Epigenetic Changes in Saccharomyces cerevisiae Alters the Aromatic Profile in Alcoholic Fermentation.

Epigenetic changes in genomics provide phenotypic modification without DNA sequence alteration. This study shows that benzoic acid, a common food additive and known histone deacetylase inhibitor (HDACi), has an epigenetic effect on Saccharomyces cerevisiae. Benzoic acid stimulated formation of epigenetic histone marks H3K4Me2, H3K27Me2, H3K18ac, and H3Ser10p in S. cerevisiae and altered their phenotypic behavior, resulting in increased production of phenylethyl alcohol and ester compounds during alcoholic fermentation using wine as a representative model system. Our study demonstrates the HDACi activity of certain dietary compounds such as sodium butyrate, curcumin and anacardic acid, suggests the potential use of these dietary compounds in altering S. cerevisiae phenotypes without altering host-cell DNA. This study highlights the potential to use common dietary compounds to exploit epigenetic modifications for various fermentation and biotechnology applications as an alternative to genetic modification. These findings indicate that benzoic acid and other food additives may have potential epigenetic effects on human gut microbiota, in which several yeast species are involved. IMPORTANCE The manuscript investigates and reports for the first time utilizing a non-GMO approach to alter the fermentation process of Pinot Noir wines. We have experimentally demonstrated that certain dietary compounds possess histone deacetylase (HDAC) inhibiting activity and can alter the wine characteristics by potentially altering yeast gene transcription, which was resulted from epigenetic effects. We have previously proposed the term "nutrifermentics" to represent this newly proposed field of research that provides insights on the effect of certain dietary compounds on microbial strains and their potential application in fermentation. This technological approach is a novel way to manipulate microorganisms for innovative food and beverage production with quality attributes catering for consumer's needs. Using a multidisciplinary approach with an emphasis on food fermentation and biotechnology, this study will be substantially useful and of broad interest to food microbiologists and biotechnologists who seek for innovative concepts with real-world application potential.

Identification and functional characterisation of an allene oxide synthase from grapevine (Vitis vinifera L. Sauvignon blanc).

Jasmonic acid (JA) is known to be an important phytohormone that orchestrates plant defence mechanisms against a range of herbivores and pathogens. Studies have suggested allene oxide synthase (AOS; E.C 4.2.1.92), the first committed step in JA biosynthesis, is essential for JA biosynthesis, yet clear evidence of its role as a biosynthetic regulatory point is lacking, in the main due to conflicting results derived from transgenic studies. However other studies lend support to a biosynthetic regulatory role for AOS. These studies have suggested that certain amino acid substitutions can increase the biosynthetic capacity of the enzyme and consequently improve pathogen tolerance in plants. To explore the role of AOS in Grapevine we isolated and functionally characterised this enzyme for the first time from Vitis vinifera L. Sauvignon blanc. The cloned AOS consisted of a single 1563 bp open reading frame. Comparative sequence analysis showed that the cloned gene (VvAOS) was highly conserved compared to those from other species. Complementation of an Arabidopsis AOS null mutant (aos) with VvAOS recovered the male sterile mutant phenotype and confirmed its function. Transcript analysis showed that VvAOS was wound responsive in leaves and was detectable in most tissues, with the highest levels of transcript in the mesocarp (pulp) of mature berries. Sub-cellular localisation of the VvAOS protein indicated that VvAOS is associated with the chloroplast membrane. Unexpectedly high levels of VvAOS transcript in complemented aos lines did not lead to predicted increases in JA. We have functionally characterised the sole AOS from Grapevine. Patterns of transcript accumulation in grapevine suggest roles in growth, development as well as an important role for JA in fruit ripening. Expression of VvAOS in Arabidopsis suggest complex epigenetic interactions between transgenic and endogenous AOS alleles, providing a possible explanation for why transgenic studies of AOS have delivered conflicting data pointing to a questionable role of AOS as a key regulatory point in JA biosynthesis.

Identification of suitable grapevine reference genes for qRT-PCR derived from heterologous species.

