Isolation and characterization of drought-related trehalose 6-phosphate-synthase gene from cultivated cotton (Gossypium hirsutum L.).

Due to the important role of cotton drought-tolerant varieties and the reported involvement in this trait of trehalose-6-phosphate-synthase, the respective gene (TPS) was isolated and characterized from cultivated cotton, Gossypium hirsutum (ZETA 2 cultivar), using a chromosome-walking technique. TPS has three exons comprising the coding region. Southern blot analysis indicated that the Gossypium genomes (A and D) contain a single copy of TPS per genome. In addition, the expression of this gene was studied in different plant tissues. Plants of the Australian cotton variety Siokra L23, known for its drought tolerance, were subjected to drought stress (using PEG 6,000 solution, for 4 h during the dark period of the day and for four consecutive days); leaves, stems and roots were collected after the end of the stress period. Total extracted RNA was examined for the presence of transcripts, in the above-mentioned tissues of stressed and well-watered plants, by reverse transcription-polymerase chain reaction (RT-PCR). The expression levels, determined semi-quantitatively, indicated that the gene was expressed in all plant tissues under both water availability conditions. However, increased expression levels of TPS were observed mainly in stressed leaves and roots compared to those of the well-watered control. This finding is in agreement with the fact that TPS participates in trehalose biosynthesis, known for its participation in stress signal transduction in higher plants.

Functional constraints of 6-phosphogluconate dehydrogenase (6-PGD) based on sequence and structural information.

The pentose phosphate cycle is considered as a major source of NADPH and pentose needed for nucleic acid biosynthesis. 6-Phosphogluconate dehydrogenase (6PGD), an enzyme participating in this cycle, catalyzes the oxidative decarboxylation of 6PGD to ribulose 5-phosphate with the subsequent release of CO2 and the reduction of NADP. We have determined the amino acid sequence of 6PGD of Bactrocera oleae and constructed a three-dimensional model based on the homologous known sheep structure. In a comparative study of 6PGD sequences from numerous species, all the conserved and variable regions of the enzyme were analyzed and the regions of functional importance were localized, in an attempt promoted also by the direct involvement of the enzyme in various human diseases. Thus, analysis of amino acid variability of 37 6PGD sequences revealed that all regions important for the catalytic activity, such as those forming the substrate and coenzyme binding sites, are highly conserved in all species examined. Moreover, several amino acid residues responsible for substrate and coenzyme specificity were also found to be identical in all species examined. The higher percentage of protein divergence is observed at two regions that accumulate mutations, located at the distant parts of the two domains of the enzyme with respect to their interface. These peripheral regions of non-functional importance are highly variable and are predicted as antigenic, thus reflecting possible regions for antibody recognition. Furthermore, locating the differences between diptera 6PGD sequences on the three-dimensional model suggests probable positions of different amino acid residues appearing at B. oleae fast, intermediate, and slow allozymic variants.

Macroevolutionary relationships of species of Drosophila melanogaster group based on mtDNA sequences.

The phylogenetic relationships among the Drosophila melanogaster group species were analyzed using approximately 1700 nucleotide-long sequences of the mitochondrial DNA. Phylogenetic analysis was performed using this region consisting of a part of the cytochrome b (cytb) coding gene, the entire coding sequences of tRNA-Leu, tRNA-Ser and the first subunit of NADH dehydrogenase (NADH1), and a part of the 16S-rRNA gene. The study of these sequences showed that this region of mtDNA is very invariable, as regards with the type of the genes that it contains, as well as the order that they are located on it. The resulting phylogenetic trees reveal a topology that separates the species into three main ancestral lines, leading to the following subgroups: (a) ananassae subgroup, (b) montium subgroup, and (c) melanogaster and Oriental subgroups. The inferred topology complements and generally agrees with previously proposed classifications based on morphological and molecular data.

Drosophila Cu,Zn superoxide dismutase gene confers resistance to paraquat in Escherichia coli.

Superoxide dismutase (SOD) is known to protect organisms from reactive oxygen metabolites. We tested the hypothesis that the Drosophila Cu,Zn SOD is capable of protecting Escherichia coli from oxidative damage caused by the herbicide paraquat. The Cu,Zn Sod gene of Drosophila sechellia was subcloned into pET-20b(+) expression vector. Transformation of E. coli with the constructed vector resulted in an overexpression of this eukaryotic superoxide dismutase, as evidenced by dramatically increased levels of the Cu,Zn SOD polypeptide in bacterial cytosolic extracts. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth and survival. Paraquat is known to promote formation of the superoxide radical anion inside cells and thus the data have been interpreted as indicating that the cloned superoxide dismutase provides protection in E. coli against damage attributable to free radicals.

Expression of selected drought-related genes and physiological response of Greek cotton varieties.

Drought-tolerant cotton varieties are very important for Greece and throughout the world. Four Greek cotton varieties (Zeta 2, Zeta 5, Korina and Eva) and an Australian variety (Siokra L23) were subjected to three water-stress levels (0.0, -0.1 and -0.3 MPa). Morphological and physiological parameters studied were plant height, total leaf area, shoot, root and total plant fresh and dry weights, stomatal resistance (SR), water potential (Ψ w), and relative water content. Siokra L23 was confirmed to be the most drought-tolerant variety based on its high SR and Ψ w , it's having the smallest total leaf area, and expression of drought-tolerance-related genes. The Greek cotton varieties were ranked from most to least drought tolerant as follows: Eva, Korina, Zeta 2, Zeta 5.Molecular responses of the cotton varieties were studied by investigating the expression of five drought-tolerance-related genes, namely, trehalose-6-P synthase, heat-shock protein calmodulin-binding homolog, late embryogenesis abundant (Lea) proteins 14A and 5D, and NAD(P)H oxidase. Reverse transcription-polymerase chain reaction was performed utilizing total RNA samples isolated after a 4-d drought treatment (i.e. at the end of the stress period). Heat-shock protein calmodulin-binding homolog was induced by water stress in drought-tolerant varieties (Eva and Siokra L23) and Zeta 2. This correlation between physiological and molecular data for this gene allows it to be used in cotton breeding programs. Trehalose-6-P synthase and NAD(P)H oxidase genes were not expressed in almost all varieties and treatments. In contrast, the Lea genes showed, with minor exceptions, expression that was independent of variety and treatment. Eva and Korina varieties should be used under conditions of water shortage, whereas Zeta varieties provide a significant advantage to the grower when planted under conditions of high water availability.

Functional constraints of the Cu,Zn superoxide dismutase in species of the Drosophila melanogaster subgroup and phylogenetic analysis.

The phylogenetic relationships among the Drosophila melanogaster subgroup species were analyzed using approximately 1550-nucleotide-long sequences of the Cu,Zn SOD gene. Phylogenetic analysis was performed using separately the whole region and the intron sequences of the gene. The resulting phylogenetic trees reveal virtually the same topology, separating the species into distinct clusters. The inferred topology generally agrees with previously proposed classifications based on morphological and molecular data. The amino acid sequences of the Cu,Zn SOD of the D. melanogaster subgroup species reveal a high-conservation pattern. Only 3.9% of the total amino acid sites are variable, and none affects the major structural elements. Comparison of the Drosophila Cu,Zn SOD amino acid sequences with the Cu,Zn SOD of Bos taurus and Xenopus laevis (whose three-dimensional structure has been elucidated) reveals conservation of all the protein's functionally important amino acids and no substitutions that dramatically change the charge or the polarity of the amino acids.