Hahsp17.6G1 is the promoter of a small heat stress protein (sHSP) from sunflower (Helianthus annuus) that is activated during zygotic embryogenesis, but which does not respond to heat stress. We report here the cloning of a transcription factor (TF), sunflower drought-responsive element binding factor 2 (HaDREB2), by one-hybrid interaction with functional cis-elements in Hahsp17.6G1. We have analyzed the functional interaction between HaDREB2 and a second transcription factor, sunflower heat stress factor A9 (HaHSFA9), which was previously assigned to the regulation of Hahsp17.6G1. HaDREB2 and HaHSFA9 synergistically trans-activate the Hahsp17.6G1 promoter in bombarded sunflower embryos. This synergistic interaction is heat stress factor (HSF) specific and requires the binding of both factors to the promoter. The C-terminal region of HaHSFA9 is sufficient for the HSF specificity. Our results represent an example of a functional interaction between members of the Apetala 2 (HaDREB2) and HSF (HaHSFA9) families of transcription factors. We suggest new roles in zygotic embryogenesis for specific members of the AP2 transcription factor family.The functional assignment of individual transcription factors (TFs) from multimember families is one of the major goals in postgenomic analyses of plants. We focus our attention on factors that are involved in the developmental induction of small heat stress protein (sHSP) genes during zygotic embryogenesis. In plants, as in other eukaryotes, HSPs of different molecular masses, including the sHSPs (17-30 kD), are expressed not only during the heat shock response but also during development in the absence of exogenous stresses (for review, see Sun et al., 2002).

BackgroundTranscription factor HaDREB2 was identified in sunflower (Helianthus annuus L.) as a drought-responsive element-binding factor 2 (DREB2) with unique properties. HaDREB2 and the sunflower Heat Shock Factor A9 (HaHSFA9) co-activated the Hahsp17.6G1 promoter in sunflower embryos. Both factors could be involved in transcriptional co-activation of additional small heat stress protein (sHSP) promoters, and thus contribute to the HaHSFA9-mediated enhancement of longevity and basal thermotolerance of seeds.ResultsWe found that overexpression of HaDREB2 in seeds did not enhance longevity. This was deduced from assays of basal thermotolerance and controlled seed-deterioration, which were performed with transgenic tobacco. Furthermore, the constitutive overexpression of HaDREB2 did not increase thermotolerance in seedlings or result in the accumulation of HSPs at normal growth temperatures. In contrast, when HaDREB2 and HaHSFA9 were conjointly overexpressed in seeds, we observed positive effects on seed longevity, beyond those observed with overexpression of HaHSFA9 alone. Such additional effects are accompanied by a subtle enhancement of the accumulation of subsets of sHSPs belonging to the CI and CII cytosolic classes.ConclusionOur results reveal the functional interdependency of HaDREB2 and HaHSFA9 in seeds. HaDREB2 differs from other previously characterized DREB2 factors in plants in terms of its unique functional interaction with the seed-specific HaHSFA9 factor. No functional interaction between HaDREB2 and HaHSFA9 was observed when both factors were conjointly overexpressed in vegetative tissues. We therefore suggest that additional, seed-specific factors, or protein modifications, could be required for the functional interaction between HaDREB2 and HaHSFA9.

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Chronic use of psychostimulants induces enduringly increased responsiveness to a subsequent psychostimulant injection and sensitivity to drug-associated cues, contributing to drug craving and relapse. Neurotensin (NT), a neuropeptide functionally linked to dopaminergic neurons, was suggested to participate in these phenomena. We and others have reported that SR 48692, an NT receptor antagonist, given in pre-or cotreatments with cocaine or amphetamine, alters some behavioral effects of these drugs in rats. However, its efficacy when applied following repeated cocaine administration remains unknown. We, therefore, evaluated the ability of SR 48692, administered after a cocaine regimen, to interfere with the expression of locomotor sensitization and conditioned place preference (CPP) in rats. We demonstrated that the expression of locomotor sensitization, induced by four cocaine injections (15 mg/kg, i.p.) every other day and a cocaine challenge 1 week later, was attenuated by a subsequent 2-week daily administration of SR 48692 (1 mg/kg, i.p.). Furthermore, the expression of cocaine-induced CPP was suppressed by a 10-day SR 48692 treatment started after the conditioning period (four 15 mg/kg cocaine injections every other day). Taken together, our data show that a chronic SR 48692 treatment given after a cocaine regimen partly reverses the expression of locomotor sensitization and CPP in the rat, suggesting that NT participates in the maintenance of these behaviors. Our results support the hypothesis that targeting neuromodulatory systems, such as the NT systems may offer new strategies in the treatment of drug addiction.

The binding properties of the G-protein coupled receptor (GPCR) serotonin 5-HT1B receptor were studied under exposure to AC (50 and 400 Hz) and DC magnetic fields (MF) in rat brain membranes. This was an attempt at replicating the positive findings of Massot et al. In saturation experiments using [3H]5-HT, 1-h exposures at 1.1 mT(rms) 50 Hz caused statistically significant increases in both the K(D) and B(max) binding parameters, from 1.74 +/- 0.3 to 4.51 +/- 0.86 nM and from 1428 +/- 205 to 2137 +/- 399 CPM, respectively, in good agreement with previous results. Exposure of the membranes at 400 Hz 0.675 mT(rms) did not elicit a larger increase in K(D) in spite of a much larger induced current density. DC fields (1.1 and 11 mT) had a lesser effect compared to AC fields at low values of K(Dsham), but decreased the affinity at higher values of K(Dsham). Modeling of the receptor-ligand-G protein interactions using the extended ternary complex model yielded good fits for all our data and that of Massot et al., showing that the AC field may act by decreasing the ability of the G-protein to alter the ligand-receptor affinity. The hypothesis is that the bipolar nature of the AC field explains the different nature of the effects observed with AC and DC exposures. These findings constitute one of the few documented pieces of evidence for cell-free effects of DC and extremely low frequency (ELF) AC MFs in the mT range.

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