Identification and validation of suitable reference genes that exhibit robust transcriptional stability across many sample types is an absolute requirement of all qRT-PCR experiments. Often, however, only small numbers of reference genes, validated across limited sample types, are available for non-model species. This points to a clear need to assess and validate a wider range of potential reference genes than is currently available. We therefore looked to test and validate a large number of potential reference genes across a wide range of tissue types and treatments to determine the applicability of these reference genes for use in grapevine and other non-model plant species. Potential reference genes were selected based on stability of gene transcription in the model plant species Arabidopsis or due to their common use in the grapevine community. The selected reference genes were analyzed across two datasets consisting of a range of either 'Sauvignon blanc' or 'Pinot noir' tissues. A total of 11 potential reference genes were screened across the two datasets. Gene stability was analyzed by GeNorm, a widely used Excel application, or an ANOVA-based method developed in red clover. Both analysis methods showed that all 11 potential reference genes are stably expressed in the datasets tested, but the rankings of gene stability differed based on the datasets and analysis method used. Furthermore, the transcript stability of these genes, initially identified in Arabidopsis and now validated in grapevine, suggests applicability across a wide range of non-model plant species in addition to their utility in grapevine.

Elevated abundance of Komagataeibacter results in a lower pH in kombucha production; insights from microbiomic and chemical analyses.

Kombucha consumption has grown rapidly worldwide in the last decade, with production at both small- and large scales. The complex fermentation process involves both bacterial and yeast species, but little is known regarding the progression of microbial development during production. We explored the microbial diversity of multiple batches across two kombucha types, i. e commercial scale versus laboratory-made (hereafter "home") kombucha brew using metabarcoding to characterize both fungal and bacterial communities. We found the microbial community of the commercial kombucha brew to be more complex than that of the home brew. Furthermore, PERMANOVA uncovered significant compositional differences between the bacterial (F = 2.68, R2 = 0.23, p = 00.001) and fungal (F = 3.18, R2 = 0.26, p = 00.006) communities between batches. For the home brew, both alpha and beta diversity analyses revealed no significant differences between all batches and replicates. When the microbial diversity of the home and commercial kombucha types were directly compared, the former had higher proportions of Ammoniphilus and Komagataeibacter. The commercial kombucha on the other hand were high in Anoxybacillus, Methylobacterium and Sphingomonas. For the fungal communities, the most dominant fungal genera detected in both kombucha types were similar. Linear model revealed significant correlations between some microorganisms and the sugars and organic acids assayed in this study. For example, rising glucose levels correlated with an increase in the relative abundance of Komagataeibacter (F = 7.115, Adj. R2 = 0.44, p = 00.0003). We believe these results contribute towards achieving a better control of the kombucha fermentation process and may assist in targeted product diversification.

Influence of climatic variation on microbial communities during organic Pinot noir wine production.

To assess the possible impact of climatic variation on microbial community composition in organic winemaking, we employed a metabarcoding approach to scrutinize the microbiome in a commercial, organic, Pinot noir wine production system that utilizes autochthonous fermentation. We assessed microbial composition across two vintages (2018 and 2021) using biological replicates co-located at the same winery. Microbial dynamics were monitored over four important fermentation time points and correlated with contemporaneous climate data. Bacterial (RANOSIM = 0.4743, p = 0.0001) and fungal (RANOSIM = 0.4738, p = 0.0001) compositions were different in both vintages. For bacteria, Lactococcus dominated the diversity associated with the 2018 vintage, while Tatumella dominated the 2021 vintage. For fungal populations, while Saccharomyces were abundant in both vintages, key differences included Starmerella, copious in the 2018 vintage; and Metschnikowia, substantive in the 2021 vintage. Ordination plots correlated the climatic variables with microbial population differences, indicating temperature as a particularly important influence; humidity values also differed significantly between these vintages. Our data illustrates how climatic conditions may influence microbial diversity during winemaking, and further highlights the effect climate change could have on wine production.

Environmental influences on microbial community development during organic pinot noir wine production in outdoor and indoor fermentation conditions.

The role of microbial diversity in influencing the organoleptic properties of wine and other fermented products is well est ablished, and understanding microbial dynamics within fermentation processes can be critical for quality assurance and product innovation. This is especially true for winemakers using spontaneous fermentation techniques, where environmental factors may play an important role in consistency of product. Here, we use a metabarcoding approach to investigate the influence of two environmental systems used by an organic winemaker to produce wines; vineyard (outdoors) and winery (indoors) to the bacterial and fungal communities throughout the duration of a spontaneous fermentation of the same batch of Pinot Noir grapes. Bacterial (RANOSIM = 0.5814, p = 0.0001) and fungal (RANOSIM = 0.603, p = 0.0001) diversity differed significantly across the fermentation stages in both systems. Members of the Hyphomicrobium genus were found in winemaking for the first time, as a bacterial genus that can survive alcoholic fermentation. Our results also indicate that Torulaspora delbrueckii and Fructobacillus species might be sensitive to environmental systems. These results clearly reflect the substantial influence that environmental conditions exert on microbial populations at every point in the process of transforming grape juice to wine via fermentation, and offer new insights into the challenges and opportunities for wine production in an ever-changing global climate.

