Genetic transformation of blue grama grass with the <Emphasis Type="BoldItalic">rolA</Emphasis> gene from <Emphasis Type="BoldItalic">Agrobacterium rhizogenes</Emphasis>: regeneration of transgenic plants involves a “hairy embryo” stage

Until recently, information about the effects of transforming plants with the rolA gene of Agrobacterium rhizogenes has been restricted mainly to dicots in which a severely wrinkled phenotype, reduced internode distances, and abnormal reproductive development were commonly observed. In this work, we analyzed the effects associated with the expression of this gene in a new genetic context: the forage grass genome. Transgenic P_35S•rolA plants of blue grama grass (Bouteloua gracilis) were obtained by a biolistic approach employing embryogenic chlorophyllic cells as the target material. Four independent transgenic lines with regeneration capacity were recovered, which showed stable integration of this transgene as demonstrated by polymerase chain reaction and Southern blot hybridization. Growth of the rolA-transformed lines under greenhouse conditions provided evidence for a new biotechnological application for the rolA gene in plants, namely, the improvement of biomass production in forage grasses. Additionally, we described here a new phenotypic marker (referred to here as the “hairy embryo” syndrome) that can be instrumental for the early identification of transformation events when transforming grasses with this gene.     

Origin and evolution of fungus farming in wood-boring Coleoptera – a palaeontological perspective

Insect–fungus mutualism is one of the better-studied symbiotic interactions in nature. Ambrosia fungi are an ecological assemblage of unrelated fungi that are cultivated by ambrosia beetles in their galleries as obligate food for larvae. Despite recently increased research interest, it remains unclear which ecological factors facilitated the origin of fungus farming, and how it transformed into a symbiotic relationship with obligate dependency. It is clear from phylogenetic analyses that this symbiosis evolved independently many times in several beetle and fungus lineages. However, there is a mismatch between palaeontological and phylogenetic data. Herein we review, for the first time, the ambrosia system from a palaeontological perspective. Although largely ignored, families such as Lymexylidae and Bostrichidae should be included in the list of ambrosia beetles because some of their species cultivate ambrosia fungi. The estimated origin for some groups of ambrosia fungi during the Cretaceous concurs with a known high diversity of Lymexylidae and Bostrichidae at that time. Although potentially older, the greatest radiation of various ambrosia beetle lineages occurred in the weevil subfamilies Scolytinae and Platypodinae during the Eocene. In this review we explore the evolutionary relationship between ambrosia beetles, fungi and their host trees, which is likely to have persisted for longer than previously supposed.     

Diversity of teiid lizards from Gran Chaco and Western Cerrado (Squamata: Teiidae)

The Gran Chaco dry forest ecoregion corresponds to the southern portion of the South America diagonal belt of open formations, being one of the most threatened subtropical woodland savannas in the world. The area is still poorly known biologically and has been suffering with impressively high forest cover loss in the last 10 years. Integrating morphological and molecular data, we detected and describe a cryptic new species of lizard genus Ameivula endemic from the eastern part of this ecoregion, the called Humid Chaco. Ameivula apipensis sp nov. is characterised by a whitish brown vertebral stripe in adults and juveniles, a lateral field without ocelli and with overlapping spot, presence of an interfrontoparietal scale in 46.2% of the specimens, 12–17 femoral pores, an hemipenis without lateral sac, five xiphisternal ribs, and by a combination of meristic features as confirmed by discriminant analysis. The new species was recovered sister to a clade from Western Cerrado in our analysis, the first phylogenetic hypothesis for the Ameivula and Glaucomastix genera based on 1977 base pairs of three mitochondrial (12S, 16S and cyt‐b) and one nuclear (c‐mos) genes, including all the recognised species at the moment. Maximum parsimony and Bayesian inference recovered the monophyly of Ameivula and Glaucomastix with strong support. Reinforcing previous studies, our results suggest the presence of additional cryptic species in Ameivula from the Western Cerrado.     

Contribution to the taxonomy and phylogeny of Sarcocapnos DC. (Fumariaceae)

 To solve problems concerning the status of the taxa described in the genus Sarcocapnos, we have conducted a study using morphological, pollen morphology (light microscopy), cytogenetic and molecular techniques. Focusing on the last technique, we have sequenced ITS-1 and ITS-2 of nuclear rDNA. The species differ basically according to 5 morphological traits (leaf shape, flower spur, corolla colour, corolla size, and crest of the stigmatic surface). The cytogenetic analyses indicated n=16 to be the standard chromosome number. The ITS analyses showed that the genus is monophyletic, defining two main well-supported clades, one containing S. saetabensis and S. enneaphylla, and one containing the rest of the species. In this second clade, S. speciosa, S. pulcherrima, and S. baetica subsp. ardalii are related, as are S. integrifolia, S. crassifolia subsp. crassifolia, and S. crassifolia subsp. atlantis; S. baetica subsp. baetica forms a trichotomy with the foregoing groups. S. speciosa is shown to be a species separate from S. crassifolia subsp. crassifolia, as in the case of S. baetica with respect to S. integrifolia. Palynologically, the parameters used enabled us to establish clear differences between the taxa, often corroborating the macromorphological and genetic data. The flower spur has been reduced several times in different groups of the genus, for which the classifications established on the basis of this trait are paraphyletic. Received July 16, 2002; accepted December 11, 2002 Published online: March 31, 2003     

Changes to the functional traits of phosphoenolpyruvate carboxylase following hybridization in C-4 halophytes

