Browsing by Author "Jacobson, Dan"

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    Constitutive expression of a grapevine polygalacturonase-inhibiting protein affects gene expression and cell wall properties in uninfected tobacco
    (BioMed Central, 2011-11) Alexandersson, Erik; Becker, John V. W.; Jacobson, Dan; Nguema-Ona, Eric; Steyn, Cobus; Denby, Katherine J.; Vivier, Melane A.
    Abstract: Background Polygalacturonase-inhibiting proteins (PGIPs) directly limit the effective ingress of fungal pathogens by inhibiting cell wall-degrading endopolygalacturonases (ePGs). Transgenic tobacco plants over-expressing grapevine (Vitis vinifera) Vvpgip1 have previously been shown to be resistant to Botrytis infection. In this study we characterized two of these PGIP over-expressing lines with known resistance phenotypes by gene expression and hormone profiling in the absence of pathogen infection. Results Global gene expression was performed by a cross-species microarray approach using a potato cDNA microarray. The degree of potential cross-hybridization between probes was modeled by a novel computational workflow designed in-house. Probe annotations were updated by predicting probe-to-transcript hybridizations and combining information derived from other plant species. Comparing uninfected Vvpgip1-overexpressing lines to wild-type (WT), 318 probes showed significant change in expression. Functional groups of genes involved in metabolism and associated to the cell wall were identified and consequent cell wall analysis revealed increased lignin-levels in the transgenic lines, but no major differences in cell wall-derived polysaccharides. GO enrichment analysis also identified genes responsive to auxin, which was supported by elevated indole-acetic acid (IAA) levels in the transgenic lines. Finally, a down-regulation of xyloglucan endotransglycosylase/hydrolases (XTHs), which are important in cell wall remodeling, was linked to a decrease in total XTH activity. Conclusions This evaluation of PGIP over-expressing plants performed under pathogen-free conditions to exclude the classical PGIP-ePG inhibition interaction indicates additional roles for PGIPs beyond the inhibition of ePGs.
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    The genes and enzymes of the carotenoid metabolic pathway in Vitis vinifera L
    (BioMed Central, 2012-06) Young, Philip R.; Lashbrooke, Justin G.; Alexandersson, Erik; Jacobson, Dan; Moser, Claudio; Velasco, Riccardo; Vivier, Melané A.
    Abstract Background Carotenoids are a heterogeneous group of plant isoprenoids primarily involved in photosynthesis. In plants the cleavage of carotenoids leads to the formation of the phytohormones abscisic acid and strigolactone, and C13-norisoprenoids involved in the characteristic flavour and aroma compounds in flowers and fruits and are of specific importance in the varietal character of grapes and wine. This work extends the previous reports of carotenoid gene expression and photosynthetic pigment analysis by providing an up-to-date pathway analysis and an important framework for the analysis of carotenoid metabolic pathways in grapevine. Results Comparative genomics was used to identify 42 genes putatively involved in carotenoid biosynthesis/catabolism in grapevine. The genes are distributed on 16 of the 19 chromosomes and have been localised to the physical map of the heterozygous ENTAV115 grapevine sequence. Nine of the genes occur as single copies whereas the rest of the carotenoid metabolic genes have more than one paralogue. The cDNA copies of eleven corresponding genes from Vitis vinifera L. cv. Pinotage were characterised, and four where shown to be functional. Microarrays provided expression profiles of 39 accessions in the metabolic pathway during three berry developmental stages in Sauvignon blanc, whereas an optimised HPLC analysis provided the concentrations of individual carotenoids. This provides evidence of the functioning of the lutein epoxide cycle and the respective genes in grapevine. Similarly, orthologues of genes leading to the formation of strigolactone involved in shoot branching inhibition were identified: CCD7, CCD8 and MAX1. Moreover, the isoforms typically have different expression patterns, confirming the complex regulation of the pathway. Of particular interest is the expression pattern of the three VvNCEDs: Our results support previous findings that VvNCED3 is likely the isoform linked to ABA content in berries. Conclusions The carotenoid metabolic pathway is well characterised, and the genes and enzymes have been studied in a number of plants. The study of the 42 carotenoid pathway genes of grapevine showed that they share a high degree of similarity with other eudicots. Expression and pigment profiling of developing berries provided insights into the most complete grapevine carotenoid pathway representation. This study represents an important reference study for further characterisation of carotenoid biosynthesis and catabolism in grapevine.
