Ancient Roman bacterium against current issues: strain Aquil_B6, Paenisporosarcina quisquiliarum, or Psychrobacillus psychrodurans?

IMPORTANCE: Since 1988, through the United States government's founding, the National Center for Biotechnology Information (NCBI) has provided an invaluable service to scientific advancement. The universality and total freedom of use if on the one hand allow the use of this database on a global level by all researchers for their valuable work, on the other hand, it has the disadvantage of making it difficult to check the correctness of all the materials present. It is, therefore, of fundamental importance for the correctness and ethics of research to improve the databases at our disposal, identifying and amending the critical issues. This work aims to provide the scientific community with a new sequence for the type strain Paenisporosarcina quisquiliarum SK 55 and broaden the knowledge of the Psychrobacillus psychrodurans species, in particular, considering the ancient strain Aquil_B6 found in an ancient Roman amphora.

Juxtaposition of the Source-to-Sink Ratio and Fruit Exposure to Solar Radiation on cv. Merlot (Vitis vinifera L.) Berry Phenolics in a Cool versus Warm Growing Region.

The grapevine source-to-sink ratio and berry exposure to solar radiation both influence grape flavonoid biosynthesis and accumulation. Here, we compared these concepts on cv. Merlot in two different growing locations (Michigan (MI) and Friuli-Venezia Giulia (FVG), IT) to understand whether the environment influences flavonoid sensitivity to these parameters. Three levels of leaf removal (LR0, LR5, LR8) were implemented at the pea-size phenological stage to compare conditions of increased cluster light exposure with a decreasing vine source-to-sink ratio on berry flavonoid accumulation. Treatments did not affect total soluble solids (TSSs) or pH, but titratable acidity (TA) was lower in LR8 at harvest in both locations. LR5 increased anthocyanins and flavonols in MI but decreased most phenolics in FVG. The decreased expression of VviLAR1 and VviF3'5'Hh during ripening supported the lower concentrations of flavan-3-ol monomers and anthocyanins in FVG. In summary, flavonoid biosynthesis and accumulation were more sensitive to solar radiation than the source-to-sink ratio, and the vineyard environment dictated whether solar radiation was beneficial or detrimental to flavonoid biosynthesis.

Draft Genome Sequences of Eight Bacilli Isolated from an Ancient Roman Amphora.

Paleomicrobiology, the study of ancient microbiological material, allows us to understand different evolutionary phenomena in bacteria. In this study, eight bacilli isolated from an ancient Roman amphora, which dates to the IV to V sec. AD, were sequenced and functionally annotated.

Influence of freezing and heating conditions on grape seed flavan-3-ol extractability, oxidation, and galloylation pattern.

In cool-climate viticulture, the short growing season can influence grape seed maturation by reducing the apparent oxidation of flavan-3-ol monomers and associated increase in seed browning. A reduction in seed maturation increases the potential extraction of flavan-3-ol monomers into wine during maceration operations, heightening bitterness. Here, we carried out a 2 × 2 factorial experiment to test the ability of freezing and heating treatments to advance maturation (decrease flavan-3-ol, improve browning) of (Vitis vinifera L.) Pinot noir and Cabernet Sauvignon seeds over a 24-h incubation period. Only freezing significantly increased seed browning in both cultivars. Subsequent correlations with seed flavan-3-ol monomer concentrations suggest that freezing enhanced the oxidation of these compounds. Interestingly, natural ripening and freezing reduced galloylated flavan-3-ol monomers to a greater extent than non-galloylated ones. This study provides new information regarding the susceptibility of flavan-3-ol monomers to freezing and heating, and also suggests that freezing can advance the maturation the seeds of under-ripe red vinifera grapes.

Stomatal responses in grapevine become increasingly more tolerant to low water potentials throughout the growing season.

