Instituto de Biología Agrícola de Mendoza

Grape pomaces (GPs) are characterized by high contents of phenolics due to an incomplete extraction during the winemaking process. These phenolics are secondary plant metabolites with potential beneficial effects on human health because of their antioxidant activity and antimicrobial, antiviral, and anti-inflammatory properties. Therefore, GP constitutes an inexpensive source for the extraction of phytochemicals that can be used in the pharmaceutical, cosmetic, and food industries. As a result of the increased attention to sustainability of agricultural practices, efforts have been made to use GP in different fields of industry. Thus, it is necessary to have efficient extraction techniques to achieve good recoveries of compounds. In this respect, sensitive and selective analytical methods have been tried for the characterization of phenolic extracts. This review summarizes the most recent developments in the extraction of polyphenols from GPs. Furthermore, the techniques used for characterization of extracts are explained, with emphasis on sample preparation, separation, and analysis of phenolics. Finally, the possible applications of GP extracts in diverse biotechnological fields are also discussed.

Production of phytohormones is one of the main mechanisms to explain the beneficial effects of plant growth-promoting rhizobacteria (PGPR) such as Azospirillum sp. The PGPRs induce plant growth and development, and reduce stress susceptibility. However, little is known regarding the stress-related phytohormone abscisic acid (ABA) produced by bacteria. We investigated the effects of Azospirillum brasilense Sp 245 strain on Arabidopsis thaliana Col-0 and aba2-1 mutant plants, evaluating the morphophysiological and biochemical responses when watered and in drought. We used an in vitro-grown system to study changes in the root volume and architecture after inoculation with Azospirillum in Arabidopsis wild-type Col-0 and on the mutant aba2-1, during early growth. To examine Arabidopsis development and reproductive success as affected by the bacteria, ABA and drought, a pot experiment using Arabidopsis Col-0 plants was also carried out. Azospirillum brasilense augmented plant biomass, altered root architecture by increasing lateral roots number, stimulated photosynthetic and photoprotective pigments and retarded water loss in correlation with incremented ABA levels. As well, inoculation improved plants seed yield, plants survival, proline levels and relative leaf water content; it also decreased stomatal conductance, malondialdehyde and relative soil water content in plants submitted to drought. Arabidopsis inoculation with A. brasilense improved plants performance, especially in drought.

Eleven bacterial strains were isolated at different soil depths from roots and rhizosphere of grapevines from a commercial vineyard. By 16S rRNA gene sequencing 10 different genera and 8 possible at species level were identified. From them, Bacillus licheniformis Rt4M10 and Pseudomonas fluorescens Rt6M10 were selected according to their characteristics as plant growth promoting rhizobacteria (PGPR). Both produced abscisic acid (ABA), indole-3-acetic acid (IAA) and the gibberellins A1 and A3 in chemically-defined medium. They also colonized roots of in vitro grown Vitis vinifera cv. Malbec plants. As result of bacterization ABA levels in 45 days-old in vitro plants were increased 76-fold by B. licheniformis and 40-fold by P. fluorescens as compared to controls. Both bacteria diminished plant water loss rate in correlation with increments of ABA. Twenty and 30 days post bacterization the plants incremented terpenes. The monoterpenes α-pinene, terpinolene, 4-carene, limonene, eucalyptol and lilac aldehyde A, and the sesquiterpenes α-bergamotene, α-farnesene, nerolidol and farnesol were assessed by gas chromatography-electron impact mass spectrometry analysis. α-Pinene and nerolidol were the most abundant (µg per g of tissue in plants bacterized with P. fluorescens). Only α-pinene, eucalyptol and farnesol were identified at low concentration in non-bacterized plants treated with ABA, while no terpenes were detected in controls. The results obtained along with others from literature suggest that B. licheniformis and P. fluorescens act as stress alleviators by inducing ABA synthesis so diminishing water losses. These bacteria also elicit synthesis of compounds of plant defense via an ABA independent mechanism.

