Response Element-binding Factor Research Articles

Comparison of cytotoxicity and expression of metal regulatory genes in zebrafish (Danio rerio) liver cells exposed to cadmium sulfate, zinc sulfate and quantum dots

Recent advances in the ability to manufacture and manipulate materials at the nanometer scale have led to increased production and use of many types of nanoparticles. Quantum dots (QDs) are small, fluorescent nanoparticles composed of a core of semiconductor material (e.g. cadmium selenide, zinc sulfide) and shells or dopants of other elements. Particle core composition, size, shell, and surface chemistry have all been found to influence toxicity in cells. The aim of this study was to compare the toxicities of ionic cadmium (Cd) and zinc (Zn) and Cd- and Zn-containing QDs in zebrafish liver cells (ZFL). As expected, Cd(2+) was more toxic than Zn(2+), and the general trend of IC50-24 h values of QDs was determined to be CdTe < CdSe/ZnS or InP/ZnS, suggesting that ZnS-shelled CdSe/ZnS QDs were more cytocompatible than bare core CdTe crystals. Smaller QDs showed greater toxicity than larger QDs. Isolated mRNA from these exposures was used to measure the expression of metal response genes including metallothionein (MT), metal response element-binding transcription factor (MTF-1), divalent metal transporter (DMT-1), zrt and irt like protein (ZIP-1) and the zinc transporter, ZnT-1. CdTe exposure induced expression of these genes in a dose dependent manner similar to that of CdSO4 exposure. However, CdSe/ZnS and InP/ZnS altered gene expression of metal homeostasis genes in a manner different from that of the corresponding Cd or Zn salts. This implies that ZnS shells reduce QD toxicity attributed to the release of Cd(2+), but do not eliminate toxic effects caused by the nanoparticles themselves.

Integrated copy number and gene expression analysis detects a CREB1 association with Alzheimer’s disease

Genetic variation, both single-nucleotide variations and copy number variations (CNV), contribute to changes in gene expression. In some cases these variations are meaningfully correlated with disease states. We hypothesized that in a genetically heterogeneous disorder such as sporadic Alzheimer's disease (AD), utilizing gene expression as a quantitative trait and CNVs as a genetic marker map within the same individuals in the context of case–control status may increase the power to detect relevant loci. Using this approach an 8-kb deletion was identified that contains a PAX6-binding site on chr2q33.3 upstream of CREB1 encoding the cAMP responsive element-binding protein1 transcription factor. The association of the CNV to AD was confirmed by a case–control association study consisting of the Texas Alzheimer Research and Care Consortium and NIA-LOAD Family Study data sets.

Expression of the α-tocopherol transfer protein gene is regulated by oxidative stress and common single-nucleotide polymorphisms

Vitamin E (α-tocopherol) is the major lipid-soluble antioxidant in most animal species. By controlling the secretion of vitamin E from the liver, the α-tocopherol transfer protein regulates whole-body distribution and levels of this vital nutrient. However, the mechanism(s) that regulates the expression of this protein is poorly understood. Here we report that transcription of the TTPA gene in immortalized human hepatocytes is induced by oxidative stress and by hypoxia, by agonists of the nuclear receptors PPARα and RXR, and by increased cAMP levels. The data show further that induction of TTPA transcription by oxidative stress is mediated by an already-present transcription factor and does not require de novo protein synthesis. Silencing of the cAMP response element-binding (CREB) transcription factor attenuated transcriptional responses of the TTPA gene to added peroxide, suggesting that CREB mediates responses of this gene to oxidative stress. Using a 1.9-kb proximal segment of the human TTPA promoter together with a site-directed mutagenesis approach, we found that single-nucleotide polymorphisms that are commonly found in healthy humans dramatically affect promoter activity. These observations suggest that oxidative stress and individual genetic makeup contribute to vitamin E homeostasis in humans. These findings may explain the variable responses to vitamin E supplementation observed in human clinical trials.

