Apiculate yeasts of the genus Hanseniaspora/Kloeckera are the main species present on mature grapes and play a significant role at the beginning of fermentation, producing enzymes and aroma compounds that expand the diversity of wine color and flavor. Ten species of the genus Hanseniaspora have been recovered from grapes and are associated in two groups: H. valbyensis, H. guilliermondii, H. uvarum, H. opuntiae, H. thailandica, H. meyeri, and H. clermontiae; and H. vineae, H. osmophila, and H. occidentalis. This review focuses on the application of some strains belonging to this genus in co-fermentation with Saccharomyces cerevisiae that demonstrates their positive contribution to winemaking. Some consistent results have shown more intense flavors and complex, full-bodied wines, compared with wines produced by the use of S. cerevisiae alone. Recent genetic and physiologic studies have improved the knowledge of the Hanseniaspora/Kloeckera species. Significant increases in acetyl esters, benzenoids, and sesquiterpene flavor compounds, and relative decreases in alcohols and acids have been reported, due to different fermentation pathways compared to conventional wine yeasts.

Interest in the use of non-Saccharomyces yeasts in winemaking has been increasing due to their positive contributions to wine quality. The non-Saccharomyces yeast Hanseniaspora vineae is an apiculate yeast that has been associated with the production of wine with good aromatic properties. However, little is known about the fermentation dynamics of H. vineae in natural must and its interaction with autochthonous yeasts. In the present study, we performed semi industrial fermentations of Macabeo and Merlot musts inoculated with either H. vineae or S. cerevisiae. The yeast population dynamics were monitored by plate culturing, PCR-DGGE and massive sequencing techniques. The results obtained with these techniques show that H. vineae was able dominate the autochthonous microbiota in Macabeo must but not in Merlot must, which exhibited a larger, more diverse yeast population. The presence of H. vineae throughout most of the Macabeo fermentation resulted in more fruity and flowery wine, as indicated by the chemical analysis of the final wines, which demonstrated a strong presence of phenyl ethyl acetate at concentrations higher than the threshold of perception and approximately 50 times more than that produced in wines fermented with S. cerevisiae. This compound is associated with fruity, floral and honey aromas.

BackgroundTeak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak.ResultsIdentification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes.ConclusionThis study proposes a first broad collection of teak tissue and organ mRNA expression data for nine selected candidate qRT-PCR reference genes. NormFinder, Bestkeeper, geNorm and Delta Ct analyses suggested that TgUbq and TgEf-1α have the highest expression stability and provided similar results when evaluating TgCAD gene expression, while the commonly used Act should be avoided.

Hanseniaspora is the main genus of the apiculate yeast group that represents approximately 70% of the grape-associated microflora. Hanseniaspora vineae is emerging as a promising species for quality wine production compared to other non-Saccharomyces species. Wines produced by H. vineae with Saccharomyces cerevisiae consistently exhibit more intense fruity flavors and complexity than wines produced by S. cerevisiae alone. In this work, genome sequencing, assembling, and phylogenetic analysis of two strains of H. vineae showed that it is a member of the Saccharomyces complex and it diverged before the whole-genome duplication (WGD) event from this clade. Specific flavor gene duplications and absences were identified in the H. vineae genome compared to 14 fully sequenced industrial S. cerevisiae genomes. The increased formation of 2-phenylethyl acetate and phenylpropanoids such as 2-phenylethyl and benzyl alcohols might be explained by gene duplications of H. vineae aromatic amino acid aminotransferases (ARO8 and ARO9) and phenylpyruvate decarboxylases (ARO10). Transcriptome and aroma profiles under fermentation conditions confirmed these genes were highly expressed at the beginning of stationary phase coupled to the production of their related compounds. The extremely high level of acetate esters produced by H. vineae compared to that by S. cerevisiae is consistent with the identification of six novel proteins with alcohol acetyltransferase (AATase) domains. The absence of the branched-chain amino acid transaminases (BAT2) and acyl coenzyme A (acyl-CoA)/ethanol O-acyltransferases (EEB1) genes correlates with H. vineae's reduced production of branched-chain higher alcohols, fatty acids, and ethyl esters, respectively. Our study provides sustenance for understanding and potentially utilizing genes that determine fermentation aromas.IMPORTANCE The huge diversity of non-Saccharomyces yeasts in grapes is dominated by the apiculate genus Hanseniaspora. Two native strains of Hanseniaspora vineae applied to winemaking because of their high oenological potential in aroma and fermentation performance were selected to obtain high-quality genomes. Here, we present a phylogenetic analysis and the complete transcriptome and aroma metabolome of H. vineae during three fermentation steps. This species produced significantly richer flavor compound diversity than Saccharomyces, including benzenoids, phenylpropanoids, and acetate-derived compounds. The identification of six Downloaded from proteins, different from S. cerevisiae ATF, with diverse acetyltransferase domains in H. vineae offers a relevant source of native genetic variants for this enzymatic activity. The discovery of benzenoid synthesis capacity in H. vineae provides a new eukaryotic model to dilucidate an alternative pathway to that catalyzed by plants' phenylalanine lyases.

Benzyl alcohol and other benzenoid-derived metabolites of particular importance in plants confer floral and fruity flavors to wines. Among the volatile aroma components in Vitis vinifera grape varieties, benzyl alcohol is present in its free and glycosylated forms. These compounds are considered to originate from grapes only and not from fermentative processes. We have found increased levels of benzyl alcohol in red Tannat wine compared to that in grape juice, suggesting de novo formation of this metabolite during vinification. In this work, we show that benzyl alcohol, benzaldehyde, p-hydroxybenzaldehyde, and p-hydroxybenzyl alcohol are synthesized de novo in the absence of grape-derived precursors by Hanseniaspora vineae. Levels of benzyl alcohol produced by 11 different H. vineae strains were 20-200 times higher than those measured in fermentations with Saccharomyces cerevisiae strains. These results show that H. vineae contributes to flavor diversity by increasing grape variety aroma concentration in a chemically defined medium. Feeding experiments with phenylalanine, tryptophan, tyrosine, p-aminobenzoic acid, and ammonium in an artificial medium were tested to evaluate the effect of these compounds either as precursors or as potential pathway regulators for the formation of benzenoid-derived aromas. Genomic analysis shows that the phenylalanine ammonia-lyase (PAL) and tyrosine ammonia lyase (TAL) pathways, used by plants to generate benzyl alcohols from aromatic amino acids, are absent in the H. vineae genome. Consequently, alternative pathways derived from chorismate with mandelate as an intermediate are discussed.