Genetic mapping of the LOSS OF PARTHENOGENESIS locus in Pilosella piloselloides and the evolution of apomixis in the Lactuceae.

Pilosella piloselloides var. praealta (syn. P. praealta; Hieracium praealtum) is a versatile model used to study gametophytic apomixis. In this system apomixis is controlled by three loci: one that controls the avoidance of meiosis (LOA), one that controls the avoidance of fertilization (LOP) and a third that controls autonomous endosperm formation (AutE). Using a unique polyhaploid mapping approach the LOP locus was mapped to a 654 kb genomic interval syntenic to linkage group 8 of Lactuca sativa. Polyhaploids form through the gametophytic action of a dominant determinant at LOP, so the mapped region represents both a functional and a physical domain for LOP in P. piloselloides. Allele sequence divergence (ASD) analysis of the PARTHENOGENESIS (PAR) gene within the LOP locus revealed that dominant PAR alleles in Pilosella remain highly similar across the genus, whilst the recessive alleles are more divergent. A previous report noted that dominant PAR alleles in both Pilosella and Taraxacum are modified by the presence of a class II transposable element (TE) in the promoter of the gene. This observation was confirmed and further extended to the related genus Hieracium. Sufficient differences were noted in the structure and location of the TE elements to conclude that TE insertional events had occurred independently in the three genera. Measures of allele crossover amongst the polyhaploids revealed that P. piloselloides is an autopolyploid species with tetrasomic inheritance. It was also noted that the dominant determinant of LOP in P. piloselloides could transmit via a diploid gamete (pollen or egg) but not via a haploid gamete. Using this information, a model is presented of how gametophytic apomixis may have evolved in several members of the Lactuceae, a tribe of the Asteraceae.

A multi-omic Nicotiana benthamiana resource for fundamental research and biotechnology.

Nicotiana benthamiana is an invaluable model plant and biotechnology platform with a ~3 Gb allotetraploid genome. To further improve its usefulness and versatility, we have produced high-quality chromosome-level genome assemblies, coupled with transcriptome, epigenome, microRNA and transposable element datasets, for the ubiquitously used LAB strain and a related wild accession, QLD. In addition, single nucleotide polymorphism maps have been produced for a further two laboratory strains and four wild accessions. Despite the loss of five chromosomes from the ancestral tetraploid, expansion of intergenic regions, widespread segmental allopolyploidy, advanced diploidization and evidence of recent bursts of Copia pseudovirus (Copia) mobility not seen in other Nicotiana genomes, the two subgenomes of N. benthamiana show large regions of synteny across the Solanaceae. LAB and QLD have many genetic, metabolic and phenotypic differences, including disparate RNA interference responses, but are highly interfertile and amenable to genome editing and both transient and stable transformation. The LAB/QLD combination has the potential to be as useful as the Columbia-0/Landsberg errecta partnership, utilized from the early pioneering days of Arabidopsis genomics to today.

Comprehensive analysis of both long and short read transcriptomes of a clonal and a seed-propagated model species reveal the prerequisites for transcriptional activation of autonomous and non-autonomous transposons in plants.

BACKGROUND: Transposable element (TE) transcription is a precursor to its mobilisation in host genomes. However, the characteristics of expressed TE loci, the identification of self-competent transposon loci contributing to new insertions, and the genomic conditions permitting their mobilisation remain largely unknown. RESULTS: Using Vitis vinifera embryogenic callus, we explored the impact of biotic stressors on transposon transcription through the exposure of the callus to live cultures of an endemic grapevine yeast, Hanseniaspora uvarum. We found that only 1.7-2.5% of total annotated TE loci were transcribed, of which 5-10% of these were full-length, and the expressed TE loci exhibited a strong location bias towards expressed genes. These trends in transposon transcription were also observed in RNA-seq data from Arabidopsis thaliana wild-type plants but not in epigenetically compromised Arabidopsis ddm1 mutants. Moreover, differentially expressed TE loci in the grapevine tended to share expression patterns with co-localised differentially expressed genes. Utilising nanopore cDNA sequencing, we found a strong correlation between the inclusion of intronic TEs in gene transcripts and the presence of premature termination codons in these transcripts. Finally, we identified low levels of full-length transcripts deriving from structurally intact TE loci in the grapevine model. CONCLUSION: Our observations in two disparate plant models representing clonally and seed propagated plant species reveal a closely connected transcriptional relationship between TEs and co-localised genes, particularly when epigenetic silencing is not compromised. We found that the stress treatment alone was insufficient to induce large-scale full-length transcription from structurally intact TE loci, a necessity for non-autonomous and autonomous mobilisation.

