Food-Related Quality of Life and Its Predictors in Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) is associated with dietary restrictions and food- and drink-driven daily life limitations. Food-related quality of life (FR-QoL) is still an under-addressed issue in IBD. AIM: We aimed to study determinants of FR-QoL in an IBD cohort, namely objective measures of disease activity. METHODS: A cross-sectional case-control study was conducted in a Tertiary Hospital, including adult patients with IBD (cases) and blood donors or subjects referred for colorectal polypectomies (controls). Participants answered an anonymous multimodal questionnaire including sociodemographic and clinical data, the validated FR-QoL-29, and the SIBDQ tools. Patients' disease activity was previously assessed by a physician using symptom-based scores and biomarkers (Harvey-Bradshaw index, partial Mayo score, fecal calprotectin). RESULTS: A total of 239 patients with IBD and 126 controls were included. Patients with active disease had poorer FR-QoL than patients in remission (80.0 [56.0-99.0] vs. 103.5 [81.0-129.9], p < 0.001). Still, patients with IBD had significantly lower FR-QoL compared with controls (99.0 [76.0-126.0] vs. 126.0 [102.8-143.0], p < 0.001), irrespective of disease activity. FR-QoL correlated with health-related quality of life, measured by SIBDQ (r = 0.490, p < 0.001), and was significantly impaired by patients' depressive humor (84.0 [61.0-112.0] vs. 108.0 [88.0-130.5], p < 0.001). Globally, FR-QoL compromise was mostly related to persistent worries about food, concerns about food-related symptoms, and life disruption due to eating and drinking. CONCLUSIONS: Patients with IBD showed significant FR-QoL impairment, irrespective of disease type and activity. Related psychosocial factors, such as the patient's affective status and fear around eating, warrant a need for a multidisciplinary approach to IBD, including tailored nutritional counseling.

Prophylactic Versus Endoscopy-driven Treatment of Crohn's Postoperative Recurrence: A Retrospective, Multicentric, European Study [PORCSE Study].

BACKGROUND AND AIMS: No consensus exists on optimal strategy to prevent postoperative recurrence [POR] after ileocaecal resection [ICR] for Crohn's disease [CD]. We compared early medical prophylaxis versus expectant management with treatment driven by findings at elective endoscopy 6-12 months after ICR. METHODS: A retrospective, multicentric, observational study was performed. CD patients undergoing first ICR were assigned to Cohort 1 if a biologic or immunomodulator was [re]started prophylactically after ICR, or to Cohort 2 if no postoperative prophylaxis was given and treatment was started as reaction to elective endoscopic findings. Primary endpoint was rate of endoscopic POR [Rutgeerts >i1]. Secondary endpoints were severe endoscopic POR [Rutgeerts i3/i4], clinical POR, surgical POR, and treatment burden during follow-up. RESULTS: Of 346 included patients, 47.4% received prophylactic postoperative treatment [proactive/Cohort 1] and 52.6% did not [reactive/Cohort 2]. Endoscopic POR [Rutgeerts >i1] rate was significantly higher in Cohort 2 [41.5% vs 53.8%, OR 1.81, p = 0.039] at endoscopy 6-12 months after surgery. No significant difference in severe endoscopic POR was found [OR 1.29, p = 0.517]. Cohort 2 had significantly higher clinical POR rates [17.7% vs 35.7%, OR 3.05, p = 0.002] and numerically higher surgical recurrence rates [6.7% vs 13.2%, OR 2.59, p = 0.051]. Cox proportional hazards regression analysis showed no significant difference in time to surgical POR of proactive versus expectant/reactive approach [HR 2.50, p = 0.057]. Quasi-Poisson regression revealed a significantly lower treatment burden for immunomodulator use in Cohort 2 [mean ratio 0.53, p = 0.002], but no difference in burden of biologics or combination treatment. CONCLUSIONS: The PORCSE study showed lower rates of endoscopic POR with early postoperative medical treatment compared with expectant management after first ileocaecal resection for Crohn's disease.

