Metabolic, biochemical and molecular profiling of Catharanthus roseus flower cultivars and transformed hairy roots for monoterpenoid indole alkaloid accumulation /

Catharanthlls rosellS (L.) G Don is a commercially significant flower species and in addition is the only source of the monoterpenoid indole alkaloids (MIA) vinblastine and vincristine, which are key pharmaceutical compounds that are used to combat a number of different cancers. Therefore, procurement of the antineoplastic agents is difficult but essential procedure. Alternatively, CatharanthllS tissue cultures have been investigated as a source of these agents; however they do not produce vindoline, which is an obligate precursor to vinblastine and vincristine. The interest in developing high MIA cultivars of Catharantlws rosellS has prompted metabolic profiling studies to determine the variability of MIA accumulation of existing flowering cultivars, with particular focus on the vindoline component ofthe pathway. Metabolic profiling studies that used high performance liquid chromatography of MIAs from seedlings and young leaf extracts from 50 different flowering cultivars showed that, except for a single low vindoline cultivar (Vinca Mediterranean DP Orchid), they all accumulate similar levels of MIAs. Further enzymatic studies with extracts from young leaves and from developing seedlings showed that the low vindoline cultivar has a IO-fold lower tabersonine-16-hydroxylase activity than those of CatharanthllS rosellS cv Little Delicata. Additionally, studies aimed at metabolic engineering ofvindoline bios}l1thesis in Catharanthus rosellS hairy root cultures have been performed by expressing the last step in vindoline biosynthesis [Dcacetylvindoline-4-0- acetyltransferase (DAT)]. Enzymatic profiling studies with transformed hairy roots have confirmed that over-expressing DAT leads to lines with high levels of O-acetyltransferase activity when compared to non-expressing hairy roots. One particular DA T over111 expressing hairy root culture (line 7) contained 200 times the OAT activity than leaves of control lines. Additional MIA analyses revealed that DAT over-expressing hairy roots have an altered alkaloid profile with significant variation in the accumulation of h6rhammericine. Further analysis of transformed hairy root line 7 suggests a correlation between the expression of OAT activity and h6rhammericine accumulation with root maturation. These studies show that metabolic and selective enzymatic profiling can enhance our ability to search for relevant MIA pathway mutants and that genetic engineering with appropriate pathway genes shows promise as a tool to modify the MIA profile of Catharanthus roseus.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

The effects of dwarfing genes on seedling root growth of wheat

Most modern wheat cultivars contain major dwarfing genes, but their effects on root growth are unclear. Near-isogenic lines (NILs) containing Rht-B1b, Rht-D1b, Rht-B1c, Rht8c, Rht-D1c, and Rht12 were used to characterize the effects of semi-dwarfing and dwarfing alleles on root growth of 'Mercia' and 'Maris Widgeon' wheat cultivars. Wheat seedlings were grown in gel chambers, soil-filled columns, and in the field. Roots were extracted and length and dry mass measured. No significant differences in root length were found between semi-dwarfing lines and the control lines in any experiment, nor was there a significant difference between the root lengths of the two cultivars grown in the field. Total root length of the dwarf lines (Rht-B1c, Rht-D1c, and Rht12) was significantly different from that of the control although the effect was dependent on the experimental methodology; in gel chambers root length of dwarfing lines was increased by; 40% while in both soil media it was decreased (by 24-33%). Root dry mass was 22-30% of the total dry mass in the soil-filled column and field experiments. Root length increased proportionally with grain mass, which varied between NILs, so grain mass was a covariate for the analysis of variance. Although total root length was altered by dwarf lines, root architecture (average root diameter, lateral root: total root ratio) was not affected by reduced height alleles. A direct effect of dwarfing alleles on root growth during seedling establishment, rather than a secondary partitioning effect, was suggested by the present experiments.

Trade-off associated with selection for increased ability to resist parasitoid attack in Drosophila melanogaster

Costs of resistance are widely assumed to be important in the evolution of parasite and pathogen defence in animals, but they have been demonstrated experimentally on very few occasions. Endoparasitoids are insects whose larvae develop inside the bodies of other insects where they defend themselves from attack by their hosts' immune systems (especially cellular encapsulation). Working with Drosophila melanogaster and its endoparasitoid Leptopilina boulardi, we selected for increased resistance in four replicate populations of flies. The percentage of flies surviving attack increased from about 0.5% to between 40% and 50% in five generations, revealing substantial additive genetic variation in resistance in the field population from which our culture was established. In comparison with four control lines, flies from selected lines suffered from lower larval survival under conditions of moderate to severe intraspecific competition.

