Differential regulation of gene products in newly synthesized Brassica napus allotetraploids is not related to protein function nor subcellular localization

BACKGROUND: Allopolyploidy is a preeminent process in plant evolution that results from the merger of distinct genomes in a common nucleus via inter-specific hybridization. Allopolyploid formation is usually related to genome-wide structural and functional changes though the underlying mechanisms operating during this "genomic shock" still remain poorly known. The aim of the present study was to investigate the modifications occurring at the proteomic level following an allopolyploidization event and to determine whether these changes are related to functional properties of the proteins. In a previous report, we applied comparative proteomics to synthetic amphiploids of Brassica napus and to its diploid progenitors B. rapa and B. oleracea. Although several hundred polypeptides displayed additivity (i.e. mid-parent values) in the amphiploids, many of them showed non-additivity. Here, we report the in silico functional characterization of the "non-additive" proteins (the ones with a non-additive pattern of regulation) in synthetic B. napus. RESULTS: The complete set of non-additive proteins (335 in the stem and 205 in the root), as well as a subset of additive polypeptides (200 per organ), was identified by mass spectrometry. Several protein isoforms were found, and most of them (~55%) displayed "different" or "opposite" patterns of regulation in the amphiploids, i.e. isoforms of the same protein showing both up-regulation and down-regulation in the synthetic B. napus compared to the mid-parent value. Components of protein complexes were identified of which ~50% also displayed "different" or "opposite" patterns of regulation in the allotetraploids. In silico functional categorization of the identified proteins was carried out, and showed that neither functional category nor metabolic pathway were systematically affected by non-additivity in the synthetic amphiploids. In addition, no subcellular compartment was found to be over- or under-represented among the proteins displaying non-additive values in the allopolyploids. CONCLUSION: Protein identification showed that functionally related polypeptides (isoforms and complex subunits) could be differentially regulated in synthetic B. napus in comparison to its diploid progenitors while such proteins are usually expected to display co-regulation. The genetic redundancy within an allopolyploid could explain why functionally related proteins could display imbalanced levels of expression. No functional category, no metabolic pathway and no subcellular localization was found to be over- or under-represented within non-additive polypeptides, suggesting that the differential regulation of gene products was not related to functional properties of the proteins. Thus, at the protein level, there is no evidence for the "genomic shock" expected in neo-polyploids and the overall topology of protein networks and metabolic pathways is conserved in synthetic allotetraploids of B. napus in comparison to its diploid progenitors B. rapa and B. oleracea

SSU1 Checkup, a Rapid Tool for Detecting Chromosomal Rearrangements Related to the SSU1 Promoter in Saccharomyces cerevisiae: An Ecological and Technological Study on Wine Yeast

Chromosomal rearrangements (CR) such as translocations, duplications and inversions play a decisive role in the adaptation of microorganisms to specific environments. In enological Saccharomyces cerevisiae strains, CR involving the promoter region of the gene SSU1 lead to a higher sulfite tolerance by enhancing the SO2 efflux. To date, three different SSU1 associated CR events have been described, including translocations XV-t-XVI and VIII-t-XVI and inversion inv-XVI. In the present study, we developed a multiplex PCR method (SSU1 checkup) that allows a rapid characterization of these three chromosomal configurations in a single experiment. Nearly 600 S. cerevisiae strains collected from fermented grape juice were genotyped by microsatellite markers. We demonstrated that alleles of the SSU1 promoter are differently distributed according to the wine environment (cellar versus vineyard) and the nature of the grape juice. Moreover, rearranged SSU1 promoters are significantly enriched among commercial starters. In addition, the analysis of nearly isogenic strains collected in wine related environments demonstrated that the inheritance of these CR shapes the genetic diversity of clonal populations. Finally, the link between the nature of SSU1 promoter and the tolerance to sulfite was statistically validated in natural grape juice containing various SO2 concentrations. The SSU1 checkup is therefore a convenient new tool for addressing population genetics questions and for selecting yeast strains by using molecular markers.Fil: Marullo, Philippe. Universite de Bordeaux; FranciaFil: Claisse, Olivier. Universite de Bordeaux; FranciaFil: Raymond Eder, María Laura. Universidad Católica de Córdoba. Instituto de Investigaciones en Recursos Naturales y Sustentabilidad José Sanchez Labrador S. J. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Recursos Naturales y Sustentabilidad José Sanchez Labrador S. J.; ArgentinaFil: Börlin, Marine. Universite de Bordeaux; FranciaFil: Feghali, Nadine. Lebanese University; LíbanoFil: Bernard, Margaux. Universite de Bordeaux; FranciaFil: Legras, Jean Luc. Université Montpellier II; FranciaFil: Albertin, Warren. Universite de Bordeaux; FranciaFil: Rosa, Alberto Luis. Universidad Católica de Córdoba. Instituto de Investigaciones en Recursos Naturales y Sustentabilidad José Sanchez Labrador S. J. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Recursos Naturales y Sustentabilidad José Sanchez Labrador S. J.; ArgentinaFil: Masneuf Pomarede, Isabelle. Universite de Bordeaux; Franci