The leaf of a deciduous species completes its life cycle in a few months. During leaf maturation, osmolyte accumulation leads to a significant reduction of the turgor loss point (ΨTLP ), a known marker for stomatal closure. Here we exposed two grapevine cultivars to drought at three different times during the growing season to explore if the seasonal decrease in leaf ΨTLP influences the stomatal response to drought. The results showed a significant seasonal shift in the response of stomatal conductance to stem water potential (gs ~Ψstem ), demonstrating that grapevines become increasingly tolerant to low Ψstem as the season progresses in coordination with the decrease in ΨTLP . We also used the SurEau hydraulic model to demonstrate a direct link between osmotic adjustment and the plasticity of gs ~Ψstem . To understand the possible advantages of gs ~Ψstem plasticity, we incorporated a seasonally dynamic leaf osmotic potential into the model that simulated stomatal conductance under several water availabilities and climatic scenarios. The model demonstrated that a seasonally dynamic stomatal closure threshold results in trade-offs: it reduces the time to turgor loss under sustained long-term drought, but increases overall gas exchange particularly under seasonal shifts in temperature and stochastic water availability. A projected hotter future is expected to lower the increase in gas exchange that plants gain from the seasonal shift in gs ~Ψstem . These findings show that accounting for dynamic stomatal regulation is critical for understanding drought tolerance.

Analysis of Non-Structural Carbohydrates and Xylem Anatomy of Leaf Petioles Offers New Insights in the Drought Response of Two Grapevine Cultivars.

In grapevine, the anatomy of xylem conduits and the non-structural carbohydrates (NSCs) content of the associated living parenchyma are expected to influence water transport under water limitation. In fact, both NSC and xylem features play a role in plant recovery from drought stress. We evaluated these traits in petioles of Cabernet Sauvignon (CS) and Syrah (SY) cultivars during water stress (WS) and recovery. In CS, the stress response was associated to NSC consumption, supporting the hypothesis that starch mobilization is related to an increased supply of maltose and sucrose, putatively involved in drought stress responses at the xylem level. In contrast, in SY, the WS-induced increase in the latter soluble NSCs was maintained even 2 days after re-watering, suggesting a different pattern of utilization of NSC resources. Interestingly, the anatomical analysis revealed that conduits are constitutively wider in SY in well-watered (WW) plants, and that water stress led to the production of narrower conduits only in this cultivar.

Increase in seed tannin extractability and oxidation using a freeze-thaw treatment in cool-climate grown red (Vitis vinifera L.) cultivars.

Grape seed maturation involves the gradual oxidation of tannins, decreasing excessive bitterness and astringency in wine. In cool climates, this process is limited by the short growing season, affecting wine quality. A "freeze-thaw" treatment on seeds of red vinifera cultivars at veraison and harvest was used to evaluate the effect of oxidation and extractability on seed phenolic fractions. Freezing increased the extraction of total phenolics and o-diphenols quantified from fractionation (fraction 1, vacuolar tannins; fraction 2, hydrogen bonded tannins; fraction 3, covalently bonded tannins), especially at harvest. Despite this, colorimetry, microscopy, oxidation reactivity index (ORI), and correlations between the color index and fractions indicated that freezing disrupted vacuole integrity, enhancing oxidation in the seed coat. In conclusion, vacuolar tannins (which are the main seed phenolics extracted during fermentation) were highly correlated with seed color change, potentially providing information for winemaking in cool climate regions.

Swift metabolite changes and leaf shedding are milestones in the acclimation process of grapevine under prolonged water stress.

BACKGROUND: Grape leaves provide the biochemical substrates for berry development. Thus, understanding the regulation of grapevine leaf metabolism can aid in discerning processes fundamental to fruit development and berry quality. Here, the temporal alterations in leaf metabolism in Merlot grapevine grown under sufficient irrigation and water deficit were monitored from veraison until harvest. RESULTS: The vines mediated water stress gradually and involving multiple strategies: osmotic adjustment, transcript-metabolite alteration and leaf shedding. Initially stomatal conductance and leaf water potential showed a steep decrease together with the induction of stress related metabolism, e.g. up-regulation of proline and GABA metabolism and stress related sugars, and the down-regulation of developmental processes. Later, progressive soil drying was associated with an incremental contribution of Ca2+ and sucrose to the osmotic adjustment concomitant with the initiation of leaf shedding. Last, towards harvest under progressive stress conditions following leaf shedding, incremental changes in leaf water potential were measured, while the magnitude of perturbation in leaf metabolism lessened. CONCLUSIONS: The data present evidence that over time grapevine acclimation to water stress diversifies in temporal responses encompassing the alteration of central metabolism and gene expression, osmotic adjustments and reduction in leaf area. Together these processes mitigate leaf water stress and aid in maintaining the berry-ripening program.