Abstract 401: Prostaglandin E2 Stimulates Renin Synthesis in Mouse Collecting Duct M-1 Cells via Ep1 Receptor Through Pkc/camp/creb Pathway

Prostaglandin E2 (PGE2) plays a major role in regulating renin expression and release by the renal juxtaglomerular (JG) cells. Recently it has been demonstrated that PGE2-dependent upregulation of renin in JG cells is mediated by activation of E prostaoind receptor type 4 (EP4) via cAMP accumulation. Renin is also produced by the principal cells of the collecting ducts (CD) and is upregulated during angiotensin II-dependent hypertension. However, the effects of PGE2 on CD renin remain unknown. Four types of receptors have been described in rat and mouse CD, EP1, EP3 and EP4. Here, we tested the hypothesis that renin is upregulated by PGE2 via activation of EP receptors in mouse CD M-1 cells. By immunostaining we confirmed the presence of EP1, EP3 and EP4 receptors, while EP2 was not detected. A dose response treatment with PGE2 showed increased levels of renin mRNA and protein with a maximum response at 1 μmol/L (mRNA: 19.3 ± 3.0; P&lt;0.05; protein: 3.01 ± 0.08 fold change; P&lt;0.05). To assess which EP receptor is involved in the renin upregulation we used specific EP receptor antagonists: ONO-8711 (EP1; 10 nmol/L), L-798106 (EP3; 10 μmol/L) and AH 23848 (EP4; 10 μmol/L). EP1 antagonist suppressed the PGE2-mediated upregulation of collecting duct renin mRNA and protein (mRNA: 1.0 ± 0.2; protein: 0.98 ± 0.13 fold change; P=NS), while EP4 antagonist only partially decreased it (mRNA: 11.2 ± 2.8; P&lt;0.05; protein: 2.81 ± 0.07 fold change; P&lt;0.05). EP3 antagonist exacerbated the PGE2 mediated-upregulation of renin (mRNA: 50.3 ± 6.0; P&lt;0.05; protein: 3.56 ± 0.08; fold change; P&lt;0.05). Because EP1 is a Gq linked receptor that activates PKC, we further assessed the effects of PKC inhibition using calphostin C and a PKCα dominant negative (DN) on renin expression. Calphostin C and PKCα-DN blunted the PGE2-induced renin upregulation. Importantly, the increases in cAMP levels and phosphorylation of the cAMP response element-binding transcription factor (CREB) mediated by PGE2 were also prevented by both treatments. The results indicate that in mouse CD cells, EP1 receptor activation upregulates renin synthesis via PKC/cAMP/CREB, suggesting that the presence of PGE2 in renal medullary tissues may contribute to the stimulation of collecting duct renin.

A Proximal Promoter Region of Arabidopsis DREB2C Confers Tissue‐specific Expression under Heat Stress

The dehydration-responsive element-binding factor 2C (DREB2C) is a member of the CBF/DREB subfamily of proteins, which contains a single APETALA2/Ethylene responsive element-binding factor (AP2/ERF) domain. To identify the expression pattern of the DREB2C gene, which contains multiple transcription cis-regulatory elements in its promoter, an approximately 1.4 kb upstream DREB2C sequence was fused to the β-glucuronidase reporter gene (GUS) and the recombinant p1244 construct was transformed into Arabidopsis thaliana (L.) Heynh. The promoter of the gene directed prominent GUS activity in the vasculature in diverse young dividing tissues. Upon applying heat stress (HS), GUS staining was also enhanced in the vasculature of the growing tissues. Analysis of a series of 5'-deletions of the DREB2C promoter revealed that a proximal upstream sequence sufficient for the tissue-specific spatial and temporal induction of GUS expression by HS is localized in the promoter region between -204 and -34 bps relative to the transcriptional start site. Furthermore, electrophoretic mobility shift assay (EMSA) demonstrated that nuclear protein binding activities specific to a -120 to -32 bp promoter fragment increased after HS. These results indicate that the TATA-proximal region and some latent trans-acting factors may cooperate in HS-induced activation of the Arabidopsis DREB2C promoter.