It has been previously found that abscisic acid (ABA) participates in the activation of grapevine leaf tissue defense against potentially damaging effects of solar ultraviolet-B radiation (UV-B), apparently by triggering biosynthesis of phenols that filter the harmful radiation and act as antioxidants. The present work studies the effect of solar UV-B and exogenously applied ABA on berry growth, sugar accumulation, and phenol (anthocyanin and nonanthocyanin) profiles across berry development and ripening of Vitis vinifera L. cv. Malbec in a vineyard at 1450 m of altitude. The grapevines were exposed to relatively high UV-B irradiation (normal sunlight; +UV-B) and also to a reduced UV-B treatment (filter exclusion; -UV-B). These two UV-B treatments were combined with weekly spray applications to the leaves and berries of 1 mM ABA (+ABA) or H(2)O (-ABA). Reduction of UV-B delayed berry development and maturation, whereas the +UV-B and +ABA combined treatment hastened berry sugar and phenol accumulation. +UV-B/+ABA treatments also reduced berry growth and decreased sugar per berry without affecting sugar concentration (°Brix) at harvest. Berry skin ABA levels were higher in the +UV-B and +ABA combined treatment, which also hastened the onset of ripening up to 20 days. Berry skin ABA levels then decreased toward harvest, implying a possible role for ABA in the control of ripening in this nonclimacteric fruit. Under both +UV-B and +ABA treatments berry skin phenols were additively increased with a change in anthocyanin and nonanthocyanin profiles and increases in the proportion of phenols with high antioxidant capacity.

Horizontal gene transfer (HGT) among flowering plant mitochondria occurs frequently and, in most cases, leads to nonfunctional transgenes in the recipient genome. Parasitic plants are particularly prone to this phenomenon, but their mitochondrial genomes (mtDNA) have been largely unexplored. We undertook a large-scale mitochondrial genomic study of the holoparasitic plant Lophophytum mirabile (Balanophoraceae). Comprehensive phylogenetic analyses were performed to address the frequency, origin, and impact of HGT. The sequencing of the complete mtDNA of L. mirabile revealed the unprecedented acquisition of host-derived mitochondrial genes, representing 80% of the protein-coding gene content. All but two of these foreign genes replaced the native homologs and are probably functional in energy metabolism. The genome consists of 54 circular-mapping chromosomes, 25 of which carry no intact genes. The likely functional replacement of up to 26 genes in L. mirabile represents a stunning example of the potential effect of rampant HGT on plant mitochondria. The use of host-derived genes may have a positive effect on the host-parasite relationship, but could also be the result of nonadaptive forces.

Abstract Natural deep eutectic solvents (NADES) is one of the hottest issues in the field of green chemistry. However, it is possible to make them greener? Herein we propose a new microwave (MW) assisted preparation demanding only seconds. The MW‐NADES were challenged with those obtained by the classical Heating & Stirring procedure in terms of green analytical metrics and physical parameters. Indeed, the following analytical applications were tested: a) extraction media for phenolics and alkaloids from plant, b) enhancers for electrochemical detection of phenolic compounds. Our results demonstrate outstanding environmental advantages as the dramatic decrease of synthesis time (the shortest time to date, 60 minutes to 20 seconds) and energy consumption (650 times lower). The analytical performance and physicochemical parameters were comparable with the traditional NADES.

Brassicaceae vegetables are important crops consumed worldwide due to their unique flavor, and for their broadly recognized functional properties, which are directly related to their phytochemical composition. Isothiocyanates (ITC) are the most characteristic compounds, considered responsible for their pungent taste. Besides ITC, these vegetables are also rich in carotenoids, phenolics, minerals, and vitamins. Consequently, Brassica’s phytochemical profile makes them an ideal natural source for improving the nutritional quality of manufactured foods. In this sense, the inclusion of functional ingredients into food matrices are of growing interest. In the present work, Brassicaceae ingredients, functionality, and future perspectives are reviewed.