Multi-Omics and Integrated Network Analyses Reveal New Insights into the Systems Relationships between Metabolites, Structural Genes, and Transcriptional Regulators in Developing Grape Berries (Vitis vinifera L.) Exposed to Water Deficit.

Grapes are one of the major fruit crops and they are cultivated in many dry environments. This study comprehensively characterizes the metabolic response of grape berries exposed to water deficit at different developmental stages. Increases of proline, branched-chain amino acids, phenylpropanoids, anthocyanins, and free volatile organic compounds have been previously observed in grape berries exposed to water deficit. Integrating RNA-sequencing analysis of the transcriptome with large-scale analysis of central and specialized metabolites, we reveal that these increases occur via a coordinated regulation of key structural pathway genes. Water deficit-induced up-regulation of flavonoid genes is also coordinated with the down-regulation of many stilbene synthases and a consistent decrease in stilbenoid concentration. Water deficit activated both ABA-dependent and ABA-independent signal transduction pathways by modulating the expression of several transcription factors. Gene-gene and gene-metabolite network analyses showed that water deficit-responsive transcription factors such as bZIPs, AP2/ERFs, MYBs, and NACs are implicated in the regulation of stress-responsive metabolites. Enrichment of known and novel cis-regulatory elements in the promoters of several ripening-specific/water deficit-induced modules further affirms the involvement of a transcription factor cross-talk in the berry response to water deficit. Together, our integrated approaches show that water deficit-regulated gene modules are strongly linked to key fruit-quality metabolites and multiple signal transduction pathways may be critical to achieve a balance between the regulation of the stress-response and the berry ripening program. This study constitutes an invaluable resource for future discoveries and comparative studies, in grapes and other fruits, centered on reproductive tissue metabolism under abiotic stress.

Grape Metabolic Response to Postveraison Water Deficit Is Affected by Interseason Weather Variability.

Postveraison water deficit is a common strategy implemented to improve fruit composition in many wine-growing regions. However, contrasting results are often reported on fruit size and composition, a challenge for generalizing the positive impact of this technique. Our research investigated the effect of water deficit (WD) imposed at veraison on Merlot grapevines, during two experimental seasons (2014-2015). In both years WD resulted in reduced carbon assimilation rates and leaf shedding. However, the treatment effect on the analyzed berry parameters varied between seasons. Modification of skin metabolites was more evident in 2015 than in 2014, despite the similar soil water content and water stress physiological parameters (gas exchange, water potential) recorded in the two experimental years. Higher solar radiation and air temperature in 2015 than in 2014 hint for the involvement of atmospheric parameters in fulfilling the potential effect of WD. Our results suggest that the interaction between water availability and weather conditions plays a crucial role in modulating the grape berry composition.

Grapevine acclimation to water deficit: the adjustment of stomatal and hydraulic conductance differs from petiole embolism vulnerability.

MAIN CONCLUSION: Drought-acclimated vines maintained higher gas exchange compared to irrigated controls under water deficit; this effect is associated with modified leaf turgor but not with improved petiole vulnerability to cavitation. A key feature for the prosperity of plants under changing environments is the plasticity of their hydraulic system. In the present research we studied the hydraulic regulation in grapevines (Vitis vinifera L.) that were first acclimated for 39 days to well-watered (WW), sustained water deficit (SD), or transient-cycles of dehydration-rehydration-water deficit (TD) conditions, and then subjected to varying degrees of drought. Vine development under SD led to the smallest leaves and petioles, but the TD vines had the smallest mean xylem vessel and calculated specific conductivity (k ts). Unexpectedly, both the water deficit acclimation treatments resulted in vines more vulnerable to cavitation in comparison to WW, possibly as a result of developmental differences or cavitation fatigue. When exposed to drought, the SD vines maintained the highest stomatal (g s) and leaf conductance (k leaf) under low stem water potential (Ψs), despite their high xylem vulnerability and in agreement with their lower turgor loss point (ΨTLP). These findings suggest that the down-regulation of k leaf and g s is not associated with embolism, and the ability of drought-acclimated vines to maintain hydraulic conductance and gas exchange under stressed conditions is more likely associated with the leaf turgor and membrane permeability.

Impact of Leaf Removal, Applied Before and After Flowering, on Anthocyanin, Tannin, and Methoxypyrazine Concentrations in 'Merlot' (Vitis vinifera L.) Grapes and Wines.

