Characterization of abiotic stress-responsive <Emphasis Type="Italic">Arabidopsis thaliana RD29A</Emphasis> and <Emphasis Type="Italic">RD29B</Emphasis> genes and evaluation of transgenes

Abiotic stresses have adverse effects on plant growth and productivity. The homologous RD29A and RD29B genes are exquisitely sensitive to various abiotic stressors. Therefore, RD29A and RD29B gene sequences have potential to confer abiotic stress resistance in crop species grown in arid and semi-arid regions. To our knowledge, no information on the physiological roles of the proteins encoded by RD29A and RD29B are available in the literature. To understand how these proteins function, we used reverse genetic approaches, including identifying rd29a and rd29b T-DNA knockout mutants, and examining the effects of complementing transgenes with the genes under control of their native promoters and chimeric genes with the native promoters swapped. Four binary vectors with the RD29A and RD29B promoters upstream of the cognate RD29A and RD29B cDNAs and as chimeric genes with noncognate promoters were used to transform rd29a and rd29b plants. Cold, drought, and salt induced both genes; the promoter of RD29A was found to be more responsive to drought and cold stresses, whereas the promoter of RD29B was highly responsive to salt stress. Morphological and physiological responses of rd29a and rd29b plants to salt stress were further investigated. Root growth, and photosynthetic properties declined significantly, while solute concentration (Ψπ), water use efficiency (WUE) and δ¹³C ratio increased under salt stress. Unexpectedly, the rd29a and rd29b knockout mutant lines maintained greater root growth, photosynthesis, and WUE under salt stress relative to control. We conclude that the RD29A and RD29B proteins are unlikely to serve directly as protective molecules.     

Interaction between two cis-acting elements,ABRE and DRE,in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses

Many abiotic stress-inducible genes contain two cis-acting elements, namely a dehydration-responsive element (DRE; TACCGACAT) and an ABA-responsive element (ABRE; ACGTGG/TC), in their promoter regions. We precisely analyzed the 120 bp promoter region (-174 to -55) of the Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments and whose 120 bp promoter region contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences. Deletion and base substitution analyses of this region showed that the DRE-core motif functions as DRE and that the DRE/DRE-core motif could be a coupling element of ABRE. Gel mobility shift assays revealed that DRE-binding proteins (DREB1s/CBFs and DREB2s) bind to both DRE and the DRE-core motif and that ABRE-binding proteins (AREBs/ABFs) bind to ABRE in the 120 bp promoter region. In addition, transactivation experiments using Arabidopsis leaf protoplasts showed that DREBs and AREBs cumulatively transactivate the expression of a GUS reporter gene fused to the 120 bp promoter region of rd29A. These results indicate that DRE and ABRE are interdependent in the ABA-responsive expression of the rd29A gene in response to ABA in Arabidopsis.     

Arabidopsis EIN2 modulates stress response through abscisic acid response pathway

The nuclear protein ETHYLENE INSENSITIVE2 (EIN2) is a central component of the ethylene signal transduction pathway in plants, and plays an important role in mediating cross-links between several hormone response pathways, including abscisic acid (ABA). ABA mediates stress responses in plants, but there is no report on the role of EIN2 on plant response to salt and osmotic stresses. Here, we show that EIN2 gene regulates plant response to osmotic and salt stress through an ABA-dependent pathway in Arabidopsis. The expression of the EIN2 gene is down-regulated by salt and osmotic stress. An Arabidopsis EIN2 null mutant was supersensitive to both salt and osmotic stress conditions. Disruption of EIN2 specifically altered the expression pattern of stress marker gene RD29B in response to the stresses, but not the stress- or ABA-responsive genes RD29A and RD22, suggesting EIN2 modulates plant stress responses through the RD29B branch of the ABA response. Furthermore, disruption of EIN2 caused substantial increase in ABA. Lastly, our data showed that mutations of other key genes in ethylene pathway also had altered sensitivity to abiotic stresses, indicating that the intact ethylene may involve in the stress response. Taken together, the results identified EIN2 as a cross-link node in ethylene, ABA and stress signaling pathways, and EIN2 is necessary to induce developmental arrest during seed germination, and seedling establishment, as well as subsequent vegetative growth, thereby allowing the survival and growth of plants under the adverse environmental conditions. Youning Wang and Chuang Liu contributed equally to this work.     

Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants

Summary We characterized the expression of genes that correspond to a cDNA clone, RD29, which is induced by desiccation, cold and high-salt conditions in Arabidopsis thaliana. Northern analysis of desiccation-induced expression revealed a two-step induction process. Early induction occurs within 20 min and secondary induction occurs 3 h after the start of desiccation. Exogenous abscisic acid (ABA) induces RD29 mRNA within 3 h. Two genes corresponding to RD29, rd29A and rd29B, are located in tandem in an 8 kb region of the Arabidopsis genome and encode hydrophilic proteins. Desiccation induces rd29A mRNA with two-step kinetics, while rd29B is induced only 3 h after the start of desiccation. The expression of both genes is stimulated about 3 h after application of ABA. It appears that rd29A has at least two cis-acting elements, one involved in the ABA-associated response to desiccation and the other induced by changes in osmotic potential. The -glucuronidase (GUS) reporter gene driven by the rd29A promoter was induced at significant levels by desiccation, cold, high-salt conditions and ABA in both transgenic Arabidopsis and tobacco. Histochemical analysis of GUS activity revealed that the rd29A promoter functions in almost all the organs and tissues of vegetative plants during water deficiency.     