The identification of melatonin in plants has inspired new investigations to understand its biological function and which endogenous and external factors control its levels in these organisms. Owing to the therapeutical and nutraceutical properties of melatonin, it should be important to develop reliable analytical methods for its quantification in vegetal matrices containing this indoleamine, such as grape and wine. The main objectives of the present study were to test whether melatonin levels fluctuate during the day in berry skins of Vitis vinifera L. cv Malbec, thereby possibly relating its abundance to its putative antioxidant function, to determine whether daylight reaching clusters negatively controls melatonin levels, and to evaluate whether total polyphenols and anthocyanins also change through a 24-hr period. Grapes were harvested throughout the day/night to determine the moment when high levels of these components are present in grapes. The presence of melatonin in grapes was evaluated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry. It is shown for the first time that melatonin levels fluctuate during the day/night cycle in plants grown under field conditions in a fruit organ of the species Vitis vinifera. We also determined that the diurnal decay of melatonin in berry skins is induced by sunlight, because covered bunches retained higher melatonin levels than exposed ones, thus explaining at least part of the basis of its daily fluctuation. Evidence of melatonin's antioxidant role in grapes is also suggested by monitoring malondialdehyde levels during the day.

BACKGROUND: Rapid and consistent genotyping is an important requirement for cultivar identification in many crop species. Among them grapevine cultivars have been the subject of multiple studies given the large number of synonyms and homonyms generated during many centuries of vegetative multiplication and exchange. Simple sequence repeat (SSR) markers have been preferred until now because of their high level of polymorphism, their codominant nature and their high profile repeatability. However, the rapid application of partial or complete genome sequencing approaches is identifying thousands of single nucleotide polymorphisms (SNP) that can be very useful for such purposes. Although SNP markers are bi-allelic, and therefore not as polymorphic as microsatellites, the high number of loci that can be multiplexed and the possibilities of automation as well as their highly repeatable results under any analytical procedure make them the future markers of choice for any type of genetic identification. RESULTS: We analyzed over 300 SNP in the genome of grapevine using a re-sequencing strategy in a selection of 11 genotypes. Among the identified polymorphisms, we selected 48 SNP spread across all grapevine chromosomes with allele frequencies balanced enough as to provide sufficient information content for genetic identification in grapevine allowing for good genotyping success rate. Marker stability was tested in repeated analyses of a selected group of cultivars obtained worldwide to demonstrate their usefulness in genetic identification. CONCLUSIONS: We have selected a set of 48 stable SNP markers with a high discrimination power and a uniform genome distribution (2-3 markers/chromosome), which is proposed as a standard set for grapevine (Vitis vinifera L.) genotyping. Any previous problems derived from microsatellite allele confusion between labs or the need to run reference cultivars to identify allele sizes disappear using this type of marker. Furthermore, because SNP markers are bi-allelic, allele identification and genotype naming are extremely simple and genotypes obtained with different equipments and by different laboratories are always fully comparable.

BACKGROUND: Ultraviolet-B radiation (UV-B, 280-315 nm) is a natural component of sunlight, which has numerous regulatory effects on plant physiology. The nature of the response to UV-B is dependent on fluence rate, dose, duration and wavelength of the UV-B treatment. Some reports have analyzed the changes in gene expression caused by UV-B light on several plant species using microarray technology. However, there is no information on the transcriptome response triggered by UV-B in grapevine. In this paper we investigate the gene expression responses of leaves from in vitro cultured Vitis vinifera cv. Malbec plants subjected to the same dose of biologically effective UV-B radiation (4.75 kJ m-2 d-1) administered at two different fluence rates (16 h at ≅ 8.25 μW cm-2, 4 h at ≅ 33 μW cm-2) using a new custom made GrapeGen Affymetrix GeneChip®. RESULTS: The number of genes modulated by high fluence rate UV-B doubled the number of genes modulated by low fluence UV-B. Their functional analyses revealed several functional categories commonly regulated by both UV-B treatments as well as categories more specifically modulated depending on UV-B fluence rate. General protective responses, namely the induction of pathways regulating synthesis of UV-B absorbing compounds such as the Phenylpropanoid pathway, the induction of different antioxidant defense systems and the activation of pathways commonly associated with pathogen defense and abiotic stress responses seem to play critical roles in grapevine responses against UV-B radiation. Furthermore, high fluence rate UV-B seemed to specifically modulate additional pathways and processes in order to protect grapevine plantlets against UV-B-induced oxidative stress, stop the cell cycle progression, and control protein degradation. On the other hand, low fluence rate UV-B regulated the expression of specific responses in the metabolism of auxin and abscisic acid as well as in the modification of cell walls that could be involved in UV-B acclimation-like processes. CONCLUSION: Our results show the UV-B radiation effects on the leaf transcriptome of grapevine (Vitis vinifera cv. Malbec) plantlets. Functional categories commonly modulated under both UV-B treatments as well as transcripts specifically regulated in an UV-B-intensity dependent way were identified. While high fluence rate UV-B had regulatory effects mainly on defense or general multiple-stress responses pathways, low fluence rate UV-B promoted the expression of genes that could be involved in UV-B protection or the amelioration of the UV-B-induced damage. This study also provides an extensive list of genes regulating multiple metabolic pathways involved in the response of grapevine to UV-B that can be used for future researches.

