Nicotianagandarela (Solanaceae), a new species of 'tobacco' highly endangered from the Quadrilátero Ferrífero in Brazil.

Nicotianagandarela Augsten & Stehmann (Solanaceae), sp. nov., a small 'tobacco' known only from one locality at Serra do Gandarela, in the state of Minas Gerais, Brazil, is described and illustrated. It is morphologically characterized by its rosulate basal leaves, red corolla with a short tube not inflated at the apex, and the peculiar habitat, a shaded site under a rocky outcrop ledge along a forested stream. Phylogenetic analyses based on a combined dataset of nuclear (ITS) and plastid (ndhF, trnLF, and trnSG) DNA sequences revealed that the species belongs to the Nicotianasect.Alatae and is sister to the clade with the remaining species in the section. A key for the identification of Brazilian species of the section is given. The unusual habitat, the small population size, and the intense pressure of mining activities in the surroundings made the species assessed as Critically Endangered (CR), needing conservation efforts to avoid its extinction.

Genetic structure is associated with phenotypic divergence in floral traits and reproductive investment in a high-altitude orchid from the Iron Quadrangle, southeastern Brazil.

Knowledge of the role of Neotropical montane landscapes in shaping genetic connectivity and local adaptation is essential for understanding the evolutionary processes that have shaped the extraordinary species diversity in these regions. In the present study, we examined the landscape genetics, estimated genetic diversity, and explored genetic relationships with morphological variability and reproductive strategies in seven natural populations of Cattleya liliputana (Orchidaceae). Nuclear microsatellite markers were used for genetic analyses. Spatial Bayesian clustering and population-based analyses revealed significant genetic structuring and high genetic diversity (He = 0.733 ± 0.03). Strong differentiation was found between populations over short spatial scales (FST = 0.138, p < 0.001), reflecting the landscape discontinuity and isolation. Monmonier´s maximum difference algorithm, Bayesian analysis on STRUCTURE and principal component analysis identified one major genetic discontinuity between populations. Divergent genetic groups showed phenotypic divergence in flower traits and reproductive strategies. Increased sexual reproductive effort was associated with rock outcrop type and may be a response to adverse conditions for growth and vegetative reproduction. Here we discuss the effect of restricted gene flow, local adaptation and phenotypic plasticity as drivers of population differentiation in Neotropical montane rock outcrops.

Molecular phylogenetics of the species-rich genus Habenaria (Orchidaceae) in the New World based on nuclear and plastid DNA sequences.

Habenaria is a large genus of terrestrial orchids distributed throughout the tropical and subtropical regions of the world. The integrity and monophyly of this genus have been under discussion for many years, and at one time or another, several genera have been either included in a broadly defined Habenaria or segregated from it. In this study, the phylogenetic relationships of the Neotropical members of the genus and selected groups of African Habenaria were investigated using DNA sequences from the nuclear internal transcribed spacer (ITS) region and the plastid matK gene sampled from 151 taxa of Habenaria from the Neotropics (ca. 51% of the total) as well as 20 species of Habenaria and Bonatea from the Old World. Bayesian and parsimony trees were congruent with each other, and in all analyses, the Neotropical species formed a highly supported group. African species of Habenaria in sections Dolichostachyae, Podandria, Diphyllae, Ceratopetalae and Bilabrellae, and the Neotropical clade formed a highly supported "core Habenaria clade", which includes the type species of the genus from the New World. The topology of the trees indicates an African origin for the Neotropical clade and the low sequence divergence among the Neotropical species suggests a recent radiation of the genus in the New World. Species of Bonatea and Habenaria sections Chlorinae and Multipartitae formed a well-supported clade that was sister to the "core Habenaria clade". The Neotropical clade consists of at least 21 well-supported subgroups, but all Neotropical sections of the current sectional classification are paraphyletic or polyphyletic and will need extensive revision and recircumscription. Most of the Neotropical subgroups formed morphologically uniform assemblage of species, but some cases of morphological divergence within subgroups and convergence between subgroups indicated that morphology alone can be misleading for inferring relationships within the genus. The genera Bertauxia, Kusibabella and Habenella, segregated from New World Habenaria, are not monophyletic and a revision of the sectional classification rather than a generic division seems most appropriate. Our results do not support an extensive generic fragmentation of Habenaria as previously suggested and will provide a framework for revising the infrageneric classification and investigating the patterns of morphological evolution and geographical distribution of the genus in the New World.

Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues.

BACKGROUND: Cotton (Gossypium spp.) is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2) family member with no previous characterization. RESULTS: Nucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2. CONCLUSIONS: uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues.

Pro domain peptide of HGCP-Iv cysteine proteinase inhibits nematode cysteine proteinases.