Hybridization is a relevant evolutionary mechanism linked to the invasiveness of plant species, but little is known about its effect on enzymatic activities in response to stress. We analyzed the effects of salinity on key mechanistic traits of phosphoenolpyruvate carboxylase (PEPC) enzyme for two hybrid taxa derived from native Spartina maritima (Curtis) Fernald and invasive Spartina densiflora Brongn. in comparison with their parental species. Parental species showed contrasted strategies at the PEPC level to cope with salinity. Spartina maritima showed its physiological optimum at 10 to 40 ppt salinity, with high PEPC activity (per unit leaf soluble protein), in contrast to the lower salinity optimum of 0.5 and 10 ppt for S. densiflora, where highest levels of PEPC apparent specific activity coincided with high light-induced activation of PEPC. Both hybrids showed constant PEPC apparent specific activity from fresh water to hypersalinity and exhibited higher net photosynthesis rates in fresh water than their parents. Spartina maritima × densiflora presented three transgressive PEPC-related traits, being the only taxon able to increase its PEPC activation in darkness at high salinity. Spartina densiflora × maritima showed most PEPC-related traits intermediate between its parents. Inheritance types operating differently in reciprocal hybrids determine key functional traits conditioning their ecological performance.     

Dynamics of protozoan and metazoan communities in a full scale wastewater treatment plant by rotating biological contactors

Performance of a full-scale wastewater treatment plant by rotating biological contactors (RBC) system was monitored during a year by physico-chemical and microbial characterisation. Six points along wastewater treatment were selected in the plant: three points along the water line (influent, sedimentation tank and effluent) and three points along RBC system (RBC1, RBC2 and RBC3). Although a large seasonal change in the values of physico-chemical parameters was observed, operation of the plant was optimal during all year (90% of removal in BOD5 and SS influent content). Microbial characterisation was approached by determining the structure and dynamics of protozoan and metazoan communities. Protozoa were the most abundant in all stages in the plant, heterotrophic flagellates being the most representative group in the water line and ciliates in the RBC system. The same seasonal preference was only observed for heterotrophic flagellates in the water line and green flagellates in the RBC system, both groups having highest abundances in summer and spring, respectively. Identification of ciliated protozoa populations rendered 58 species of ciliates in the plant. Most of these species are typical of aerobic wastewater treatment systems except three of them, which are cited for the first time in this type of ecosystems: Chaenea stricta, Holosticha mancoidea and Oxytricha lanceolata. Along the water line 34 species were identified, and half of them only appeared occasionally (once in all the study), while along the RBC system biofilms 55 species were observed, and the majority appeared permanently in this system. Our results indicate that the type of habitat, rather than the physico-chemical water parameters, was the primary factor in determining the different distribution of protozoan and metazoan communities in the plant. In RBC biofilms, the structure of ciliate protozoa community was found to be quite sensitive to changes in physico-chemical parameters, mainly to organic loading (BOD5) variations.     

Effects of culture medium and auxins on growth of adventitious root cultures of Cuphea aequipetala Cav. and their ability to produce antioxidant compounds

Cuphea aequipetala Cav. (Lythraceae), a species highly valued for its medicinal properties, is threatened in the wild. To provide an alternative source of material for production of bioactive compounds, we established adventitious root cultures of C. aequipetala and determined their phenolic compounds contents and antioxidant activity. Cultures were initiated from root tips of in vitro C. aequipetala plantlets and were grown in B5 or SH culture medium containing either indole butyric acid (IBA) or α-naphthalene acetic acid at 0, 5 or 10 µM. The maximum root biomass (1.6 g/L dry mass (DM) per L medium) was recorded after 14 days of growth in B5 + 5 µM IBA. Roots in B5 medium remained green, whereas they tended to oxidize in SH medium. The highest contents of total phenolic compounds (9.1 ± 0.1 µg gallic acid equivalents/g DM) and flavonoids (37.5 ± 0.7 µg quercetin equivalents/g DM) were in roots grown in B5 + 5 µM IBA after 14 days of growth. Root cultures accumulated mainly flavan-3-ols, whereas roots or leaves from whole plants accumulated mainly flavonols. We analyzed the antioxidant properties of root extracts using in vitro assays. Roots grown in B5 medium showed stronger free-radical scavenging activity than that of roots grown in SH medium. Our results show that adventitious root cultures of C. aequipetala are a promising system for research on antioxidant compounds biosynthesis and for scaled-up production of useful biological materials.     

Alpha-fetoprotein in tumours derived from cystic and simple embryoid bodies.

Cellular aggregates called embryoid bodies (EB) have been obtained from the experimental teratocarcinoma (TC) 0TT6050. Two morphological types of EB can be differentiated, which are injected subcutaneously into isogenic 129/Sv mice. The tumors are collected 20 and 30 days after EB injection and processed histologically, and immunohistochemically with anti-alpha-fetoprotein (alpha-FP) antibodies. Our results indicate that the histological pattern of the tumors is related to the degree of morphological organization of the EB used.     

A structurally unique Fusobacterium nucleatum tannase provides detoxicant activity against gallotannins and pathogen resistance

Colorectal cancer pathogenesis and progression is associated with the presence of Fusobacterium nucleatum and the reduction of acetylated derivatives of spermidine, as well as dietary components such as tannin-rich foods. We show that a new tannase orthologue of F. nucleatum (TanBF_nn) has significant structural differences with its Lactobacillus plantarum counterpart affecting the flap covering the active site and the accessibility of substrates. Crystallographic and molecular dynamics analysis revealed binding of polyamines to a small cavity that connects the active site with the bulk solvent which interact with catalytically indispensable residues. As a result, spermidine and its derivatives, particularly N⁸-acetylated spermidine, inhibit the hydrolytic activity of TanBF_nn and increase the toxicity of gallotannins to F. nucleatum. Our results support a model in which the balance between the detoxicant activity of TanBF_nn and the presence of metabolic inhibitors can dictate either conducive or unfavourable conditions for the survival of F. nucleatum.