Overexpression of Arabidopsis ABF3 gene confers enhanced tolerance to drought and heat stress in creeping bentgrass

The Arabidopsis, abscisic acid responsive element-binding factor 3, ABF3 is known to play an important role in stress responses via regulating the expression of stress-responsive genes. In this study, we introduced pCAMBIA3301 vector harboring the ABF3 gene into creeping bentgrass (Agrostis stolonifera) through Agrobacterium-mediated transformation in order to develop a stress-tolerant variety of turfgrass. After transformation, putative transgenic plants were selected using the herbicide resistance assay. Genomic integration of the transgene was confirmed by genomic PCR and Southern blot analysis, and gene expression was validated by northern blot analysis. Under drought-stressed condition, the transgenic plants overexpressing ABF3 displayed significantly enhanced drought tolerance with higher water content and slower water loss rate than the control plants. Furthermore, the stomata of the ABF3 transgenic plants closed more than those of wild-type creeping bentgrass plants, under both non-stressed and ABA treatment conditions. In addition, the transgenic plants showed enhanced tolerance to heat stress. These results suggest that the overexpression of the ABF3 gene in creeping bentgrass might enhance survival in water-limiting and high temperature environments through increased stomatal closure and reduced water losses.

P4‐114: Integrated copy number and gene expression analysis detects a CREB1 association with Alzheimer's disease

Genetic variation, both single-nucleotide variations and copy number variations (CNV), contribute to changes in gene expression. In some cases these variations are meaningfully correlated with disease states. We hypothesized that in a genetically heterogeneous disorder such as sporadic Alzheimer’s disease (AD), utilizing gene expression as a quantitative trait and CNVs as a genetic marker map within the same individuals in the context of case–control status may increase the power to detect relevant loci. Using this approach an 8-kb deletion was identified that contains a PAX6-binding site on chr2q33.3 upstream of CREB1 encoding the cAMP responsive element-binding protein1 transcription factor. The association of the CNV to AD was confirmed by a case–control association study consisting of the Texas Alzheimer Research and Care Consortium and NIA-LOAD Family Study data sets. Translational Psychiatry (2012) 2, e192; doi:10.1038/tp.2012.119; published online 20 November 2012

Isolation and characterization of a dehydration responsive element binding factor MsDREBA5 in Malus sieversii Roem.

A dehydration responsive element binding (DREB) protein homologous gene, designated MsDREBA5 (GenBank ID: JQ655768), was isolated from Malus sieversii Roem. Based on similarity with AP2 domains, MsDREBA5 was cladeded into the A-5 subgroup in DREB subfamily in AP2/ERF family. Subcellular localization analyses revealed that MsDREBA5 was subcellularly localized into the nucleus when transiently expressed in onion epidermis cells. The MsDREBA5 protein might show no transcriptional activation ability. The 1624-bp-long promoter contained putative cis-acting elements and transcription factor binding motifs, including elements predicted to respond to abiotic stresses. MsDREBA5 was expressed in almost all the tissues and organs examined under normal conditions. Additionally, expression of MsDREBA5 was induced by cold, drought and exogenously applied abscisic acid, but down-regulated by salt stress. Transgenic Arabidopsis plants overexpressing MsDREBA5 appeared to down-regulate the expression levels of the stress-induced effecter genes under drought stress. Our findings may provide an important basis for further studies of the acclimation and tolerance of M. sieversii to abiotic stresses.

