Genetic control of flowering in spotted gum, Corymbia citriodora subsp. variegata and C. maculata

Genetically controlled asynchrony in anthesis is an effective barrier to gene flow between planted and native forests. We investigated the degree of genetically controlled variation in the timing of key floral developmental stages in a major plantation species in subtropical Australia, Corymbia citriodora subsp. variegata K.D. Hill and L.A.S Johnson, and its relative C. maculata K.D. Hill and L.A.S. Johnson. Flowering observations were made in a common garden planting at Bonalbo in northern New South Wales in spring on 1855 trees from eight regions over three consecutive years, and monthly on a subset of 208 trees for 12 months. Peak anthesis time was stable over years and observations from translocated trees tended to be congruent with the observations in native stands, suggesting strong genetic control of anthesis time. A cluster of early flowering provenances was identified from the north-east of the Great Dividing Range. The recognition of a distinct flowering race from this region accorded well with earlier evidence of adaptive differentiation of populations from this region and geographically-structured genetic groupings in C. citriodora subsp. variegata. The early flowering northern race was more fecund, probably associated with its disease tolerance and greater vigour. Bud abundance fluctuated extensively at the regional level across 3 years suggesting bud abundance was more environmentally labile than timing of anthesis. Overall the level of flowering in the planted stand (age 12 years) was low (8–12% of assessed trees with open flowers), and was far lower than in nearby native stands. Low levels of flowering and asynchrony in peak anthesis between flowering races of C. citriodora subsp. variegata may partially mitigate a high likelihood of gene flow where the northern race is planted in the south of the species range neighbouring native stands

Novel bradykinins and their precursor cDNAs from European yellow-bellied toad (Bombina variegata) skin

Two novel bradykinin-related peptides (Ala3,Thr6)-bradykinin and (Val1,Thr3,Thr6)-bradykinin, were identified by a systematic sequencing study of peptides in the defensive skin secretion of the yellow-bellied toad, Bombina variegata. These peptides are the first amphibian skin bradykinins to exhibit amino acid substitutions at the Pro3 position of the bradykinin nonapeptide. Previously reported bradykinins from other Bombina species were not detected. Respective precursor cDNAs, designated BVK-1 and BVK-2, respectively, were cloned from a skin library by 3'- and 5'-RACE reactions. BVK-1 contained an open-reading frame of 97 amino acids encoding a single copy of (Ala3,Thr6)-bradykinin and similarly, the open-reading frame of BVK-2 consisted of 96 amino acids encoding a single copy of (Val1,Thr3,Thr6)-bradykinin. Synthetic replicates of each novel bradykinin were found to be active on mammalian arterial and small intestinal smooth muscle preparations. The structural diversity of bradykinins in amphibian defensive skin secretions may be related to defence against specific predators.

Identification and molecular cloning of novel trypsin inhibitors from the dermal venom of the Oriental fire-bellied toad (Bombina orientalis) and the European yellow-bellied toad (Bombina variegata).

The structural diversity of polypeptides in amphibian skin secretion probably reflects different roles in dermal regulation or in defense against predators. Here we report the structures of two novel trypsin inhibitor analogs, BOTI and BVTI, from the dermal venom of the toads, Bombina orientalis and Bombina variegata. Cloning of their respective precursors was achieved from lyophilized venom cDNA libraries for the first time. Amino acid alignment revealed that both deduced peptides, consisting of 60 amino acid residues, including 10 cysteines and the reactive center motif, -CDKKC-, can be affirmed as structural homologs of the trypsin inhibitor from Bombina bombina skin.

Construction of cDNA libraries from trifluoroacetic acid-solvated amphibian skin secretions: molecular cloning of multiple bombinin-like peptide precursor transcripts from a library of yellow-bellied toad (Bombina variegata) secretion

Amphibian skin secretions are renowned as complex mixtures of bioactive peptides many of which are analogues of endogenous regulatory peptides. While skin secretions can be obtained non-invasively for peptidome analysis, parallel studies on the granular gland transcriptome required specimen sacrifice. The aim of the present study was to analyse archived skin secretions to determine the robustness of bioactive peptide precursor-encoding polyadenylated mRNAs in an attempt to extract maximum molecular information from rare samples. A range of solvated skin secretion samples were examined after lyophilisation for their potential to generate viable and comprehensive cDNA libraries based upon polyadenylated mRNA capture and amplification/cloning using appropriate commercial kits. Here we present unequivocal data that the granular gland transcriptome persists in a PCR amenable format even after storage for as long as 12 years in 0.1%(v/v) aqueous trifluoroacetic acid (TFA). We used a pooled skin secretion sample (2 ml) from the yellow-bellied toad, Bombina variegata (n = 14), containing the equivalent of 5 mg/ml of lyophilised skin secretion, that had been used in part for peptide isolation purposes in 1998 and had been stored at - 20 °C since that time. In the first cloning experiment, 12 different bombinin-like peptide precursor cDNAs were cloned encoding 17 different bombinins, the majority of which were novel. Subsequently, bombesin and bradykinin-related peptide precursor transcripts have been cloned successfully. These data illustrate the unexpected stability/longevity of the transcriptome in these secretions — a finding with implications for both this field of research and for the wider field of molecular biology.

Peptide IC-20, encoded by skin kininogen-1 of the European yellow-bellied toad, Bombina variegata, antagonizes bradykinin-induced arterial smooth muscle relaxation

The objectives were to determine if the skin secretion of the European yellow-bellied toad (Bombina variegata), in common with other related species, contains a bradykinin inhibitor peptide and to isolate and structurally characterize this peptide. Materials and Methods: Lyophilized skin secretion obtained from this toad was subjected to reverse phase HPLC fractionation with subsequent bioassay of fractions for antagonism of the bradykinin activity using an isolated rat tail artery smooth muscle preparation. Subsequently, the primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy, and molecular cloning, following which a synthetic replicate was chemically synthesised for bioassay. Results: A single peptide of molecular mass 2300.92 Da was resolved in HPLC fractions of skin secretion and its primary structure determined as IYNAIWP-KH-NK-KPGLL-. Database interrogation with this sequence indicated that this peptide was encoded by skin kininogen-1 previously cloned from B. variegata. The blank cycles were occupied by cysteinyl (C) residues and the peptide was located toward the C-terminus of the skin kininogen, and flanked N-terminally by a classical -KR- propeptide convertase processing site. The peptide was named IC-20 in accordance (I = N-terminal isoleucine, C = C-terminal cysteine, 20 = number of residues). Like the natural peptide, its synthetic replicate displayed an antagonism of bradykinin-induced arterial smooth muscle relaxation. Conclusion: IC-20 represents a novel bradykinin antagonizing peptide from amphibian skin secretions and is the third such peptide found to be co-encoded with bradykinins within skin kininogens.