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    GSA-PCA : gene set generation by principal component analysis of the Laplacian matrix of a metabolic network
    (BioMed Central, 2012-08) Jacobson, Dan; Emerton, Guy
    Abstract Background Gene Set Analysis (GSA) has proven to be a useful approach to microarray analysis. However, most of the method development for GSA has focused on the statistical tests to be used rather than on the generation of sets that will be tested. Existing methods of set generation are often overly simplistic. The creation of sets from individual pathways (in isolation) is a poor reflection of the complexity of the underlying metabolic network. We have developed a novel approach to set generation via the use of Principal Component Analysis of the Laplacian matrix of a metabolic network. We have analysed a relatively simple data set to show the difference in results between our method and the current state-of-the-art pathway-based sets. Results The sets generated with this method are semi-exhaustive and capture much of the topological complexity of the metabolic network. The semi-exhaustive nature of this method has also allowed us to design a hypergeometric enrichment test to determine which genes are likely responsible for set significance. We show that our method finds significant aspects of biology that would be missed (i.e. false negatives) and addresses the false positive rates found with the use of simple pathway-based sets. Conclusions The set generation step for GSA is often neglected but is a crucial part of the analysis as it defines the full context for the analysis. As such, set generation methods should be robust and yield as complete a representation of the extant biological knowledge as possible. The method reported here achieves this goal and is demonstrably superior to previous set analysis methods.
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    Identifying genes that impact on aroma profiles produced by Saccharomyces cerevisiae and the production of higher alcohols
    (Springer-Verslag, 2011-05) Styger, Gustav; Jacobson, Dan; Bauer, Florian
    During alcoholic fermentation, many volatile aroma compounds are formed by Saccharomyces cerevisiae, including esters, fatty acids, and higher alcohols. While the metabolic network that leads to the formation of these compounds is reasonably well mapped, surprisingly little is known about specific enzymes involved in specific reactions, the regulation of the network, and the physiological roles of individual pathways within the network. Furthermore, different yeast strains tend to produce significantly different aroma profiles. These differences are of tremendous biotechnological interest, since producers of alcoholic beverages such as wine and beer are searching for means to diversify and improve their product range. Various factors such as the redox, energy, and nutritional balance of a cell have previously been suggested to directly or indirectly affect and regulate the network. To gain a better understanding of the regulations and physiological role of this network, we screened a subset of the EUROSCARF strain deletion library for genes that, when deleted, would impact most significantly on the aroma profile produced under fermentative conditions. The 10 genes whose deletion impacted most significantly on higher alcohol production were selected and further characterized to assess their mode of action within or on this metabolic network. This is the first description of a large-scale screening approach using aroma production as the primary selection criteria, and the data suggest that many of the identified genes indeed play central and direct roles within the aroma production network of S. cerevisiae.
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    Many saccharomyces cerevisiae cell wall protein encoding genes are coregulated by Mss11, but cellular adhesion phenotypes appear only flo protein dependent
    (Genetics Society of America, 2012-01) Bester, Michael C.; Jacobson, Dan; Bauer, Florian
    The outer cell wall of the yeast Saccharomyces cerevisiae serves as the interface with the surrounding environment and directly affects cell2cell and cell2surface interactions. Many of these interactions are facilitated by specific adhesins that belong to the Flo protein family. Flo mannoproteins have been implicated in phenotypes such as flocculation, substrate adhesion, biofilm formation, and pseudohyphal growth. Genetic data strongly suggest that individual Flo proteins are responsible for many specific cellular adhesion phenotypes. However, it remains unclear whether such phenotypes are determined solely by the nature of the expressed FLO genes or rather as the result of a combination of FLO gene expression and other cell wall properties and cell wall proteins. Mss11 has been shown to be a central element of FLO1 and FLO11 gene regulation and acts together with the cAMP-PKA-dependent transcription factor Flo8. Here we use genome-wide transcription analysis to identify genes that are directly or indirectly regulated by Mss11. Interestingly, many of these genes encode cell wall mannoproteins, in particular, members of the TIR and DAN families. To examine whether these genes play a role in the adhesion properties associated with Mss11 expression, we assessed deletion mutants of these genes in wild-type and flo11D genetic backgrounds. This analysis shows that only FLO genes, in particular FLO1/10/11, appear to significantly impact on such phenotypes. Thus adhesion-related phenotypes are primarily dependent on the balance of FLO gene expression.