Interaction of Osmotic Stress, Temperature, and Abscisic Acid in the Regulation of Gene Expression in Arabidopsis

The impact of simultaneous environmental stresses on plants and how they respond to combined stresses compared with single stresses is largely unclear. By using a transgene (RD29A-LUC) consisting of the firefly luciferase coding sequence (LUC) driven by the stress-responsive RD29A promoter, we investigated the interactive effects of temperature, osmotic stress, and the phytohormone abscisic acid (ABA) in the regulation of gene expression in Arabidopsis seedlings. Results indicated that both positive and negative interactions exist among the studied stress factors in regulating gene expression. At a normal growth temperature (22°C), osmotic stress and ABA act synergistically to induce the transgene expression. Low temperature inhibits the response to osmotic stress or to combined treatment of osmotic stress and ABA, whereas low temperature and ABA treatments are additive in inducing transgene expression. Although high temperature alone does not activate the transgene, it significantly amplifies the effects of ABA and osmotic stress. The effect of multiple stresses in the regulation of RD29A-LUC expression in signal transduction mutants was also studied. The results are discussed in the context of cold and osmotic stress signal transduction pathways.     

Isolation and characterization of two <Emphasis Type="Italic">DREB1</Emphasis> genes encoding dehydration-responsive element binding proteins in chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis>)

Two novel DREB (dehydration-responsive element-binding protein) genes, designated as CiDREB1A and CiDREB1B, were cloned from chicory (Cichorium intybus). Both of these genes contained a conserved EREBP/AP2 domain and were classified into the A-1 subgroup of the DREB subfamily based on phylogenetic analysis. Quantitative real-time PCR analysis revealed that low temperature, but not ABA, greatly induced the expression of both CiDREB1 genes, suggesting that these genes are involved in ABA-independent stress signaling pathways. A subcellular localization assay revealed that both CiDREBs localized to the nucleus. In addition, we showed by yeast one-hybrid analysis that these two CiDREB proteins bind to the DRE motif of RD19A. These results suggest that CiDREB1A and CiDREB1B are important regulators of stress-responsive signaling in chicory.     

A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress.

Two genes, rd29A and rd29B, which are closely located on the Arabidopsis genome, are differentially induced under conditions of dehydration, low temperature, high salt, or treatment with exogenous abscisic acid (ABA). It appears that rd29A has at least two cis-acting elements, one involved in the ABA-associated response to dehydration and the other induced by changes in osmotic potential, and that rd29B contains at least one cis-acting element that is involved in ABA-responsive, slow induction. We analyzed the rd29A promoter in both transgenic Arabidopsis and tobacco and identified a novel cis-acting, dehydration-responsive element (DRE) containing 9 bp, TACCGACAT, that is involved in the first rapid response of rd29A to conditions of dehydration or high salt. DRE is also involved in the induction by low temperature but does not function in the ABA-responsive, slow expression of rd29A. Nuclear proteins that specifically bind to DRE were detected in Arabidopsis plants under either high-salt or normal conditions. Different cis-acting elements seem to function in the two-step induction of rd29A and in the slow induction of rd29B under conditions of dehydration, high salt, or low temperature.     

Overexpression of two chrysanthemum DgDREB1 group genes causing delayed flowering or dwarfism in Arabidopsis

We isolated 13 DREB1 (dehydration responsive element binding factor 1) genes from chrysanthemum and further divided them into three groups, DgDREB1A, DgDREB1B and DgDREB1C, based on the phylogenetic analysis. Each group showed their unique expression patterns under cold, dehydration and salt stress conditions. Arabidopsis plants overexpressing DgDREB1A (1A plants) exhibited significantly stronger tolerance to freezing and drought than those overexpressing DgDREB1B (1B plants) and the control plants. In addition, 1A plants showed delayed flowering, but not dwarfism; while 1B plants showed dwarfism, but not delayed flowering. In 1A plants, the expression of three stress-related DREB1-downstream genes, COR47, COR15A, and RD29A, was strongly induced while the expression of CO and FT, two photoperiod responsive flowering-time genes, was inhibited. In 1B plants, the expression of GA2ox7, a GA-deactivation enzyme gene, was dramatically enhanced. The results above strongly suggest that members from different DgDREB1 groups may have distinct effects on plant development: DgDREB1A may be involved in photoperiod-related flowering-time determination and DgDREB1B in GA-mediated plant development.