The development and accumulation of secondary metabolites in grapes determine wine color, taste, and aroma. This study aimed to investigate the effect of leaf removal before flowering, a practice recently introduced to reduce cluster compactness and Botrytis rot, on anthocyanin, tannin, and methoxypyrazine concentrations in 'Merlot' grapes and wines. Leaf removal before flowering was compared with leaf removal after flowering and an untreated control. No effects on tannin and anthocyanin concentrations in grapes were observed. Both treatments reduced levels of 3-isobutyl-2-methoxypyrazine (IBMP) in the grapes and the derived wines, although the after-flowering treatment did so to a greater degree in the fruit specifically. Leaf removal before flowering can be used to reduce cluster compactness, Botrytis rot, and grape and wine IBMP concentration and to improve wine color intensity but at the expense of cluster weight and vine yield. Leaf removal after flowering accomplishes essentially the same results without loss of yield.

Transcriptome and metabolite profiling reveals that prolonged drought modulates the phenylpropanoid and terpenoid pathway in white grapes (Vitis vinifera L.).

BACKGROUND: Secondary metabolism contributes to the adaptation of a plant to its environment. In wine grapes, fruit secondary metabolism largely determines wine quality. Climate change is predicted to exacerbate drought events in several viticultural areas, potentially affecting the wine quality. In red grapes, water deficit modulates flavonoid accumulation, leading to major quantitative and compositional changes in the profile of the anthocyanin pigments; in white grapes, the effect of water deficit on secondary metabolism is still largely unknown. RESULTS: In this study we investigated the impact of water deficit on the secondary metabolism of white grapes using a large scale metabolite and transcript profiling approach in a season characterized by prolonged drought. Irrigated grapevines were compared to non-irrigated grapevines that suffered from water deficit from early stages of berry development to harvest. A large effect of water deficit on fruit secondary metabolism was observed. Increased concentrations of phenylpropanoids, monoterpenes, and tocopherols were detected, while carotenoid and flavonoid accumulations were differentially modulated by water deficit according to the berry developmental stage. The RNA-sequencing analysis carried out on berries collected at three developmental stages-before, at the onset, and at late ripening-indicated that water deficit affected the expression of 4,889 genes. The Gene Ontology category secondary metabolic process was overrepresented within up-regulated genes at all the stages of fruit development considered, and within down-regulated genes before ripening. Eighteen phenylpropanoid, 16 flavonoid, 9 carotenoid, and 16 terpenoid structural genes were modulated by water deficit, indicating the transcriptional regulation of these metabolic pathways in fruit exposed to water deficit. An integrated network and promoter analyses identified a transcriptional regulatory module that encompasses terpenoid genes, transcription factors, and enriched drought-responsive elements in the promoter regions of those genes as part of the grapes response to drought. CONCLUSION: Our study reveals that grapevine berries respond to drought by modulating several secondary metabolic pathways, and particularly, by stimulating the production of phenylpropanoids, the carotenoid zeaxanthin, and of volatile organic compounds such as monoterpenes, with potential effects on grape and wine antioxidant potential, composition, and sensory features.

Characterization of major ripening events during softening in grape: turgor, sugar accumulation, abscisic acid metabolism, colour development, and their relationship with growth.

Along with sugar accumulation and colour development, softening is an important physiological change during the onset of ripening in fruits. In this work, we investigated the relationships among major events during softening in grape (Vitis vinifera L.) by quantifying elasticity in individual berries. In addition, we delayed softening and inhibited sugar accumulation using a mechanical growth-preventing treatment in order to identify processes that are sugar and/or growth dependent. Ripening processes commenced on various days after anthesis, but always at similarly low elasticity and turgor. Much of the softening occurred in the absence of other changes in berry physiology investigated here. Several genes encoding key cell wall-modifying enzymes were not up-regulated until softening was largely completed, suggesting softening may result primarily from decreases in turgor. Similarly, there was no decrease in solute potential, increase in sugar concentration, or colour development until elasticity and turgor were near minimum values, and these processes were inhibited when berry growth was prevented. Increases in abscisic acid occurred early during softening and in the absence of significant expression of the V. vinifera 9-cis-epoxycarotenoid dioxygenases. However, these increases were coincident with decreases in the abscisic acid catabolite diphasic acid, indicating that initial increases in abscisic acid may result from decreases in catabolism and/or exogenous import. These data suggest that softening, decreases in turgor, and increases in abscisic acid represent some of the earliest events during the onset of ripening. Later, physical growth, further increases in abscisic acid, and the accumulation of sugar are integral for colour development.