Combined omics approaches expose metabolite-microbiota correlations in grape berries of three cultivars of Douro wine region.

This study hypothesized the existence of cultivar-associated correlations between grape berry metabolites and its microbial residents, in Douro wine region. Integrated metabolomics with metabarcoding showed that the microbial biodiversity is not associated to berry sugar concentration, but closely connected to the profile of amino acids, flavonoids and wax compounds, which drove cultivar differentiation together with the prevalence of pathogenic fungi, yeasts and bacteria, mainly Dothideomycetes and Gammaproteobacteria. Over 7000 metabolite-microbiota correlations with ρ >|0.99| exposed a core of 15 metabolites linked to 11 microbial taxa. Serine, oxalate, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, gallic acid, germanicol, sitosterol and erythrodiol correlated negatively to the abundance of most taxa, including Alternaria, Aureobasidium, Pseudopithomyces, Pseudomonas and Sphingomonas. In contrast, phenylalanine, asparagine, alanine, (epi)gallocatechin and procyanidin gallate mediated positive metabolite-OTU correlations. E. necator and A. carbonarius correlated negatively with stigmasterol and amyrin. Complex fungi-bacteria relationships ruled by Dothideomycetes and Alphaproteobacteria further suggest tight host-microbe interactions at the carposphere.

The restructuring of grape berry waxes by calcium changes the surface microbiota.

The observation that exogenous Ca shifted the polyphenolic profile of grape berries and conferred a glossy appearance to mature fruits led us to hypothesize that the composition of grape berry waxes and thus surface microbiota are modified. In two cultivars sharing the same microclimate, the triterpenoid and steroid profile of berry cuticle was characterized by a targeted metabolomic approach, and surface microbial communities were surveyed by ITS and 16S metabarcoding. Results showed that Ca strongly decreased the levels of oleanolic acid, while steroids and neutral triterpenoids were affected in a cultivar-dependent manner. A total of 174 fungi and 192 bacteria OTUs were identified, with Dothideomycetes, Leotiomycetes, Alphaproteobacteria and Gammaproteobacteria comprising the most abundant classes. Ca decreased fungi biodiversity, favoring the growth of Basidiomycetes, and shifting fungi-bacteria relationships. Metabolite-microbiota networks revealed a tight relationship between microbial communities and triterpenoid components of fruit waxes, mainly stigmasterol, tremulone and oleanolic acid.

Exogenous Calcium Delays Grape Berry Maturation in the White cv. Loureiro While Increasing Fruit Firmness and Flavonol Content.

Vineyard calcium (Ca) sprays have been increasingly used by grape growers to improve fruit firmness and thus maintain quality, particularly in periods of heavy rains and hail. The observation that Ca visibly modified berry size, texture, and color in the most prominent white cultivar of the DOC region 'Vinhos Verdes', cultivar (cv.) Loureiro, led us to hypothesize that Ca induced metabolic rearrangements that resulted in a substantial delay in fruit maturation. Targeted metabolomics by ultra-performance liquid chromatography coupled to mass spectrometry and directed transcriptomics were thus combined to characterize the metabolic and transcriptional profiles of cv. Loureiro berries that, together with firmness, °Brix, and fruit weight measurements, allowed to obtain an integrated picture of the biochemical and structural effects of Ca in this cultivar. Results showed that exogenous Ca decreased amino acid levels in ripe berries while upregulating PAL1 expression, and stimulated the accumulation of caftaric, coutaric, and fertaric acids. An increase in the levels of specific stilbenoids, namely E-piceid and E-ω-viniferin, was observed, which correlated with the upregulation of STS expression. Trace amounts of anthocyanins were detected in berries of this white cultivar, but Ca treatment further inhibited their accumulation. The increased berry flavonol content upon Ca treatment confirmed that Ca delays the maturation process, which was further supported by an increase in fruit firmness and decrease in weight and °Brix at harvest. This newly reported effect may be specific to white cultivars, a topic that deserves further investigation.

