Design innovation for disability in Kasese : final technical report

Imported wheelchairs are unable to function given the state of roads and infrastructure, nor are repairs possible. The project focus was to assist CanUgan in designing a hand operated tricycle to be built locally in Kasese, western Uganda. The overall objective was to involve various stakeholders in designing suitable devices for persons with disabilities to be built and maintained by local tradespeople. The project is described with details and references to prototypes, outcomes, and spin-off projects; the research expanded to include a series of income-generating inventions envisioned by the end users through their participation in the project, and produced locally

Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants

The flavonoid-derived proanthocyanidins (PAs) are one class of the major defence phenolics in poplar leaves. Transcriptional activation of PA biosynthetic genes, resulting in PA accumulation in leaves, was detected following infection by the fungal Marssonina brunnea f.sp. multigermtubi using digital gene expression analysis. In order to study PA biosynthesis and its induction by fungi, a putative leucoanthocyanidin reductase gene, PtrLAR3, was isolated from Populus trichocarpa. Sequence comparison of PtrLAR3 with other known leucoanthocyanidin reductase proteins revealed high amino acid sequence similarity. Semi-quantitative reverse-transcription (RT) PCR and quantitative real-time PCR analysis demonstrated that PtrLAR3 was expressed in various tissues and the highest level of expression was observed in roots. Overexpression of PtrLAR3 in Chinese white poplar (Populus tomentosa Carr.) led to a significant plant-wide increase in PA levels. In vitro assays showed that crude leaf extracts from 35S:PtrLAR3 transformants were able to inhibit significantly the hyphal growth of M. brunnea f.sp. multigermtubi compared to the extracts from control plants. The transgenic 35S:PtrLAR3 poplar plants displayed a significant (P < 0.05) reduction in their disease symptoms compared with the control. RT-PCR analysis showed that PtrLAR3 expression was up-regulated in all transformants. These results suggested that constitutive expression of endogenous PtrLAR3 could be exploited to improve resistance to fungal pathogens in poplar

Reference gene validation for quantitative RT-PCR during biotic and abiotic stresses in Vitis vinifera

Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studiesinfo:eu-repo/semantics/publishedVersio

The R2R3-MYB Transcription Factor Gene Family in Maize

MYB proteins comprise a large family of plant transcription factors, members of which perform a variety of functions in plant biological processes. To date, no genome-wide characterization of this gene family has been conducted in maize (Zea mays). In the present study, we performed a comprehensive computational analysis, to yield a complete overview of the R2R3-MYB gene family in maize, including the phylogeny, expression patterns, and also its structural and functional characteristics. The MYB gene structure in maize and Arabidopsis were highly conserved, indicating that they were originally compact in size. Subgroup-specific conserved motifs outside the MYB domain may reflect functional conservation. The genome distribution strongly supports the hypothesis that segmental and tandem duplication contribute to the expansion of maize MYB genes. We also performed an updated and comprehensive classification of the R2R3-MYB gene families in maize and other plant species. The result revealed that the functions were conserved between maize MYB genes and their putative orthologs, demonstrating the origin and evolutionary diversification of plant MYB genes. Species-specific groups/subgroups may evolve or be lost during evolution, resulting in functional divergence. Expression profile study indicated that maize R2R3-MYB genes exhibit a variety of expression patterns, suggesting diverse functions. Furthermore, computational prediction potential targets of maize microRNAs (miRNAs) revealed that miR159, miR319, and miR160 may be implicated in regulating maize R2R3-MYB genes, suggesting roles of these miRNAs in post-transcriptional regulation and transcription networks. Our comparative analysis of R2R3-MYB genes in maize confirm and extend the sequence and functional characteristics of this gene family, and will facilitate future functional analysis of the MYB gene family in maize

Transgenic upregulation of the condensed tannin pathway in poplar leads to a dramatic shift in leaf palatability for two tree-feeding Lepidoptera