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    Proteomic analysis of grape berry cell cultures reveals that developmentally regulated ripening related processes can be studied using cultured cells
    (Public Library of Science (PLOS), 2011-02) Sharathchandra, Ramaschandra G.; Stander, Charmaine; Jacobson, Dan; Ndimba, Bongani; Vivier, Melane A.
    Background: This work describes a proteomics profiling method, optimized and applied to berry cell suspensions to evaluate organ-specific cultures as a platform to study grape berry ripening. Variations in berry ripening within a cluster(s) on a vine and in a vineyard are a major impediment towards complete understanding of the functional processes that control ripening, specifically when a characterized and homogenous sample is required. Berry cell suspensions could overcome some of these problems, but their suitability as a model system for berry development and ripening needs to be established first. Methodology/Principal Findings: In this study we report on the proteomic evaluation of the cytosolic proteins obtained from synchronized cell suspension cultures that were established from callus lines originating from green, véraison and ripe Vitis vinifera berry explants. The proteins were separated using liquid phase IEF in a Microrotofor cell and SDS PAGE. This method proved superior to gel-based 2DE. Principal component analysis confirmed that biological and technical repeats grouped tightly and importantly, showed that the proteomes of berry cultures originating from the different growth/ripening stages were distinct. A total of twenty six common bands were selected after band matching between different growth stages and twenty two of these bands were positively identified. Thirty two % of the identified proteins are currently annotated as hypothetical. The differential expression profile of the identified proteins, when compared with published literature on grape berry ripening, suggested common trends in terms of relative abundance in the different developmental stages between real berries and cell suspensions. Conclusions: The advantages of having suspension cultures that accurately mimic specific developmental stages are profound and could significantly contribute to the study of the intricate regulatory and signaling networks responsible for berry development and ripening. © 2011 Sharathchandra et al.
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    Proteomics and transcriptomics of the BABA-induced resistance response in potato using a novel functional annotation approach
    (BioMed Central, 2014-04) Bengtsson, Therese; Weighill, Deborah; Proux-Wera, Estelle; Levander, Fredrik; Resjo, Svante; Burra, Dharani Dhar; Moushib, Laith Ibrahim; Hedley, Pete E.; Liljeroth, Erland; Jacobson, Dan; Alexandersson, Erik; Andreasson, Erik
    Abstract Background Induced resistance (IR) can be part of a sustainable plant protection strategy against important plant diseases. β-aminobutyric acid (BABA) can induce resistance in a wide range of plants against several types of pathogens, including potato infected with Phytophthora infestans. However, the molecular mechanisms behind this are unclear and seem to be dependent on the system studied. To elucidate the defence responses activated by BABA in potato, a genome-wide transcript microarray analysis in combination with label-free quantitative proteomics analysis of the apoplast secretome were performed two days after treatment of the leaf canopy with BABA at two concentrations, 1 and 10 mM. Results Over 5000 transcripts were differentially expressed and over 90 secretome proteins changed in abundance indicating a massive activation of defence mechanisms with 10 mM BABA, the concentration effective against late blight disease. To aid analysis, we present a more comprehensive functional annotation of the microarray probes and gene models by retrieving information from orthologous gene families across 26 sequenced plant genomes. The new annotation provided GO terms to 8616 previously un-annotated probes. Conclusions BABA at 10 mM affected several processes related to plant hormones and amino acid metabolism. A major accumulation of PR proteins was also evident, and in the mevalonate pathway, genes involved in sterol biosynthesis were down-regulated, whereas several enzymes involved in the sesquiterpene phytoalexin biosynthesis were up-regulated. Interestingly, abscisic acid (ABA) responsive genes were not as clearly regulated by BABA in potato as previously reported in Arabidopsis. Together these findings provide candidates and markers for improved resistance in potato, one of the most important crops in the world.