Transcriptional responses of Hypericum perforatum cells to Agrobacterium tumefaciens and differential gene expression in dark glands.

Hypericum perforatum L. (St. John's wort) is a well-known medicinal plant that possesses secondary metabolites with beneficial pharmacological properties. However, improvement in the production of secondary metabolites via genetic manipulation is a challenging task as H. perforatum remains recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, the transcripts of key genes involved in several plant defence responses (secondary metabolites, RNA silencing, reactive oxygen species (ROS) and specific defence genes) were investigated in H. perforatum suspension cells inoculated with A. tumefaciens by quantitative real-time PCR. Results indicated that key genes from the xanthone, hypericin and melatonin biosynthesis pathways, the ROS-detoxification enzyme HpAOX, as well as the defence genes Hyp-1 and HpPGIP, were all upregulated to rapidly respond to A. tumefaciens elicitation in H. perforatum. By contrast, expression levels of genes involved in hyperforin and flavonoid biosynthesis pathways were markedly downregulated upon A. tumefaciens elicitation. In addition, we compared the expression patterns of key genes in H. perforatum leaf tissues with and without dark glands, a major site of secondary metabolite production. Overall, we provide evidence for the upregulation of several phenylpropanoid pathway genes in response to elicitation by Agrobacterium, suggesting that production of secondary metabolites could modulate H. perforatum recalcitrance to A. tumefaciens-mediated transformation.

Calcium and methyl jasmonate cross-talk in the secondary metabolism of grape cells.

In grape cell cultures cv. Gamay Fréaux var. Teinturier, Ca was shown to decrease cell pigmentation through the inhibition of anthocyanin biosynthesis, while stimulating stilbenoids accumulation. Because methyl jasmonate (MeJA) is a well-known inducer of secondary metabolism in grape cells, and Ca antagonizes its stimulatory effect over several enzymes of core metabolic branches, in the present study we hypothesized that Ca and MeJA signaling pathways interact to regulate specific secondary metabolism routes. Grape cultured cells were elicited with MeJA or with MeJA + Ca and an UPLC-MS-based targeted metabolomic method was implemented to characterize their polyphenolic profiles. Results were compared with the profile of cells elicited with Ca only, previously reported. Data was complemented with gene expression analysis, allowing the assembly of a metabolic map that unraveled routes specifically regulated by both elicitors. MeJA + Ca specifically boosted E-resveratrol and E-ε-viniferin levels by 180% and 140%, respectively, in comparison to cells treated with MeJA only, while the stimulatory effect of MeJA over flavonoid synthesis was inhibited by Ca. In parallel, Ca downregulated most flavonoid pathway genes, including LAR1, ANS, BAN and ANR. Ca was able to mimic or potentiate the effect of MeJA on the expression of JA signaling genes, including JAR1, PIN and PR10. Transcript/metabolite correlation networks exposed the central influence of FLS1,STS,CDPK17 and COI1 in polyphenolic biosynthetic routes. This study highlights the potential of the MeJA-Ca combination for diverting polyphenolic metabolism towards the production of specific metabolites of interest, highly relevant in a biotechnological perspective.

Vineyard calcium sprays reduce the damage of postharvest grape berries by stimulating enzymatic antioxidant activity and pathogen defense genes, despite inhibiting phenolic synthesis.

