Isabel Rodrigues Gerhardt

The effect of arcelin-1 on the structure of the midgut of bruchid larvae and immunolocalization of the arcelin protein

Some wild accessions of the common bean (Phaseolus vulgaris) contain a family of proteins called arcelins, that are toxic to the larvae of certain bruchid species. Among the six allelic variants of arcelin tested so far, arcelin-5 and arcelin-1 confer the highest level of resistance against the Mexican bean weevil, Zabrotes subfasciatus. The same proteins are not toxic to the bean weevil, Acanthoscelides obtectus, which is also a serious pest of cultivated beans. Arcelins belong to the bean lectin family that includes phytohemaggutinins and alpha-amylase inhibitors. Although homologous to lectins, arcelins are themselves only very weak lectins, and their binding properties have not been clearly established. The toxic properties of arcelins may be related to their recognition of and interaction with the glycoproteins and other constituents of the membranes along the digestive tract of insects. Since arcelin-1 was shown to have growth inhibitory effects for the larvae of Z. subfasciatus but not of A. obtectus, we examined the effect of an arcelin-1 containing diet on the structure of the cells that line the intestinal tract of the larvae of these two bruchid species, and used antibodies against arcelin to examine the distribution of arcelin within the cells and tissues. Here we show that dietary arcelin-1 caused an alteration of the gut structure and the penetration of arcelin into the haemolymph in Z. subfasciatus but not in A. obtectus. These results lead us to suggest that arcelins exert their toxic effect by severely damaging the epithelial cells.

Molecular characterization of a new arcelin-5 gene.

Arcelins are insecticidal proteins found in some wild accessions of the common bean, Phaseolus vulgaris. They are grouped in six allelic variants and arcelin-5 is the variant with the highest inhibitory effect on the development of Zabrotes subfasciatus larvae. Characterization of the protein and its genes resulted in the identification of three polypeptides and the isolation of two genes that encode the Arc5a and Arc5b polypeptides. Here we describe a new gene, Arc5-III. The protein it encodes has 81% amino acid identity with the derived amino acid sequences of Arc5-I and Arc5-II. The Arc5-III gene is highly expressed in developing seeds and at a much lower level in roots. Data obtained by a combination of two-dimensional gel electrophoresis, protein sequencing and MALDI-TOF mass spectrometry analysis support the conclusion that Arc5-III encodes a polypeptide present in Arc5c band. Using ion-exchange chromatography, three fractions containing arcelin-5 polypeptides were eluted by increasing the salt concentration. The three fractions contain various amounts of the three arc-5 polypeptides and inhibit the growth of Zabrotes subfasciatus larvae differentially, suggesting differences in insecticidal activity among the arcelin-5 isoforms.

Indirect organogenesis from leaf explants of Eucalyptus benthamii x Eucalyptus dunnii and shoot multiplication

Background In Brazil, especially in the Southern region, stresses caused by cold and eventual frost are those that exert the most negative effect on the productivity of Eucalyptus spp. The genetic transformation techniques may contribute to forestry improvement programs in order to obtain genotypes expressing new interesting characteristics. They allow shortening the long breeding cycles and avoiding manipulation of adult trees. Their efficiency depends on establishment of regeneration procedures that allow the development of shoots from the transformed tissues. E. benthamiix E. dunnii hybrids have shown superiority to their parents concerning growth and frost tolerance [1], but no information about their in vitro organogenesis has been reported in the literature. The objective of this study was to evaluate the effect of some factors of culture medium on indirect organogenesis and shoot multiplication of anE. benthammi x E. dunnii clone. Methods In vitroestablished shoots,provided by EMBRAPA-Florestas (Colombo, PR, Brazil), were used as explant source. Cultures were maintained under white fluorescent tubes providing a photon flux density (PFD) of approximately 20 μmol m -2 s -1 , a 16-h photoperiod and a temperature of 25±2 °C. The cultures were performed in glass flasks containing 25 mL MS [2] medium supplemented with 1.11µM BA and sealed with rigid polypropylene caps. For the indirect organogenesis, leaves were excised from shoots at the petiole base, split into two halves and inoculated in culture media. The cultures were done in Petri dishes kept in a growth chamber in the dark throughout the experiment. The statistical design was performed in a factorial scheme (2:2:2) and a comparison was done between two culture media (MS-N/2, with half concentration of potassium and ammonium nitrates, and JADS [3] with 0.1 µM NAA, with and without PVP-40 (250 mg L -1 ) and two TDZ concentrations (0.1 and 0.5 µM). After 70 days, the percentages of explants forming callus, oxidized explants, explants producing anthocyanin, explants forming buds and shoots, and the number of shoots per explant were evaluated. For the multiplication test, the statistical design was performed in a factorial scheme (3:2) with three culture media (MS, WPM [4] and JADS, with 1.11µM BA) and two subcultures (28 and 56 days after the initial culture period). The analyzed variables for each subculture were: percentage of oxidation, of explants showing chlorosis, fresh weight and number of shoots. Results and discussion Regarding the oxidation, the higher rates (100%) were observed on JADS medium in presence or absence of PVP-40 and on MS-N/2 medium (68.3%) in presence of PVP-40. However, the JADS medium showed the highest percentage of callus formation (83.3%). In MS-N/2 medium the highest percentage of callus formation (55%) was found in the presence of PVP-40 and 0.5µM