An ASIFT-based local registration method for satellite imagery

Imagery registration is a fundamental step, which greatly affects later processes in image mosaic, multi-spectral image fusion, digital surface modelling, etc., where the final solution needs blending of pixel information from more than one images. It is highly desired to find a way to identify registration regions among input stereo image pairs with high accuracy, particularly in remote sensing applications in which ground control points (GCPs) are not always available, such as in selecting a landing zone on an outer space planet. In this paper, a framework for localization in image registration is developed. It strengthened the local registration accuracy from two aspects: less reprojection error and better feature point distribution. Affine scale-invariant feature transform (ASIFT) was used for acquiring feature points and correspondences on the input images. Then, a homography matrix was estimated as the transformation model by an improved random sample consensus (IM-RANSAC) algorithm. In order to identify a registration region with a better spatial distribution of feature points, the Euclidean distance between the feature points is applied (named the S criterion). Finally, the parameters of the homography matrix were optimized by the Levenberg–Marquardt (LM) algorithm with selective feature points from the chosen registration region. In the experiment section, the Chang’E-2 satellite remote sensing imagery was used for evaluating the performance of the proposed method. The experiment result demonstrates that the proposed method can automatically locate a specific region with high registration accuracy between input images by achieving lower root mean square error (RMSE) and better distribution of feature points.

Molecular and phenotypic characterization of the alternative seasonal growth habit and flowering time in barley (Hordeum vulgare ssp. vulgare L.)

Barley can be classified into three major agronomic types, based on its seasonal growth habit (SGH): spring, winter and alternative. Winter varieties require exposure to vernalization to promote subsequent flowering and are autumn-sown. Spring varieties proceed to flowering in the absence of vernalization and are sown in the spring. The ‘alternative’ (also known as ‘facultative’) SGH is only loosely defined and can be sown in autumn or spring. Here, we investigate the molecular genetic basis of alternative barley. Analysis of the major barley vernalization (VRN-H1, VRN-H2) and photoperiod (PPD-H1, PPD-H2) response genes in a collection of 386 varieties found alternative SGH to be characterized by specific allelic combinations. Spring varieties possessed spring loci at one or both of the vernalization response loci, combined with long-day non-responsive ppd-H1 alleles and wild-type alleles at the short-day photoperiod response locus, PPD-H2. Winter varieties possessed winter alleles at both vernalization loci, in combination with the mutant ppd-H2 allele conferring delayed flowering under short-day photoperiods. In contrast, all alternative varieties investigated possessed a single spring allele (either at VRN-H1 or at VRN-H2) combined with mutant ppd-H2 alleles. This allelic combination is found only in alternative types and is diagnostic for alternative SGH in the collection studied. Analysis of flowering time under controlled environment found alternative varieties flowered later than spring control lines, with the difference most pronounced under short-day photoperiods. This work provides genetic characterization of the alternative SGH phenotype, allowing precise manipulation of SGH and flowering time within breeding programmes, and provides the molecular tools for classification of all three SGH categories within national variety registration processes.

Heritability of corticosterone response and changes in life history traits during selection in the zebra finch

Vertebrates respond to environmental stressors through the neuro-endocrine stress response, which involves the production of glucocorticoids. We have selected independent, duplicate divergent lines of zebra finches for high, low and control corticosterone responses to a mild stressor. This experiment has shown that over the first four generations, the high lines have demonstrated a significant realized heritability of about 20%. However, the low lines have apparently not changed significantly from controls. This asymmetry in response is potentially because of the fact that all birds appear to be showing increased adaptation to the environment in which they are housed, with significant declines in corticosterone response in control lines as well as low lines. Despite the existence of two- to threefold difference in mean corticosterone titre between high and low lines, there were no observed differences in testosterone titre in adult male birds from the different groups. In addition, there were no consistent, significant differences between the lines in any of the life history variables measured – number of eggs laid per clutch, number of clutches or broods produced per pair, number of fledglings produced per breeding attempt, nor in any of egg, nestling and fledgling mortality. These results highlight the fact that the mechanisms that underlie variation in the avian physiological system can be modified to respond to differences between environments through selection. This adds an additional level of flexibility to the avian physiological system, which will allow it to respond to environmental circumstances.