Fatty acids-stress attenuates gluconeogenesis induction and glucose production in primary hepatocytes

BackgroundHepatic gluconeogenesis tightly controls blood glucose levels in healthy individuals, yet disorders of fatty acids (FAs) oxidation are characterized by hypoglycemia. We studied the ability of free-FAs to directly inhibit gluconeogenesis, as a novel mechanism that elucidates the hypoglycemic effect of FAs oxidation defects.MethodsPrimary rat hepatocytes were pre-treated with FAs prior to gluconeogenic stimuli with glucagon or dexamethasone and cAMP.ResultsPre-treatment with 1 mM FAs (mixture of 2:1 oleate:palmitate) for 1 hour prior to gluconeogenic induction, significantly decreases the induced expression of the gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6pase) as well as the induced glucose production by the cells. The inhibitory effect of FAs upon gluconeogenesis is abolished when pre-treatment is elongated to 18 hours, allowing clearance of FAs into triglycerides by the cells. Replacement of palmitate with the non-metabolic fatty acid 2-bromopalmitate inhibits esterification of FAs into triglycerides. Accordingly, the increased exposure to unesterified-FAs allows their inhibitory effect to be extended even when pre-treatment is elongated to 18 hours. Similar changes were caused by FAs to the induction of peroxisome-proliferator-activated receptor-γ coactivator 1α (PGC1α) expression, indicating this transcriptional coactivator as the mediating link of the effect. This inhibitory effect of FAs upon gluconeogenic induction is shown to involve reduced activation of cAMP response element-binding (CREB) transcription factor.ConclusionsThe present results demonstrate that free-FAs directly inhibit the induced gluconeogenic response in hepatocytes. Hence, high levels of free-FAs may attenuate hepatic gluconeogenesis, and liver glucose output.

An effective strategy for increasing the radiosensitivity of Human lung Cancer cells by blocking Nrf2-dependent antioxidant responses

Radiotherapy and chemotherapeutic agents can effectively induce apoptosis through generation of reactive oxygen species (ROS). Cancer cells frequently express high levels of ROS-scavenging enzymes, which confer resistance to ROS-mediated cell death. Keap1 (Kelch-like ECH-associated protein 1) sequesters and promotes the degradation of the antioxidant response element-binding transcription factor Nrf2 (nuclear factor erythroid-2-related factor 2). In non-small-cell lung cancer (NSCLC) cell lines and NSCLC patients, Keap1 is often present as a biallelic mutant that results in constitutive activation of Nrf2 function, which contributes to cytoprotection against oxidative stress and xenobiotics. To identify small molecules that inhibit antioxidant responses and increase apoptotic death after radiotherapy, we screened a chemical library containing 8000 synthetic compounds using a cell-based luciferase assay system. 4-(2-Cyclohexylethoxy)aniline (IM3829) inhibited the increase in Nrf2-binding activity and expression of the Nrf2 target genes induced by treatment with tertiary butylhydroquinone or radiation. Combined treatment with IM3829 and radiation significantly inhibited clonogenic survival of H1299, A549, and H460 lung cancer cells. IM3829 significantly increased ROS accumulation in irradiated cells compared with cells exposed to radiation alone and led to apoptotic cell death, as confirmed by caspase-3 and PARP cleavage. In mice bearing H1299 or A549 lung cancer xenografts, IM3829 together with radiation inhibited tumor growth more effectively than radiation alone. Our findings suggest that IM3829 could be a promising radiosensitizer in lung cancer patients, particularly those with high expression of Nrf2.

Delta subclass HD-Zip proteins and a B-3 AP2/ERF transcription factor interact with promoter elements required for expression of the Arabidopsis cytochrome c oxidase 5b-1 gene

We have identified transcription factors that interact with a promoter region involved in expression of the Arabidopsis thaliana COX5b-1 gene, which encodes an isoform of the cytochrome c oxidase zinc binding subunit. Elements with the core sequence ATCATT, involved in induction by sugars, are recognized both in vitro and in one-hybrid assays in yeast by HD-Zip proteins from the delta subclass and, though less efficiently, by the trihelix transcription factor GT-3b. DistalB-like elements (CCACTTG), required for induction by abscisic acid (ABA), interact with ESE1, a member of the B-3 subgroup of AP2/ERF transcription factors. The HD-Zip protein Athb-21 and ESE1 are able to interact in yeast two-hybrid assays with the ABA responsive element binding factor AREB2/ABF4, which binds to a G-box absolutely required for expression of the COX5b-1 gene. Overexpression of the identified transcription factors in plants produces an increase in COX5b-1 transcript levels. Moreover, these factors are able to induce the expression of a reporter gene located in plants under the control of the relevant COX5b-1 promoter regions required for expression. Analysis of promoter regions of COX5b genes from different plant species suggests that the identified transcription factors were recruited for the regulation of COX5b gene expression at different stages during the evolution of dicot plants.