transcription factors required for anthocyanin pigmentation in the berry skin. Loss of heterozygosity and decreased copy number delimited interspersed monosomic and disomic regions in the right arm of linkage groups 2 and 5. At least 11 validated clustered breakpoints involving intrachromosomal and interchromosomal translocations between three linkage groups flanked the deleted fragments, which, according to segregation analyses, are phased in a single copy of each of the affected chromosomes. These hallmarks, along with the lack of homology between breakpoint joins and the randomness of the order and orientation of the rearranged fragments, are all consistent with a chromothripsis-like pattern generated after chromosome breakage and illegitimate rejoining. This unbalanced genome reshuffling has additional consequences in reproductive development. In TB, lack of sexual transmission of rearranged chromosomes associates with low gamete viability, which compromises fruit set and decreases fruit production. Our findings show that catastrophic genome rearrangements arise spontaneously and stabilize during plant somatic growth. These dramatic rearrangements generate new interesting phenotypes that can be selected for the improvement of vegetatively propagated plant species.

Phytochrome B (phyB) can adjust morphological and physiological responses according to changes in the red:far-red (R:FR) ratio. phyB-driven acclimation of plants to open environments (high R:FR ratio) increases carbon gain at the expense of increased water loss. This behaviour alleviates stressful conditions generated by an excess of light, but increases the chances of desiccation. Here we evaluated how phyB modulates this drought-tolerance response by comparing wild-type Arabidopsis thaliana adult plants to the null phyB in response to water shortage. phyB wilted before the wild type, and this was due to phyB maintaining open stomata under a reduction in soil water availability. Although phyB presented enhanced ABA levels under well-watered conditions, this mutant was less sensitive than the wild type in diminishing stomatal conductance in response to exogenous ABA application. Reduced sensitivity to ABA in phyB correlated with a lower expression of ABCG22, which encodes a putative ABA influx transporter, and PYL5, which encodes a soluble ABA receptor. Furthermore, the expression of RAB18 and RD29A, both typical ABA-induced genes, was lower in phyB than the wild type after ABA treatment. We propose that phyB contributes to the acclimation of plants to open environments by enhancing ABA sensitivity when soil water becomes limiting.

SCOPE: This study evaluated the capacity of dietary catechin (C), quercetin (Q), and the combination of both (CQ), to attenuate adipose inflammation triggered by high fructose (HFr) consumption in rats and by tumor necrosis factor alpha (TNF-α) in 3T3-L1 adipocytes. METHODS AND RESULTS: In rats, HFr consumption for 6 wk caused dyslipidemia, insulin resistance, reduced plasma adiponectin, adiposity, and adipose tissue inflammation. Dietary supplementation with 20 mg/kg/day of C, Q, and CQ improved all these parameters. In 3T3-L1 adipocytes, C and Q attenuated TNF-α-induced elevated protein carbonyls, increased proinflammatory cytokine expression (MCP-1, resistin), and decreased adiponectin. The protective effects of C and Q on adipose inflammation are in part associated with their capacity to (i) decrease the activation of the mitogen-activated kinases (MAPKs) JNK and p38; and (ii) prevent the downregulation of PPAR-γ. In summary, C and Q, and to a larger extent the combination of both, attenuated adipose proinflammatory signaling cascades and regulated the balance of molecules that improve (adiponectin) or impair (TNF-α, MCP-1, resistin) insulin sensitivity. CONCLUSION: Together, these findings suggest that dietary Q and C may have potential benefits in mitigating MetS-associated adipose inflammation, oxidative stress, and insulin resistance.