Metabolic, organoleptic and transcriptomic impact of saccharomyces cerevisiae genes involved in the biosynthesis of linear and substituted esters

Esters constitute a broad family of volatile compounds impacting the organoleptic properties of many beverages, including wine and beer. They can be classified according to their chemical structure. Higher alcohol acetates differ from fatty acid ethyl esters, whereas a third group, substituted ethyl esters, contributes to the fruitiness of red wines. Derived from yeast metabolism, the biosynthesis of higher alcohol acetates and fatty acid ethyl esters has been widely investigated at the enzymatic and genetic levels. As previously reported, two pairs of esterases, respectively encoded by the paralogue genes ATF1 and ATF2, and EEB1 and EHT1, are mostly involved in the biosynthesis of higher alcohol acetates and fatty acid ethyl esters. These esterases have a moderate effect on the biosynthesis of substituted ethyl esters, which depend on mono-acyl lipases encoded by MGL2 and YJU3. The functional characterization of such genes helps to improve our understanding of substituted ester metabolism in the context of wine alcohol fermentation. In order to evaluate the overall sensorial impact of esters, we attempted to produce young red wines without esters by generating a multiple esterase-free strain (Δatf1, Δatf2, Δeeb1, and Δeht1). Surprisingly, it was not possible to obtain the deletion of MGL2 in the Δatf1/Δatf2/Δeeb1/Δeht1 background, highlighting unsuspected genetic incompatibilities between ATF1 and MGL2. A preliminary RNA-seq analysis depicted the overall effect of the Δatf1/Δatf2/Δeeb1/Δeht1 genotype that triggers the expression shift of 1124 genes involved in nitrogen and lipid metabolism, but also chromatin organization and histone acetylation. These findings reveal unsuspected regulatory roles of ester metabolism in genome expression for the first time

Front Microbiol

Chromosomal rearrangements (CR) such as translocations, duplications and inversions play a decisive role in the adaptation of microorganisms to specific environments. In enological strains, CR involving the promoter region of the gene lead to a higher sulfite tolerance by enhancing the SO efflux. To date, three different associated CR events have been described, including translocations XV-t-XVI and VIII-t-XVI and inversion inv-XVI. In the present study, we developed a multiplex PCR method ( checkup) that allows a rapid characterization of these three chromosomal configurations in a single experiment. Nearly 600 strains collected from fermented grape juice were genotyped by microsatellite markers. We demonstrated that alleles of the promoter are differently distributed according to the wine environment (cellar versus vineyard) and the nature of the grape juice. Moreover, rearranged promoters are significantly enriched among commercial starters. In addition, the analysis of nearly isogenic strains collected in wine related environments demonstrated that the inheritance of these CR shapes the genetic diversity of clonal populations. Finally, the link between the nature of promoter and the tolerance to sulfite was statistically validated in natural grape juice containing various SO concentrations. The checkup is therefore a convenient new tool for addressing population genetics questions and for selecting yeast strains by using molecular markers

High intraspecific variation of the cell surface physico-chemical and bioadhesion properties in Brettanomyces bruxellensis

Brettanomyces bruxellensis is the most damaging spoilage yeast in the wine industry because of its negative impact on the wine organoleptic qualities. The strain persistence in cellars over several years associated with recurrent wine contamination suggest specific properties to persist and survive in the environment through bioadhesion phenomena. In this work, the physico-chemical surface properties, morphology and ability to adhere to stainless steel were studied both on synthetic medium and on wine. More than 50 strains representative of the genetic diversity of the species were considered. Microscopy techniques made it possible to highlight a high morphological diversity of the cells with the presence of pseudohyphae forms for some genetic groups. Analysis of the physico-chemical properties of the cell surface reveals contrasting behaviors: most of the strains display a negative surface charge and hydrophilic behavior while the Beer 1 genetic group has a hydrophobic behavior. All strains showed bioadhesion abilities on stainless steel after only 3 h with differences in the concentration of bioadhered cells ranging from 2.2 × 102 cell/cm2 to 7.6 × 106 cell/cm2. Finally, our results show high variability of the bioadhesion properties, the first step in the biofilm formation, according to the genetic group with the most marked bioadhesion capacity for the beer group. © 2023 The Author