A PARTHENOGENESIS allele from apomictic dandelion can induce egg cell division without fertilization in lettuce.

Apomixis, the clonal formation of seeds, is a rare yet widely distributed trait in flowering plants. We have isolated the PARTHENOGENESIS (PAR) gene from apomictic dandelion that triggers embryo development in unfertilized egg cells. PAR encodes a K2-2 zinc finger, EAR-domain protein. Unlike the recessive sexual alleles, the dominant PAR allele is expressed in egg cells and has a miniature inverted-repeat transposable element (MITE) transposon insertion in the promoter. The MITE-containing promoter can invoke a homologous gene from sexual lettuce to complement dandelion LOSS OF PARTHENOGENESIS mutants. A similar MITE is also present in the promoter of the PAR gene in apomictic forms of hawkweed, suggesting a case of parallel evolution. Heterologous expression of dandelion PAR in lettuce egg cells induced haploid embryo-like structures in the absence of fertilization. Sexual PAR alleles are expressed in pollen, suggesting that the gene product releases a block on embryogenesis after fertilization in sexual species while in apomictic species PAR expression triggers embryogenesis in the absence of fertilization.

Functional Characterization of the Grapevine γ-Glutamyl Transferase/Transpeptidase (E.C. 2.3.2.2) Gene Family Reveals a Single Functional Gene Whose Encoded Protein Product Is Not Located in Either the Vacuole or Apoplast.

γ-glutamyl transferases/transpeptidases (E.C. 2.3.2.2, GGTs) are involved in the catabolism of many compounds that are conjugated to glutathione (GSH), which have a variety of roles. GSH can act as storage and transport vehicle for reduced sulfur; it is involved in the detoxification of xenobiotics and also acts as a redox buffer by utilizing its thiol residue to protect against reactive oxygen species, which accumulate in response to biotic and abiotic stress. Furthermore, many distinctive flavor and aroma compounds in Sauvignon blanc wines originate from odorless C5- and C6-GSH conjugates or their GGT catabolized derivatives. These precursors are then processed into their volatile forms by yeast during fermentation. In many plant species, two or more isoforms of GGTs exist that target GSH-conjugates to either the apoplast or the vacuole. A bioinformatics approach identified multiple GGT candidates in grapevine (Vitis vinifera). However, only a single candidate, VvGGT3, has all the conserved residues needed for GGT activity. This is intriguing given the variety of roles of GSH and GGTs in plant cells. Characterization of VvGGT3 from cv. Sauvignon blanc was then undertaken. The VvGGT3 transcript is present in roots, leaves, inflorescences, and tendril and at equal abundance in the skin, pulp, and seed of mature berries and shows steady accumulation over the course of whole berry development. In addition, the VvGGT3 transcript in whole berries is upregulated upon Botrytis cinerea infection as well as mechanical damage to leaf tissue. VvGGT3-GFP fusion proteins transiently over-expressed in onion cells were used to study subcellular localization. To confirm VvGGT3 activity and localization in vivo, the fluorescent γ-glutamyl-7-amido-4-methylcoumarin substrate was added to Nicotiana benthamiana leaves transiently over-expressing VvGGT3. In combination, these results suggest that the functional VvGGT3 is associated with membrane-like structures. This is not consistent with its closely related functionally characterized GGTs from Arabidopsis, radish and garlic.

Tools and Strategies for Long-Read Sequencing and De Novo Assembly of Plant Genomes.