Transcriptional regulation of anthocyanin biosynthesis in ripening fruits of grapevine under seasonal water deficit.

Anthocyanin biosynthesis is strongly up-regulated in ripening fruit of grapevines (Vitis vinifera L.) grown under drought conditions. We investigated the effects of long-term water deficit on the expression of genes coding for flavonoid and anthocyanin biosynthetic enzymes and related transcription factors, genes sensitive to endogenous [sugars, abscisic acid (ABA)] and environmental (light) stimuli connected to drought stress, and genes developmentally regulated in ripening berries. Total anthocyanin content has increased at harvest in water-stressed (WS) fruits by 37-57% in two consecutive years. At least 84% of the total variation in anthocyanin content was explained by the linear relationship between the integral of mRNA accumulation of the specific anthocyanin biosynthetic gene UDP-glucose : flavonoid 3-O-glucosyltransferase (UFGT) and metabolite content during time series from véraison through ripening. Chalcone synthase (CHS2, CHS3) and flavanone 3-hydroxylase (F3H) genes of the flavonoid pathway showed high correlation as well. Genes coding for flavonoid 3',5'-hydroxylase (F3'5'H) and O-methyltransferase (OMT) were also up-regulated in berries from dehydrated plants in which anthocyanin composition enriched in more hydroxylated and more methoxylated derivatives such as malvidin and peonidin, the grape anthocyanins to which human gastric bilitranslocase displays the highest affinity. The induction in WS plants of structural and regulatory genes of the flavonoid pathway and of genes that trigger brassinosteroid hormonal onset of maturation suggested that the interrelationships between developmental and environmental signalling pathways were magnified by water deficit which actively promoted fruit maturation and, in this context, anthocyanin biosynthesis.

Colour variation in red grapevines (Vitis vinifera L.): genomic organisation, expression of flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase genes and related metabolite profiling of red cyanidin-/blue delphinidin-based anthocyanins in berry skin.

BACKGROUND: Structural genes of the phenyl-propanoid pathway which encode flavonoid 3'- and 3',5'-hydroxylases (F3'H and F3'5'H) have long been invoked to explain the biosynthesis of cyanidin- and delphinidin-based anthocyanin pigments in the so-called red cultivars of grapevine. The relative proportion of the two types of anthocyanins is largely under genetic control and determines the colour variation among red/purple/blue berry grape varieties and their corresponding wines. RESULTS: Gene fragments of VvF3'H and VvF3'5'H, that were isolated from Vitis vinifera 'Cabernet Sauvignon' using degenerate primers designed on plant homologous genes, translated into 313 and 239 amino acid protein fragments, respectively, with up to 76% and 82% identity to plant CYP75 cytochrome P450 monooxygenases. Putative function was assigned on the basis of sequence homology, expression profiling and its correlation with metabolite accumulation at ten different ripening stages. At the onset of colour transition, transcriptional induction of VvF3'H and VvF3'5'H was temporally coordinated with the beginning of anthocyanin biosynthesis, the expression being 2-fold and 50-fold higher, respectively, in red berries versus green berries. The peak of VvF3'5'H expression was observed two weeks later concomitantly with the increase of the ratio of delphinidin-/cyanidin-derivatives. The analysis of structural genomics revealed that two copies of VvF3'H are physically linked on linkage group no. 17 and several copies of VvF3'5'H are tightly clustered and embedded into a segmental duplication on linkage group no. 6, unveiling a high complexity when compared to other plant flavonoid hydroxylase genes known so far, mostly in ornamentals. CONCLUSION: We have shown that genes encoding flavonoid 3'- and 3',5'-hydroxylases are expressed in any tissues of the grape plant that accumulate flavonoids and, particularly, in skin of ripening red berries that synthesise mostly anthocyanins. The correlation between transcript profiles and the kinetics of accumulation of red/cyanidin- and blue/delphinidin-based anthocyanins indicated that VvF3'H and VvF3'5'H expression is consistent with the chromatic evolution of ripening bunches. Local physical maps constructed around the VvF3'H and VvF3'5'H loci should help facilitate the identification of the regulatory elements of each isoform and the future manipulation of grapevine and wine colour through agronomical, environmental and biotechnological tools.