Engineering Advantages, Challenges and Status of Sugarcane and other Sugar-Based Biomass Resources

Sugarcane (Saccharum spp.) is a highly productive tropical stem crop that has been cultivated for its high sugar content for hundreds of years. In recent times, sugarcane has been the focus of several programs aiming at the production of fuel ethanol. Compared to starch-based sources such as corn, production of ethanol from sugarcane has obvious advantages due to the amount of photosynthate accumulated during the crop cycle and the low production costs of sugarcane. The rise of cellulosic ethanol technologies will allow the conversion of part of the sugarcane lignocellulosic materials into ethanol, thus maximizing the utilization of this crop as a biofuel feedstock. Despite the rapid progress made in recent years, breeding and biotechnology have been hampered by the complex nature of sugarcane genetics and physiology. Biotechnology and marker-assisted breeding have great potential for generating cultivars and optimizing the utilization of sugarcane sucrose and lignocellulosic materials as a source of fuel ethanol. Other sugar-producing plants, such as sweet sorghum and sugar beet, are also potential biofuel sources, especially in water-limited and temperate areas, respectively, where sugarcane cultivation is not economically viable.

Overexpression of walldof transcription factor increases secondary wall deposition and alters carbon partitioning in poplar

Background Of the total worldwide biomass produced by land plants, 70% is represented by cell walls [1]. In angiosperm trees, the fixed carbon is accumulated mainly in the secondary walls of xylem cells, which turns xylem cell wall formation a major carbon sink in trees[2]. The understanding of mechanisms that regulate carbon flux to the synthesis of cell wall components is a fundamental goal to maximize the amount of lignocellulosic biomass. Several families of transcription factors have been shown to be important regulators of secondary wall biosynthesis, but how plants control and direct carbon partitioning among wall components still remains unclear [3,4]. In an attempt to identify transcription factors that participate in the regulation of carbon flux into secondary wall formation, a systematic search for poplar transcription factors expressed in a wood-preferred manner was performed. We have used the collection of over 400,000 ESTs from Populus sp. available at GenBank to determine the tissue-specific pattern of genes encoding transcription factors. Among these, a DOF (DNA-binding with one finger) domain transcription factor family member (which we named WALLDOF) that presents a cambium-preferred expression profiling was selected for further characterization. DOF proteins are plant-specific transcription factors suggested to participate in the regulation of biological processes such as light-regulated gene expression, photosynthetic carbon assimilation, accumulation of seed-storage proteins, germination, response to phytohormones, guard cell-specific gene expression, flavonoid metabolism, and lipid biosynthesis [5,6]. In this work, we report that overexpression of WALLDOF in transgenic poplar is capable of increasing secondary wall deposition and altering carbon partitioning in poplar stems.

Use of kanamycin for selection of Eucalyptus saligna genetically transformed plants

Background Several factors may affect the genetic transformation efficiency of woody species. One factor is the use of an efficient selective agent that inhibits the development of non-transformed cells and just allows the development of transformed tissues. The most used selection agent is the neomycin phosphotransferase II (NPTII) gene, which confers resistance to aminoglycoside antibiotics kanamycin, neomycin and G-418 [1]. The selective agent concentration in culture medium may have influence on shoot regeneration and high concentrations may promote adverse effects on organogenic potential [2]. The kanamycin effects in Eucalyptus are variable and depend on the species and genotypes [3]. The purpose of this study was to evaluate the effect of kanamycin concentration on transformation efficiency for Eucalyptus saligna cotyledons after co-culture with Agrobacterium tumefaciens.

In vitro shoot organogenesis from Eucalyptus sp. leaf explants

Background In vitro organogenesis is one of the key techniques associated with genetic transformation, as it determines the successful regeneration of transgenic plants after co-cultivation with bacteria. Therefore, the development of efficient regeneration protocols is the most critical step in developing genetic transformation. Protocols have been developed for several species of eucalyptus. In most of the studies cotyledon, hypocotyl and leaf segments of plants cultivated in vitro are used as explants [1]. Several eucalyptus functional genomics projects have used Populus species as a model for it counts with well established regeneration and transformation protocols. However, the use of Eucalyptus clones as model plants could be more adequate, if clones with high regeneration rates as those obtained for Populus could be found. The aim of this study was to evaluate several variables in the in vitro organogenesis from leaves of an Eucalyptus sp. clone maintained in vitro at Embrapa Forestry.