Gene expression combined with gene set enrichment analysis to identify markers of vinorelbine efficacy in breast cancer patients.

e21099 Background: Vinorelbine (V) induces mitotic arrest and apoptosis but there are limited data on its effect on gene expression in breast cancer clinical setting. Methods: 43 adult female patients with pathologically confirmed breast cancer and locally advanced or metastatic disease were treated with V 25 mg/m2 days 1, 8, 15 of a 28-day cycle. Gene expression was assessed in archival FFPE tissue using the microarray-based DASL assay (cDNA-mediated Annealing, Selection extension and Ligation) and correlated with time-to-progression (TTP). Using a Gene Set Enrichment Analysis (GSEA), groups of genes that share a common molecular function, chromosomal location, or regulation were identified in patients classified as having either a short (S) (n=25) or a long (L) (n=18) time to progression (TTP) divided by the median (72 days). The GSEA software ( http://www.broadinstitute.org/gsea/index.jsp ) was used for the analysis. Results: GSEA focusing on genes grouped according to similar a) molecular function: 16 out of a set of 43 genes involved in histone binding were enriched in group S (p = 0.002), consistent with higher expression in group S of HIST3H2BB and HIST1H3I as well as a nuclear transcription factor promoting their expression. b) transcription factors: 14 out of 47 genes were enriched in group S (p = 0.004) and corresponds to genes with promoter regions that match c-fos serum response element-binding transcription factor that modulates, for example, ABCC1 and ABCB1 (P-gp/MDR1) solute carriers. c) chromosomal location: in group S, genes were enriched on chromosome 11q21 (20 out of 45 genes p = 0.004) and on chromosome 12p12 (14 out of 22 genes p = 0.002). Conclusions: a) the up-regulation of histone binding genes is consonant with recent discovery of high affinity V binding to histones b) the role of P-gp/MDR1 in V transport is well known c) our observations on chromosome 11q21 and12p12 are novel. DASL expression combined with GSEA highlights gene sets that correlate with clinical outcome and may lead to predictive markers of V efficacy. Further confirmatory analysis is needed due to the limitation of small sample size and multiple comparisons.

Isodesacetyluvaricin, an Annonaceous Acetogenin, Specifically Inhibits Gene Expression of Cyclooxygenase-2

Cyclooxygenase-2 (COX-2) is an inducible isoform of the enzyme responsible for the synthesis of several inflammatory mediators. In a search for phytochemicals with anti-inflammatory activity, the COX-2 inhibitory activity of 15 typical Annonaceous acetogenins was examined. Isodesacetyluvaricin (1), from the Formosan tropical fruit tree Annona glabra, exhibited the most potent activity. Reverse transcription PCR was used to test the effect of 1 on epidermal growth factor-stimulated expression of COX-2 in cultures of A431 human epidermoid carcinoma cells. Three hours after exposure to 1 (5 μM), A431 cells had barely detectable levels of COX-2 mRNA. A corresponding but smaller decline in the COX-2 protein appeared on using Western blots. Lipopolysaccharide-stimulated expression of COX-2 in Raw 264.7 mouse leukemic monocyte-macrophages showed a similar decrease. Luciferase assays revealed that cells exposed to 1 had reduced activities of two COX-2 promoter-transcription factors: cAMP response element-binding factor and nuclear factor of activated T-cells. Compound 1 did not affect cell proliferation, as measured by a colorimetric assay, or intracellular store-operated calcium influx, as determined by fluorescence imaging. Thus, 1 may serve as a lead compound for targeting inflammatory diseases as well as angiogenesis and cancer metastasis.

