Abstract. Autophagy triggered by carbohydrate starvation was characterized at both biochemical and structural levels, with the aim to identify reliable and easily detectable marker(s) and to investigate the factors controlling this process. Incubation of suspension cells in sucrose-free culture medium triggered a marked degradation of the membrane polar lipids, including phospholipids and galactolipids. In contrast, the total amounts of sterols, which are mainly associated with plasmalemma and tonoplast membranes, remained constant. In particular, phosphatidylcholine decreased, whereas phosphodiesters including glycerylphosphorylcholine transiently increased, and phosphorylcholine (P-Cho) steadily accumulated. P-Cho exhibits a remarkable metabolic inertness and therefore can be used as a reliable biochemical marker reflecting the extent of plant cell autophagy. Indeed, whenever P-Cho accumulated, a massive regression of cytoplasm was noticed using EM. Double membrane-bounded vacuoles were formed in the peripheral cytoplasm during sucrose starvation and were eventually expelled into the central vacuole, which increased in volume and squeezed the thin layer of cytoplasm spared by autophagy.The biochemical marker P-Cho was used to investigate the factors controlling autophagy. P-Cho did not accumulate when sucrose was replaced by glycerol or by pyruvate as carbon sources. Both compounds entered the cells and sustained normal rates of respiration. No recycling back to the hexose phosphates was observed, and cells were rapidly depleted in sugars and hexose phosphates, without any sign of autophagy. On the contrary, when pyruvate (or glycerol) was removed from the culture medium, P-Cho accumulated without a lag phase, in correlation with the formation of autophagic vacuoles. These results strongly suggest that the supply of mitochondria with respiratory substrates, and not the decrease of sucrose and hexose phosphates, controls the induction of autophagy in plant cells starved in carbohydrates.

Sex pheromones are intraspecific chemical signals that are crucial for mate attraction and discrimination. In Drosophila melanogaster, the predominant hydrocarbons on the cuticle of mature female and male flies are radically different and tend to stimulate or inhibit male courtship, respectively. This sexual difference depends largely upon the number of double bonds (one in males and two in females) added by desaturase enzymes. A mutation was caused by a PGal4 transposon inserted in the desat1 gene that codes for the desaturase crucial for setting these double bonds. Homozygous mutant flies produced 70-90% fewer sex pheromones than control flies, and the pheromonal difference between the sexes was almost abolished. A total of 134 excision alleles were induced by pulling out all or a part of the transposon. The pheromonal profile was generally rescued in excision alleles with a completely or largely removed transposon whereas it remained mutant in alleles with a larger piece of the transposon. Five desat1 transcripts were detected during larval-to-adult development. Their levels were precisely quantified in 24-hr-old adults, a critical period for the production of sex pheromones. Three transcripts significantly varied between control females and males; however, the predominant transcript showed no difference. In mutant flies, the predominant transcript was highly decreased with the two sexually dimorphic transcripts.These two transcripts were also absent in the sibling species D. simulans, which shows no sexually dimorphic hydrocarbons. We also induced a larval-lethal allele that lacked all transcripts and failed to complement the defective hydrocarbon phenotype of mutant alleles.

The regulatory sequences of the Drosophila ACP65A cuticle gene were analyzed in vivo in transgenic flies, using both fusion genes constructs and transposase-mediated deletions within a P element containing ACP65A regulatory sequences fused to the lacZ gene (deletion scanning). The sequences located between -594 and +161 are sufficient to confer both temporal and spatial expression specificities, indicating the presence of tissue-specific enhancers and response elements to hormone-induced factors. In addition, timing of expression and tissue-specificity appear to be controlled by distinct cis-regulatory elements, which suggests the existence of independent hormonal and tissue-specific signaling pathways. Gain and loss of function studies also implicate DHR38, the Drosophila homolog of the vertebrate NGFI-B-type nuclear receptors, as an important activator of the ACP65A gene.

Five PCR fragments corresponding to a part of the DNA-binding domain of different hormone nuclear receptors were isolated from Tenebrio molitor mRNAs. The sequence identity of three of them with known Drosophila nuclear receptors strongly suggests that they are the Tenebrio orthologs of seven-up, DHR3 and b -FTZ-F1, and thus named Tmsvp, TmHR3 and TmFTZ-F1. The full-length sequences of the other two were established. TmHR78 is either a new receptor of the DHR78 family or the same gene which has evolved rapidly, particularly in the E domain. TmGRF belongs to the GCNF1 family and its in vitro translated product binds to the extended half site TCAAGGTCA with high affinity. The periods of expression of the corresponding transcripts in epidermal cells during Tenebrio metamorphosis were analyzed as a function of 20-hydroxyecdysone titers measured in the hemolymph of the animals taken for RNA extraction. Comparison of the expression profiles of these nuclear receptors with those observed during Drosophila metamorphosis revealed similar temporal correlations as a function of ecdysteroid variations, which further supported the sequence identity data for TmSVP, TmHR3, TmFTZ-F1 and TmHR78.Keywords: ecdysone; GCNF1; insect; metamorphosis; nuclear receptors.The pioneering work on chromosomal puffing induced by ecdysteroid in Drosophila salivary gland cells [1] gave rise to the idea that the molting hormone ecdysone triggered a regulatory cascade of gene expression. During the last decade, this hypothesis has been confirmed by molecular studies identifying the ecdysone receptor (EcR) [2] and several ecdysone-induced orphan members of the nuclear hormone receptor superfamily in Drosophila [3], and by developmental studies showing that many of these puff gene products are expressed hierarchically at various times after ecdysone action [4±6]. Such studies also suggest that in different tissues, the combinatorial expression cascade of these ecdysone-induced receptors may be different, thus contributing to the specificity of response to the hormone.The identification of several orthologs of Drosophila nuclear hormone receptors in other insect species supports the idea that the regulatory cascade found in the fruit fly could be conserved throughout this class. However, differences occur at the structural and transcriptional levels among different insect species studied so far [3], which could explain the differences observed in their life-cycles. Indeed, even in the apparently homogenous group of holometabolous insects, the metamorphic events present such striking differences in timing and cell fate among different orders that it is of interest to compare the cascade of molecular events triggered by ecdysone described for Drosophila with that of other metamorphosing insects. The identification of Drosophila EcR homologs in several other holometabolous species [7±15] supports this molecular diversity hypothesis. However, less is known concerning the homologs of other nuclear receptors characterized in Drosophila and thus it seems in...