acquired freezing tolerance and plant resistance to chilling under certain conditions. LOS2 was isolated through positional cloning and shown to encode an enolase in the glycolytic pathway. In animal cells, enolase has also been known to function as a transcription factor that represses the expression of c‐myc by binding to the c‐myc gene promoter. LOS2 fused to green fluorescent protein is targeted to the nucleus as well as to the cytoplasm. LOS2/enolase protein can bind to the cis‐element of the human c‐myc gene promoter and to the gene promoter of STZ/ZAT10, a zinc finger transcriptional repressor from Arabidopsis. STZ/ZAT10 expression is induced rapidly and transiently by cold in the wild type, and this induction is stronger and more sustained in the los2 mutant. Furthermore, the expression of a RD29A‐LUC reporter gene is repressed significantly by STZ/ZAT10 in transient expression assays in Arabidopsis leaves. Our results demonstrate that cold‐responsive gene transcription in plants is controlled by a bi‐functional enolase.

An Arabidopsis thaliana mutant, cryophyte, was isolated and found to have an enhanced cold stress-induction of the master regulator of cold tolerance, C-repeat binding factor 2 (CBF2), and its downstream target genes. The mutant is more tolerant to chilling and freezing stresses but is more sensitive to heat stress. Under warm but not cold growth temperatures, the mutant has a reduced stature and flowers earlier. Under long day conditions, flowering of the mutant is insensitive to vernalization. The mutant is also hypersensitive to the phytohormone abscisic acid. The mutation was found in a DEAD box RNA helicase gene that is identical to the previously identified low expression of osmotically responsive genes 4 (LOS4) locus, which was defined by the los4-1 mutation that reduces cold regulation of CBFs and their target genes and renders Arabidopsis plants chilling sensitive. We show evidence suggesting that the CRYOPHYTE/LOS4 protein may be enriched in the nuclear rim. In situ poly(A) hybridization indicates that the export of poly(A) þ RNAs is blocked in the cryophyte/los4-2 mutant at warm or high temperatures but not at low temperatures, whereas the los4-1 mutation weakens mRNA export at both low and warm temperatures. These results demonstrate an important role of the CRYOPHYTE/LOS4 RNA helicase in mRNA export, plant development, and stress responses.

To study low-temperature signaling in plants, we previously screened for cold stress response mutants using bioluminescent Arabidopsis plants that express the firefly luciferase reporter gene driven by the stress-responsive RD29A promoter. Here, we report on the characterization and cloning of one mutant, frostbite1 ( fro1 ), which shows reduced luminescence induction by cold. fro1 plants display reduced cold induction of stress-responsive genes such as RD29A , KIN1 , COR15A , and COR47 . fro1 leaves have a reduced capacity for cold acclimation, appear water-soaked, leak electrolytes, and accumulate reactive oxygen species constitutively. FRO1 was isolated through positional cloning and found to encode a protein with high similarity to the 18-kD Fe-S subunit of complex I (NADH dehydrogenase, EC 1.6.5.3) in the mitochondrial electron transfer chain. Confocal imaging shows that the FRO1:green fluorescent protein fusion protein is localized in mitochondria. These results suggest that cold induction of nuclear gene expression is modulated by mitochondrial function.