Front Microbiol

Brettanomyces bruxellensis is the main spoilage microbial agent in red wines. The use of fungal chitosan has been authorized since 2009 as a curative treatment to eliminate this yeast in conventional wines and in 2018 in organic wines. As this species is known to exhibit great genetic and phenotypic diversity, we examined whether all the strains responded the same way to chitosan treatment. A collection of 53 strains of was used. In the conditions of the reference test, all were at least temporarily affected by the addition of chitosan to wine, with significant decrease of cultivable population. Some (41%) were very sensitive and no cultivable yeast was detected in wine or lees after 3 days of treatment, while others (13%) were tolerant and, after a slight drop in cultivability, resumed growth between 3 and 10 days and remained able to produce spoilage compounds. There were also many strains with intermediate behavior. The strain behavior was only partially linked to the strain genetic group. This behavior was little modulated by the physiological state of the strain or the dose of chitosan used (within the limits of the authorized doses). On the other hand, for a given strain, the sensitivity to chitosan treatment was modulated by the chitosan used and by the properties of the wine in which the treatment was carried out.Recherches sur l’origine et les effets secondaires des propriétés stabilisantes du chitosane fongique dans le vi

Régulation de l'expression des gènes dupliqués chez les polyploïdes (approche protéomique appliquée à l'analyse de Brassicacées autopolyploïdes et allopolyploïdes)

LA POLYPLOÏDIE EST UN PHENOMENE D'IMPORTANCE CAPITALE : LA PLUPART DES PLANTES SONT DES POLYPLOÏDES OU DES PALEOPOLYPLOÏDES, DONT DE NOMBREUSES PLANTES D'INTERET AGRONOMIQUE. IL EST DONC DE PREMIERE IMPORTANCE POUR LA GENETIQUE EVOLUTIVE ET L'AMELIORATION DES PLANTES DE COMPRENDRE COMMENT UNE PLANTE FONCTIONNE AVEC UNE INFORMATION GENETIQUE REDONDANTE. DANS UN CONTEXTE D'AUTOPOLYPLOÏDIE OU D'ALLOPOLYPLOÏDIE LES OBJECTIFS DE CETTE THESE ETAIENT I- DE DECRIRE L'EXPRESSION DES GENES HOMOLOGUES CHEZ UN POLYPLOÏDE PAR RAPPORT AUX DIPLOÏDES CORRESPONDANTS II- DE CARACTERISER LES GENES CIBLES DES PHENOMENES DE REGULATION QUI ACCOMPAGNENT LA POLYPLOÏDISATION III- DE PRECISER LES MECANISMES GENETIQUES OU EPIGENETIQUES A L'ŒUVRE LES PROFILS D'EXPRESSION D'UNE SERIE AUTOPOLYPLOÏDE DE BRASSICA OLERACEA (HA-, DI- ET TETRA- PLOÏDE) ONT ETE ANALYSES A L'AIDE D'UNE APPROCHE DE PROTEOMIQUE COMPARATIVE. NOS RESULTATS INDIQUENT QUE LE DOUBLEMENT LE COLZA TETRAPLOÏDE B. NAPUS, RE-SYNTHETISE A PARTIR DE SES PROGENITEURS DIPLOÏDES B. OLERACEA ET B. RAPA, A ETE UTILISE COMME MODELE D'ALLOPOLYPLOÏDIE. NOUS AVONS TESTE L'HYPOTHESE DE L'ADDITVITE DES GENOMES (SOUS LAQUELLE UN AMPHIPLOÏDE PRESENTE UN PROFIL D'EXPRESSION INTERMEDIAIRE A SES PARENTS DIPLOÏDES). NOTRE APPROCHE PROTEOMIQUE REVELE DE TRES NOMBREUX ECARTS A L'ADDITIVITE (26-39%). LA COMPARAISON D'AMPHIPLOÏDES INDEPENDANTS MONTRE QUE PLUS DE 98% DES VARIATIONS OBSERVEES SONT REPRODUCITBLES, ILLUSTRANT LE CARACTERE NON-STOCHASTIQUE DES MECANISMES A L'ORIGINE DE CELLES-CI. LES PROTEINES DONT L'ABONDANCE VARIE SONT EN COURS D'IDENTIFICATION.POLYPLOIDISATION IS A MAJOR EVOLUTIONARY PROCESS IN EUKARYOTES MOST FLOWERING PLANTS ARE POLYPLOIDS OR PALEOPOLYPLOIDS, INCLUDING MAJOR CROP SPECIES UNDERSTANDING THE MECHANISMS OF REGULATION OF DUPLICATED GENE EXPRESSION IN POLYPLOIDS IS THUS ESSENTIAL FOR EVOLUTIONARY GENETICS AND PLANT BREEDING. THE PURPOSES OF THESE THESIS WERE I- TO DESCRIBE HOMOLOGOUS GENE EXPRESSION IN AN AUTOPOLYPLOID (IN WHICH IHE CHROMOSOME SETS ORIGINATED FROM THE SAME SPECIES) AND IN AN ALLOPOLYPLOID MODEL (IN WHICH THE HOMEOLOGOUS CHROMOSOME SETS DERIVED FROM MORE THAN ONE SPECIES THROUGH HYBRIDIZATION); II- TO CHARACTERIZE THE GENES TARGETED BY DIFFERENTIAL GENE REGULATION DURING POLYPLOIDISAIION AND III- T0 PRECISE THE UNDERLYING MECHANISMS. WE FIRST STUDIED A BRASSICA OLERACEA AUTOPOLYPLOIDY SERIES INVOLVING HAPLOID, DIPLOID AND TETRAPLOID CABBAGES. GENE EXPRESSION WAS INVESTIGATED AT THE PROTEIN LEVEL USING COMPARATIVE PROTEOMICS. OUR RESULTS INDICATED THAT GENOME DOUBLING DID NOT ALTER SIGNIFICANTLY THE PROTEOMES OF GREEN TISSUES IN B. OLERACEA. TO STUDY GENE EXPRESSION DURING THE EARLY STEPS OF ALLOPOLYPLOID FORMATION, THE OILSEED RAPE B. NAPUS ALLOTETRAPLOID MODEL WAS CHOSEN. COMPARATIVE PROTEOMICS WAS APPLIED TO NEO-SYNTHESIZED B. NAPUS AND ITS DIPLOID PROGENITORS B. RAPA AND B. OLERACEA. SEVERAL DEVIATIONS FROM THE ADDITIVITY HYPOTHESIS (PREDICTING A MIDPARENT PROTEOME FOR THE AMPHIPLOIDS) WERE OBSERVED (26-39% OF POLYPEPTIDES) NON-STOCHASTIC GENE EXPRESSION RE-PATTERNING WAS FOUND SINCE 98% OF THE DETECTED VARIATIONS WERE REPRODUCIBLE IN FOUR INDEPENDENTLY CREATED AMPHIPLOIDS THE IDENTIFICATION OF THE PROTEINS DISPLAYING NON-ADDITIVE PATTERNS IS UNDER PROCESS.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Molecular Diagnosis of Brettanomyces bruxellensis’ Sulfur Dioxide Sensitivity Through Genotype Specific Method