Function of the airway epithelium in asthma.

Function of the airway epithelium in asthma.

Melanogenesis Inhibitor(s) fromPhyla nodifloraExtract

Overexpression of tyrosinase can cause excessive production of melanin and lead to hyperpigmentation disorders, including melasma and freckles. Recently, agents obtained from plants are being used as alternative medicines to downregulate tyrosinase synthesis and decrease melanin production. Phyla nodiflora Greene (Verbenaceae) is used as a folk medicine in Taiwanese for treating and preventing inflammatory diseases such as hepatitis and dermatitis. However, the antimelanogenesis activity and molecular biological mechanism underlying the activity of the methanolic extract of P. nodiflora (PNM) have not been investigated to date. Our results showed that PNM treatment was not cytotoxic and significantly reduced the cellular melanin content and tyrosinase activity in a dose-dependent manner (P < 0.05). Further, PNM exhibited a significant antimelanogenesis effect (P < 0.05) by reducing the levels of phospho-cAMP response element-binding protein and microphthalmia-associated transcription factor (MITF), inhibiting the synthesis of tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2, and decreasing the cellular melanin content. Moreover, PNM significantly activated the phosphorylation of mitogen-activated protein kinases, including phospho-extracellular signal-regulated kinase, c-Jun N-terminal kinase, and phospho-p38, and inhibited the synthesis of MITF, thus decreasing melanogenesis. These properties suggest that PNM could be used as a clinical and cosmetic skin-whitening agent to cure and/or prevent hyperpigmentation.

Ammonia increases nitric oxide, free Zn2+, and metallothionein mRNA expression in cultured rat astrocytes

Ammonia is a major player in the pathogenesis of hepatic encephalopathy (HE) and affects astrocyte function by triggering a self-amplifying cycle between osmotic and oxidative stress. We recently demonstrated that hypoosmotic astrocyte swelling rapidly stimulates nitric oxide (NO) production and increases intracellular free Zn(2+) concentration ([Zn(2+)](i)). Here we report effects of ammonia on [Zn(2+)](i) homeostasis and NO synthesis. In cultured rat astrocytes, NH(4)Cl (5 mm) increased within 6 h both cytosolic and mitochondrial [Zn(2+)]. The [Zn(2+)](i) increase was transient and was mimicked by the nonmetabolizable CH(3)NH(3)Cl, and it was dependent on NO formation, as evidenced by the sensitivity toward the nitric oxide synthase inhibitor N(G)-monomethyl-l-arginine. The NH(4)Cl-induced NO formation was sensitive to the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester and increases in both NO and [Zn(2+)](i) were blocked by the N-methyl-d-aspartate receptor antagonist MK-801. The NH(4)Cl-triggered increase in [Zn(2+)](i) was followed by a Zn(2+)-dependent nuclear appearance of the metal response element-binding transcription factor and metallothionein messenger RNA (mRNA) induction. Metallothionein mRNA was also increased in vivo in rat cerebral cortex 6 h after an NH(4)Ac challenge. NH(4)Cl increased peripheral-type benzodiazepine receptor (PBR) protein expression, whereas PBR mRNA levels were decreased in a Zn(2+)-independent manner. The Zn(2+)-dependent upregulation of metallothionein following ammonia intoxication may reflect a cytoprotective response, whereas the increase in PBR expression may augment HE development.