Transgenic hybrid aspen (Populus tremula x tremuloides) overexpressing the MYB134 tannin regulatory gene show dramatically enhanced condensed tannin (proanthocyanidin) levels, as well as shifts in other phenolic metabolites. A series of insect bioassays with forest tent caterpillars (Malacosoma disstria) and gypsy moth (Lymantria dispar) caterpillars was carried out to determine how this metabolic shift affects food preference and performance of generalist tree-feeding lepidopterans. Both species showed a distinct preference for the high-tannin MYB134 overexpressor plants, and L. dispar performance was enhanced relative to controls. L. dispar reached greater pupal weight and showed reduced time to pupation when reared on the MYB134 overexpressing poplar. These results were unexpected since enhanced condensed tannin levelswere predicted to act as feeding deterrents. However, the data may be explained by the observed decrease in the salicinoids (phenolic glycosides) salicortin and tremulacin that accompanied the upregulation of the condensed tannins in the transgenics. We conclude that for these two lepidopteran species, condensed tannin levels are unlikely to be a major determinant of caterpillar food preference or performance. However, our experiments show that overexpression of a single regulatory gene in transgenic aspen can have a significant impact on herbivorous insects

The Wound-, Pathogen-, and Ultraviolet B-Responsive MYB134 Gene Encodes an R2R3 MYB Transcription Factor That Regulates Proanthocyanidin Synthesis in Poplar1[W][OA]

In poplar (Populus spp.), the major defense phenolics produced in leaves are the flavonoid-derived proanthocyanidins (PAs) and the salicin-based phenolic glycosides. Transcriptional activation of PA biosynthetic genes leading to PA accumulation in leaves occurs following herbivore damage and mechanical wounding as well as infection by the fungal biotroph Melampsora medusae. In this study, we have identified a poplar R2R3 MYB transcription factor gene, MYB134, that exhibits close sequence similarity to the Arabidopsis (Arabidopsis thaliana) PA regulator TRANSPARENT TESTA2 and that is coinduced with PA biosynthetic genes following mechanical wounding, M. medusae infection, and exposure to elevated ultraviolet B light. Overexpression of MYB134 in poplar resulted in transcriptional activation of the full PA biosynthetic pathway and a significant plant-wide increase in PA levels, and electrophoretic mobility shift assays showed that recombinant MYB134 protein is able to bind to promoter regions of PA pathway genes. MYB134-overexpressing plants exhibited a concomitant reduction in phenolic glycoside concentrations and other minor alterations to levels of small phenylpropanoid metabolites. Our data provide insight into the regulatory mechanisms controlling stress-induced PA metabolism in poplar, and the identification of a regulator of stress-responsive PA biosynthesis constitutes a valuable tool for manipulating PA metabolism in poplar and investigating the biological functions of PAs in resistance to biotic and abiotic stresses

Poplar MYB115 and MYB134 transcription factors regulate proanthocyanidin synthesis and structure

The accumulation of proanthocyanidins is regulated by a complex of transcription factors composed of R2R3 MYB, basic helix-loop-helix, and WD40 proteins that activate the promoters of biosynthetic genes. In poplar (genus Populus), MYB134 is known to regulate proanthocyanidin biosynthesis by activating key flavonoid genes. Here, we characterize a second MYB regulator of proanthocyanidins, MYB115. Transgenic poplar overexpressing MYB115 showed a high-proanthocyanidin phenotype and reduced salicinoid accumulation, similar to the effects of MYB134 overexpression. Transcriptomic analysis of MYB115- and MYB134-overexpressing poplar plants identified a set of common up-regulated genes encoding proanthocyanidin biosynthetic enzymes and several novel uncharacterized MYB transcriptional repressors. Transient expression experiments demonstrated the capacity of both MYB134 and MYB115 to activate flavonoid promoters, but only in the presence of a basic helix-loop-helix cofactor. Yeast two-hybrid experiments confirmed the direct interaction of these transcription factors. The unexpected identification of dihydromyricetin in leaf extracts of both MYB115- and MYB134-overexpressing poplar led to the discovery of enhanced flavonoid B-ring hydroxylation and an increased proportion of prodelphinidins in proanthocyanidin of the transgenics. The dramatic hydroxylation phenotype of MYB115 overexpressors is likely due to the up-regulation of both flavonoid 3′,5′-hydroxylases and cytochrome b5. Overall, this work provides new insight into the complexity of the gene regulatory network for proanthocyanidin synthesis in poplar