Cysteine proteinases (CPs) are synthesized as zymogens and converted to mature proteinase forms by proteolytic cleavage and release of their pro domain peptides. A cDNA encoding a papain-like CP, called hgcp-Iv, was isolated from a Heterodera glycines J2 cDNA library, expressed and utilized to assess the ability of its propeptide to inhibit proteinase in its active form. The hgcp-Iv cDNA sequence encodes a polypeptide of 374 amino acids with the same domain organization as other cathepsin L-like CPs, including a hydrophobic signal sequence and a pro domain region. HGCP-Iv, produced in Escherichia coli as a fusion protein with thioredoxin, degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin and is inhibited by E-64, a substrate and inhibitor commonly used for functional characterization of CPs. Recombinant propeptides of HGCP-Iv, expressed in E. coli, presented high inhibitory activity in vitro towards its cognate enzyme and proteinase activity of Meloidogyne incognita females, suggesting its usefulness in inhibiting nematode CPs in biological systems. Cysteine proteinases from other species produced no noticeable activity.

A diverse family of serine proteinase genes expressed in cotton boll weevil (Anthonomus grandis): implications for the design of pest-resistant transgenic cotton plants.

Fourteen different cDNA fragments encoding serine proteinases were isolated by reverse transcription-PCR from cotton boll weevil (Anthonomus grandis) larvae. A large diversity between the sequences was observed, with a mean pairwise identity of 22% in the amino acid sequence. The cDNAs encompassed 11 trypsin-like sequences classifiable into three families and three chymotrypsin-like sequences belonging to a single family. Using a combination of 5' and 3' RACE, the full-length sequence was obtained for five of the cDNAs, named Agser2, Agser5, Agser6, Agser10 and Agser21. The encoded proteins included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Southern blotting analysis suggested that one or two copies of these serine proteinase genes exist in the A. grandis genome. Northern blotting analysis of Agser2 and Agser5 showed that for both genes, expression is induced upon feeding and is concentrated in the gut of larvae and adult insects. Reverse northern analysis of the 14 cDNA fragments showed that only two trypsin-like and two chymotrypsin-like were expressed at detectable levels. Under the effect of the serine proteinase inhibitors soybean Kunitz trypsin inhibitor and black-eyed pea trypsin/chymotrypsin inhibitor, expression of one of the trypsin-like sequences was upregulated while expression of the two chymotrypsin-like sequences was downregulated.

Molecular cloning of alpha-amylases from cotton boll weevil, Anthonomus grandis and structural relations to plant inhibitors: an approach to insect resistance.

Anthonomus grandis, the cotton boll weevil, causes severe cotton crop losses in North and South America. Here we demonstrate the presence of starch in the cotton pollen grains and young ovules that are the main A. grandis food source. We further demonstrate the presence of alpha-amylase activity, an essential enzyme of carbohydrate metabolism for many crop pests, in A. grandis midgut. Two alpha-amylase cDNAs from A. grandis larvae were isolated using RT-PCR followed by 5' and 3' RACE techniques. These encode proteins with predicted molecular masses of 50.8 and 52.7kDa, respectively, which share 58% amino acid identity. Expression of both genes is induced upon feeding and concentrated in the midgut of adult insects. Several alpha-amylase inhibitors from plants were assayed against A. grandis alpha-amylases but, unexpectedly, only the BIII inhibitor from rye kernels proved highly effective, with inhibitors generally active against other insect amylases lacking effect. Structural modeling of Amylag1 and Amylag2 showed that different factors seem to be responsible for the lack of effect of 0.19 and alpha-AI1 inhibitors on A. grandis alpha-amylase activity. This work suggests that genetic engineering of cotton to express alpha-amylase inhibitors may offer a novel route to A. grandis resistance.

Effects of black-eyed pea trypsin/chymotrypsin inhibitor on proteolytic activity and on development of Anthonomus grandis.

The cotton boll weevil Anthonomus grandis (Boheman) is one of the major pests of cotton (Gossypium hirsutum L.) in tropical and sub-tropical areas of the New World. This feeds on cotton floral fruits and buds causing severe crop losses. Digestion in the boll weevil is facilitated by high levels of serine proteinases, which are responsible for the almost all proteolytic activity. Aiming to reduce the proteolytic activity, the inhibitory effects of black-eyed pea trypsin/chymotrypsin inhibitor (BTCI), towards trypsin and chymotrypsin from bovine pancreas and from midguts of A. grandis larvae and adult insects were analyzed. BTCI, purified from Vigna unguiculata (L.) seeds, was highly active against different trypsin-like proteinases studied and moderately active against the digestive chymotrypsin of adult insects. Nevertheless, no inhibitory activity was observed against chymotrypsin from A. grandis larval guts. To test the BTCI efficiency in vivo, neonate larvae were reared on artificial diet containing BTCI at 10, 50 and 100 microM. A reduction of larval weight of up to approximately 54% at the highest BTCI concentration was observed. At this concentration, the insect mortality was 65%. This work constitutes the first observation of a Bowman-Birk type inhibitor active in vitro and in vivo toward the cotton boll weevil A. grandis. The results of bioassays strongly suggest that BTCI may have potential as a transgene protein for use in engineered crop plants modified for heightened resistance to the cotton boll weevil.