Whole blood genome-wide expression profiling and network analysis suggest MELAS master regulators

Background: The heteroplasmic mitochondrial DNA (mtDNA) mutation A3243G causes the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome as one of the most frequent mitochondrial diseases. The process of reconfiguration of nuclear gene expression profile to accommodate cellular processes to the functional status of mitochondria might be a key to MELAS disease manifestation and could contribute to its diverse phenotypic presentation.Objective: To determine master regulatory protein networks and disease-modifying genes in MELAS syndrome.Methods: Analyses of whole blood transcriptomes from 10 MELAS patients using a novel strategy by combining classic Affymetrix oligonucleotide microarray profiling with regulatory and protein interaction network analyses.Results: Hierarchical cluster analysis elucidated that the relative abundance of mutant mtDNA molecules is decisive for the nuclear gene expression response. Further analyses confirmed not only transcription factors already known to be involved in mitochondrial diseases (such as TFAM), but also detected the hypoxia-inducible factor 1 complex, nuclear factor Y and cAMP responsive element-binding protein-related transcription factors as novel master regulators for reconfiguration of nuclear gene expression in response to the MELAS mutation. Correlation analyses of gene alterations and clinico-genetic data detected significant correlations between A3243G-induced nuclear gene expression changes and mutant mtDNA load as well as disease characteristics. These potential disease-modifying genes influencing the expression of the MELAS phenotype are mainly related to clusters primarily unrelated to cellular energy metabolism, but important for nucleic acid and protein metabolism, and signal transduction.Discussion: Our data thus provide a framework to search for new pathogenetic concepts and potential therapeutic approaches to treat the MELAS syndrome.

Vitis CBF1 and Vitis CBF4 differ in their effect on Arabidopsis abiotic stress tolerance, development and gene expression

Plants growing in temperate regions encode several C-repeat binding factor/dehydration responsive element binding factors (CBF/DREB1) and the question is whether these transcription factors have different functions. In this study, Arabidopsis transformed with grape CBF1 (VrCBF1) or grape CBF4 (VrCBF4) were characterized. Electrolyte leakage assays showed that the freezing tolerance of transgenic lines was correlated with the level of VrCBF expression irrespective of the type of CBF, while drought tolerance was most increased by VrCBF1. VrCBF overexpression coincided with an increase in the expression of the cold-regulated genes AtCOR15a, AtRD29A, AtCOR6.6 and AtCOR47. In addition, the development of grape CBF overexpressing plants was seen to be altered and resulted in dwarf plants which flowered later and had thicker rosette leaves with a higher stomatal density. Analysis of gene expression showed that these morphological changes may be because of an increase in the expression of AtRGL3 in VrCBF4 lines or AtGA2ox7 in VrCBF1 lines, and AtFLC in both. In addition, the results show for the first time that CBFs can positively affect the expression of AtICE1/SCREAM1, the gene that is known to induce AtCBF3 expression. The difference in gene induction by VrCBF1 compared with VrCBF4 suggests that these CBFs have different regulons.

Serum response factor (c-fos serum response element-binding transcription factor; SRF)

Serum response factor (c-fos serum response element-binding transcription factor; SRF)

Overexpression of HARDY, an AP2/ERF gene from Arabidopsis, improves drought and salt tolerance by reducing transpiration and sodium uptake in transgenic Trifolium alexandrinum L.

Trifolium alexandrinum L. was transformed with the Arabidopsis HARDY gene that belongs to the stress-related AP2/ERF (APETALA2/ethylene responsive element binding factors) superfamily of transcription factors. The fresh weights of the transgenic lines L2 and L3 were improved by 42 and 55% under drought stress and by 38 and 95% under salt stress compared to the wild type, respectively. The dry weights were similarly improved. Overexpression of HARDY improved the instantaneous water use efficiency (WUE) under drought stress by reducing transpiration (E) and under salt stress by improving photosynthesis (A), through reducing Na+ accumulation in leaves, and reducing E. However, HARDY improved the growth of drought-stressed transgenic plants as compared to the wild type by delaying water depletion from soil and preventing rapid decline in A. L2 and L3 had thicker stems and in case of L3, more xylem rows per vascular bundle, which may have made L3 more resistant to lodging in the field. Field performance of L2 and L3 under combined drought and salt stress was significantly better than that of the wild type in terms of fresh and dry weights (40%, 46% and 31%, 40%, respectively). The results provide further evidence for the efficiency of overexpression of a single gene in improving tolerance to abiotic stress under field conditions.