The commercial release of third-generation sequencing technologies (TGSTs), giving long and ultra-long sequencing reads, has stimulated the development of new tools for assembling highly contiguous genome sequences with unprecedented accuracy across complex repeat regions. We survey here a wide range of emerging sequencing platforms and analytical tools for de novo assembly, provide background information for each of their steps, and discuss the spectrum of available options. Our decision tree recommends workflows for the generation of a high-quality genome assembly when used in combination with the specific needs and resources of a project.

Comparisons of controlled environment and vineyard experiments in Sauvignon blanc grapes reveal similar UV-B signal transduction pathways for flavonol biosynthesis.

UV-B radiation is an environmental challenge affecting a number of metabolic functions in plants. Plants protect themselves from this potentially damaging radiation through synthesising UV-absorbing compounds such as flavonoids. This study aims to investigate the effect of UV-B on flavonoid biosynthesis in Sauvignon blanc grapes. In particular, a comparison has been made between controlled environment (CE) and vineyard trials to better understand molecular mechanisms of low/high fluence UV-B responses and how the results relate to each other in the context of flavonoid biosynthesis. Following exposure to supplemental UV-B in the CE, both flavonols and gene expression exhibited UV-B induced response. Flavonols, particularly quercetin/kaempferol 3-O-glycosides were increased at distinct stages of berry development. All genes measured showed a significant developmental regulation. VvFLS4, VvCHS1, VvMYB12, VvHY5 and PR (VvTL1 and VvChi4A/4B) increased due to UV-B in the CE experiments. However, PR were not responsive to the natural UV-B fluence in vineyard but were significantly induced at later stages of development. Overall, despite very different conditions in the CE and vineyard the majority of UV-B induced responses are similar. Only PR activities in the CE cabinets reflect a higher fluence stress response that is not reflected in the natural lower UV-B fluence environment.

Intraspecific differences in long-term drought tolerance in perennial ryegrass.

Lolium perenne L. (perennial ryegrass) is the most important pasture grass species in temperate regions of the world. However, its growth is restricted in summer dry environments. Germplasm screening can be used to identify accessions or individual plants for incorporation into breeding programs for drought tolerance. We selected nine perennial ryegrass accessions from different global origins and from a range of climatic and environmental conditions. In addition, the perennial ryegrass cultivar 'Grasslands Impact' was chosen as a reference. The accessions were grown for 360 days in a controlled environment through six consecutive drought stress and recovery cycles. We observed intraspecific differences in drought stress responsiveness for shoot biomass and survival from the third stress cycle. An accession from Norway had 50% more shoot dry matter than the next best-performing accession after six drought cycles. Compared with the reference cultivar 'Grasslands Impact', shoot dry matter of the accession from Norway was more than seven times higher after six drought cycles, indicating superior performance of this ecotype under drought stress. Drought tolerance was characterized by osmotic adjustment and higher relative leaf water content at low soil moisture levels. Furthermore, the findings of this study identify solute potential as an early predictor of drought stress tolerance. These intraspecific differences can be used in breeding programs for the development of drought-tolerant perennial ryegrass cultivars.

The Apoplastic Secretome of Trichoderma virens During Interaction With Maize Roots Shows an Inhibition of Plant Defence and Scavenging Oxidative Stress Secreted Proteins.

In Nature, almost every plant is colonized by fungi. Trichoderma virens is a biocontrol fungus which has the capacity to behave as an opportunistic plant endophyte. Even though many plants are colonized by this symbiont, the exact mechanisms by which Trichoderma masks its entrance into its plant host remain unknown, but likely involve the secretion of different families of proteins into the apoplast that may play crucial roles in the suppression of plant immune responses. In this study, we investigated T. virens colonization of maize roots under hydroponic conditions, evidencing inter- and intracellular colonization by the fungus and modifications in root morphology and coloration. Moreover, we show that upon host penetration, T. virens secretes into the apoplast an arsenal of proteins to facilitate inter- and intracellular colonization of maize root tissues. Using a gel-free shotgun proteomics approach, 95 and 43 secretory proteins were identified from maize and T. virens, respectively. A reduction in the maize secretome (36%) was induced by T. virens, including two major groups, glycosyl hydrolases and peroxidases. Furthermore, T. virens secreted proteins were mainly involved in cell wall hydrolysis, scavenging of reactive oxygen species and secondary metabolism, as well as putative effector-like proteins. Levels of peroxidase activity were reduced in the inoculated roots, suggesting a strategy used by T. virens to manipulate host immune responses. The results provide an insight into the crosstalk in the apoplast which is essential to maintain the T. virens-plant interaction.