Leaf epidermal peels of Arabidopsis (Arabidopsis thaliana) mutants lacking either phototropins 1 and 2 (phot1 and phot2) or cryptochromes 1 and 2 (cry1 and cry2) exposed to a background of red light show severely impaired stomatal opening responses to blue light. Since phot and cry are UV-A/blue light photoreceptors, they may be involved in the perception of the blue light-specific signal that induces the aperture of the stomatal pores. In leaf epidermal peels, the blue light-specific effect saturates at low irradiances; therefore, it is considered to operate mainly under the low irradiance of dawn, dusk, or deep canopies. Conversely, we show that both phot1 phot2 and cry1 cry2 have reduced stomatal conductance, transpiration, and photosynthesis, particularly under the high irradiance of full sunlight at midday. These mutants show compromised responses of stomatal conductance to irradiance. However, the effects of phot and cry on photosynthesis were largely nonstomatic. While the stomatal conductance phenotype of phot1 phot2 was blue light specific, cry1 cry2 showed reduced stomatal conductance not only in response to blue light, but also in response to red light. The levels of abscisic acid were elevated in cry1 cry2. We conclude that considering their effects at high irradiances cry and phot are critical for the control of transpiration and photosynthesis rates in the field. The effects of cry on stomatal conductance are largely indirect and involve the control of abscisic acid levels.

Grapevine berry quality for winemaking depends on complex and dynamic relationships between the plant and the environment. Winemakers around the world are demanding a better understanding of the factors that influence berry growth and development. In the last decades, an increment in air temperature, CO 2 concentration and dryness occurred in wine-producing regions, affecting the physiology and the biochemistry of grapevines, and by consequence the berry quality. The scientific community mostly agrees in a further raise as a result of climate change during the rest of the century. As a consequence, areas most suitable for viticulture are likely to shift into higher altitudes where mean temperatures are suitable for grape cultivation. High altitude can be defined as the minimum altitude at which the grapevine growth and development are differentially affected. At these high altitudes, the environments are characterized by high thermal amplitudes and great solar radiations, especially ultraviolet-B (UV-B). This review summarizes the environmental contribution of global high altitude-related climatic variables to the grapevine physiology and wine composition, for a better evaluation of the possible establishment of vineyards at high altitude in climate change scenarios.

Meeting global food demands for a growing human population with finite natural resources is a major challenge. Aquaculture and agriculture are critical to satisfy food requirements, yet suffer significant losses from bacterial diseases. Therefore, there is an urgent need to develop novel antimicrobial strategies, which is heightened by increasing antibiotic resistance. Bacteriophages (phages) are viruses that specifically infect bacteria, and phage-derived therapies are promising treatments in the fight against bacterial diseases. Here, we describe multiple ways that phages and phage-based technologies can be used as antimicrobials. Antimicrobial activity can be achieved through lysis of targeted bacteria by virulent phages or lytic enzymes. Alternatively, phages can be engineered for the delivery of lethal genes and other cargoes to kill bacteria and to manipulate the bacterial response to conventional antibiotics. We also briefly highlight research exploring phages as potential biocontrol agents with examples from agriculture and aquaculture.

We previously demonstrated that solar ultraviolet-B (UV-B) radiation levels in high altitude vineyards improve berry quality in Vitis vinifera cv. Malbec, but also reduce berry size and yield, possibly as a consequence of increased oxidative damage and growth reductions (lower photosynthesis). The defense mechanisms toward UV-B signal and/or evoked damage promote production of antioxidant secondary metabolites instead of primary metabolites. Purportedly, the UV-B effects will depend on tissues developmental stage and interplay with other environmental conditions, especially stressful situations. In this work, grapevines were exposed to high solar UV-B (+UV-B) and reduced (by filtering) UV-B (-UV-B) treatments during three consecutive seasons, and the effects of UV-B, developmental stages and seasons on the physiology were studied, i.e. growth, tissues morphology, photosynthesis, photoprotective pigments, proline content and antioxidant capacity of leaves. The +UV-B reduced photosynthesis and stomatal conductance, mainly through limitation in gas exchange, reducing plant's leaf area, net carbon fixation and growth. The +UV-B augmented leaf thickness, and also the amounts of photoprotective pigments and proline, thereby increasing the antioxidant capacity of leaves. The defense mechanisms triggered by + UV-B reduced lipid peroxidation, but they were insufficient to protect the photosynthetic pigments per leaf dry weight basis. The +UV-B effects depend on tissues developmental stage and interplay with other environmental conditions such as total radiation and air temperatures.