Ozbot and haptics : remote surveillance to physical presence

This paper reports on robotic and haptic technologies and capabilities developed for the law enforcement and defence community within Australia by the Centre for Intelligent Systems Research (CISR). The OzBot series of small and medium surveillance robots have been designed in Australia and evaluated by law enforcement and defence personnel to determine suitability and ruggedness in a variety of environments. Using custom developed digital electronics and featuring expandable data busses including RS485, I2C, RS232, video and Ethernet, the robots can be directly connected to many off the shelf payloads such as gas sensors, x-ray sources and camera systems including thermal and night vision. Differentiating the OzBot platform from its peers is its ability to be integrated directly with haptic technology or the 'haptic bubble' developed by CISR. Haptic interfaces allow an operator to physically 'feel' remote environments through position-force control and experience realistic force feedback. By adding the capability to remotely grasp an object, feel its weight, texture and other physical properties in real-time from the remote ground control unit, an operator's situational awareness is greatly improved through Haptic augmentation in an environment where remote-system feedback is often limited.

Are voluntary wheel running and open-field behavior correlated in mice? Different answers from comparative and artificial selection approaches

Voluntary wheel running and open-field behavior are probably the two most widely used measures of locomotion in laboratory rodents. We tested whether these two behaviors are correlated in mice using two approaches: the phylogenetic comparative method using inbred strains of mice and an ongoing artificial selection experiment on voluntary wheel running. After taking into account the measurement error and phylogenetic relationships among inbred strains, we obtained a significant positive correlation between distance run on wheels and distance moved in the open-field for both sexes. Thigmotaxis was negatively correlated with distance run on wheels in females but not in males. By contrast, mice from four replicate lines bred for high wheel running did not differ in either distance covered or thigmotaxis in the open field as compared with mice from four non-selected control lines. Overall, results obtained in the selection experiment were generally opposite to those observed among inbred strains. Possible reasons for this discrepancy are discussed.

Orientação de imagens CBERS-2B usando o modelo rigoroso de colinearidade com dados orbitais

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito da seleção quanto ao peso corporal nas características reprodutivas em linhagens de frangos de corte

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Orientação semi-automática de imagens CBERS usando rodovias como controle de campo

In this paper is proposed a methodology for semiautomatic CBERS image orientation using roads as ground control. It is based on an iterative strategy involving three steps. In the first step, an operator identifies on the image the ground control roads and supplies along them a few seed points, which could be sparsely and coarsely distributed. These seed points are used by the dynamic programming algorithm for extracting the ground control roads from the image. In the second step, it is established the correspondences between points describing the ground control roads and the corresponding ones extracted from the image. In the last step, the corresponding points are used to orient the CBERS image by using the DLT (Direct Linear Transformation). The two last steps are iterated until the convergence of the orientation process is verified. Experimental results showed that the proposed methodology was efficient with several test images. In all cases the orientation process converged. Moreover, the estimated orientation parameters allowed the registration of check roads with pixel accuracy or better.

Running behavior and its energy cost in mice selectively bred for high voluntary locomotor activity

Locomotion is central to behavior and intrinsic to many fitnesscritical activities (e.g., migration, foraging), and it competes with other life-history components for energy. However, detailed analyses of how changes in locomotor activity and running behavior affect energy budgets are scarce. We quantified these effects in four replicate lines of house mice that have been selectively bred for high voluntary wheel running (S lines) and in their four nonselected control lines (C lines). We monitored wheel speeds and oxygen consumption for 24-48 h to determine daily energy expenditure (DEE), resting metabolic rate (RMR), locomotor costs, and running behavior (bout characteristics). Daily running distances increased roughly 50%-90% in S lines in response to selection. After we controlled for body mass effects, selection resulted in a 23% increase in DEE in males and a 6% increase in females. Total activity costs (DEE - RMR) accounted for 50%-60% of DEE in both S and C lines and were 29% higher in S males and 5% higher in S females compared with their C counterparts. Energetic costs of increased daily running distances differed between sexes because S females evolved higher running distances by running faster with little change in time spent running, while S males also spent 40% more time running than C males. This increase in time spent running impinged on high energy costs because the majority of running costs stemmed from postural costs (the difference between RMR and the zero-speed intercept of the speed vs. metabolic rate relationship). No statistical differences in these traits were detected between S and C females, suggesting that large changes in locomotor behavior do not necessarily effect overall energy budgets. Running behavior also differed between sexes: within S lines, males ran with more but shorter bouts than females. Our results indicate that selection effects on energy budgets can differ dramatically between sexes and that energetic constraints in S males might partly explain the apparent selection limit for wheel running observed for over 15 generations. © 2009 by The University of Chicago. All rights reserved.

Orientação semi-automática de imagens CBERS usando rodovias como controle de campo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Automatic Orientation of Multi-Scale Terrestrial Images for 3D Reconstruction

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)