The yeast species Brettanomyces bruxellensis is associated with important economic losses due to red wine spoilage. The most common method to prevent and/or control B. bruxellensis spoilage in winemaking is the addition of sulfur dioxide into must and wine. However, recently, it was reported that some B. bruxellensis strains could be tolerant to commonly used doses of SO2. In this work, B. bruxellensis response to SO2 was assessed in order to explore the relationship between SO2 tolerance and genotype. We selected 145 isolates representative of the genetic diversity of the species, and from different fermentation niches (roughly 70% from grape wine fermentation environment, and 30% from beer, ethanol, tequila, kombucha, etc.). These isolates were grown in media harboring increasing sulfite concentrations, from 0 to 0.6 mg.L-1 of molecular SO2. Three behaviors were defined: sensitive strains showed longer lag phase and slower growth rate and/or lower maximum population size in presence of increasing concentrations of SO2. Tolerant strains displayed increased lag phase, but maximal growth rate and maximal population size remained unchanged. Finally, resistant strains showed no growth variation whatever the SO2 concentrations. 36% (52/145) of B. bruxellensis isolates were resistant or tolerant to sulfite, and up to 43% (46/107) when considering only wine isolates. Moreover, most of the resistant/tolerant strains belonged to two specific genetic groups, allowing the use of microsatellite genotyping to predict the risk of sulfur dioxide resistance/tolerance with high reliability (>90%). Such molecular diagnosis could help the winemakers to adjust antimicrobial techniques and efficient spoilage prevention with minimal intervention

Food microbiol.

Brettanomyces bruxellensis is the most common spoilage wine yeast which can provoke great economic damage to the wine industry due to the production of undesirable odors. The capacity of the species to adapt in various environmental conditions offers a selective advantage that is reflected by intraspecific variability at genotypic and phenotypic level. In this study, microsatellite analysis of 22 strains isolated from Greek wine revealed the existence of distinct genetic subgroups that are correlated with their geographical origin. The response of these strains to increasing levels of sulfur dioxide confirmed the presence of both sensitive and tolerant strains, which belong to distinguished genetic clusters. The genetic categorization of B. bruxellensis strains could be used by the winemakers as a diagnostic tool regarding sulfur dioxide sensitivity