Several studies have shown the presence of melatonin and related compounds in grapes and wines. The latter provides evidence of the possibility to enhance the nutraceutical properties of premium wines. However, there are many external factors that can influence the levels of this indolamine in grape and wines. In this study, the monitoring of melatonin and its tentatively identified isomer was carried out during the entire winemaking process in Vitis vinifera cv. Malbec by ultra high-performance liquid chromatography-tandem mass spectrometry. Laboratory and pilot studies were carried out to elucidate the role of grape, yeasts, and tryptophan in the evolution of the indolamines during the fermentation process. Melatonin was detected in grape extract within the range 120-160 ng/g while its isomer was found in musts and finished wines. Our results demonstrate that Saccaromyces cervisiae plays a decisive role in contributing to the content of melatonin and its isomer in wine.

Under conditions of competition for light, which lead to the inactivation of the photoreceptor phytochrome B (phyB), the growth of shade-intolerant plants is promoted and the accumulation of direct anti-herbivore defenses is down-regulated. Little is known about the effects of phyB on emissions of volatile organic compounds (VOCs), which play a major role as informational cues in indirect defense. We investigated the effects of phyB on direct and indirect defenses in tomato (Solanum lycopersicum) using two complementary approaches to inactivate phyB: illumination with a low red to far-red ratio, simulating competition, and mutation of the two PHYB genes present in the tomato genome. Inactivation of phyB resulted in low levels of constitutive defenses and down-regulation of direct defenses induced by methyl jasmonate (MeJA). Interestingly, phyB inactivation also had large effects on the blends of VOCs induced by MeJA. Moreover, in two-choice bioassays using MeJA-induced plants, the predatory mirid bug Macrolophus pygmaeus preferred VOCs from plants in which phyB was inactivated over VOCs from control plants. These results suggest that, in addition to repressing direct defense, phyB inactivation has consequences for VOC-mediated tritrophic interactions in canopies, presumably attracting predators to less defended plants, where they are likely to find more abundant prey.

Grape quality for winemaking depends on sugar accumulation and metabolism in berries. Abscisic acid (ABA) and gibberellins (GAs) have been reported to control sugar allocation in economically important crops, although the mechanisms involved are still unknown. The present study tested if ABA and gibberellin A3 (GA3) enhance carbon allocation in fruits of grapevines by modifying phloem loading, phloem area and expression of sugar transporters in leaves and berries. Pot-grown Vitis vinifera cv. Malbec plants were sprayed with ABA and GA3 solutions. The amount of soluble sugars in leaves and berries related to photosynthesis were examined at three points of berry growth: pre-veraison, full veraison and post-veraison. Starch levels and amylase activity in leaves, gene expression of sugar transporters in leaves and berries and phloem anatomy were examined at full veraison. Accumulation of glucose and fructose in berries was hastened in ABA-treated plants at the stage of full veraison, which was correlated with enhancement of Vitis vinifera HEXOSE TRANSPORTER 2 (VvHT2) and Vitis vinifera HEXOSE TRANSPORTER 6 (VvHT6) gene expression, increases of phloem area and sucrose content in leaves. On the other hand, GA3 increased the quantity of photoassimilates delivered to the stem thus increasing xylem growth. In conclusion, stimulation of sugar transport by ABA and GA3 to berries and stems, respectively, was due to build-up of non-structural carbohydrates in leaves, modifications in phloem tissue and modulation in gene expression of sugar transporters.