Calcium supplements have been increasingly used for decay prevention, sanitation and nutritional enrichment of fruits, as more environmentally friendly alternatives to fungicides. However, little is known on the effects of these supplements on grape berry biochemical and molecular properties during storage. In this study, we addressed the hypothesis that the application of calcium chloride (CaCl2) in grapevines throughout the fruiting season reduces damage (and decay) of postharvest grape berries, through several biochemical and transcriptional modifications in sugar transport, secondary metabolism, antioxidant activity, cell wall organization and pathogen defense. Results showed that calcium (Ca) treatments in cv. "Vinhão" vines increased fruit Ca content and significantly decreased fruit damage by 60%, 10-d after storage at 4 °C. Grape berries from Ca-treated vines displayed lower levels of total phenolics and anthocyanins, compared to control fruits, corroborating the downregulation of PAL1 and STS which resulted in decreased non-enzymatic antioxidant capacity estimated by FRAP assay. In contrast, a strong upregulation of CAT1, ASPX1, ASPX3, GLPX1, CSD3 and CSD6 encoding antioxidant enzymes was observed. Accordingly, catalase enzyme activity was stimulated, significantly reducing hydrogen peroxide (H2O2) levels by 36%. The overexpression of the cell wall and pathogen defense genes PME, PGIP, PIN and PR1 likely contributed to the reduction in fruit rot. This work suggested that preharvest Ca treatment is an efficient agronomical strategy that prolongs the shelf life of grape berries through modifications at molecular and biochemical levels, bringing further insight on the benefits and drawbacks of preharvest Ca applications on postharvest fruit quality attributes.

Overexpression of polygalacturonase-inhibiting protein (PGIP) gene from Hypericum perforatum alters expression of multiple defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens in tobacco.

Hypericum perforatum L is a remarkable source of high-value secondary metabolites with increasing applications in pharmaceutical industry. However, improvement in the production of secondary metabolites through genetic engineering is a demanding task, as H. perforatum is not amenable to Agrobacterium tumefaciens-mediated transformation. In this study, we identified a Polygalacturonase-inhibiting protein (PGIP) gene from a subtractive cDNA library of A. tumefaciens-treated H. perforatum suspension cells. The role of HpPGIP in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum overexpressing HpPGIP alone or fused at the N-terminus to Phenolic oxidative coupling protein (Hyp-1), a gene that positively modulates resistance to A. tumefaciens. Furthermore, virus-induced gene silencing was employed to knock down the expression of the PGIP homologous in N. benthamiana. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in both HpPGIP and Hyp-1-PGIP transgenic plants, as assessed by GUS staining assays. However, silencing of PGIP in N. benthamiana increased the resistance to A. tumefaciens rather than susceptibility, which correlated with induction of pathogenesis-related proteins (PRs). The expression of core genes involved in several defense pathways was also analyzed in transgenic tobacco plants. Overexpression of HpPGIP led to up-regulation of key genes involved in hormone signaling, microRNA-based gene silencing, homeostasis of reactive oxygen species, and the phenylpropanoid pathway. Overexpression of Hyp-1-PGIP seemed to enhance the effect of PGIP on the expression of most genes analyzed. Moreover, HpPGIP was detected in the cytoplasm, nucleus and the plasma membrane or cell wall by confocal microscopy. Overall, our findings suggest HpPGIP modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

The grapevine CAX-interacting protein VvCXIP4 is exported from the nucleus to activate the tonoplast Ca2+/H+ exchanger VvCAX3.

MAIN CONCLUSION: The nuclear-localized CAX-interacting protein VvCXIP4 is exported to the cytosol after a Ca2+ pulse, to activate the tonoplast-localized Ca2+/H+ exchanger VvCAX3. Vacuolar cation/H+ exchangers (CAXs) have long been recognized as 'housekeeping' components in cellular Ca2+ and trace metal homeostasis, being involved in a range of key cellular and physiological processes. However, the mechanisms that drive functional activation of the transporters are largely unknown. In the present study, we investigated the function of a putative grapevine CAX-interacting protein, VvCXIP4, by testing its ability to activate VvCAX3, previously characterized as a tonoplast-localized Ca2+/H+ exchanger. VvCAX3 contains an autoinhibitory domain that drives inactivation of the transporter and thus, is incapable of suppressing the Ca2+-hypersensitive phenotype of the S. cerevisiae mutant K667. In this study, the co-expression of VvCXIP4 and VvCAX3 in this strain efficiently rescued its growth defect at high Ca2+ levels. Flow cytometry experiments showed that yeast harboring both proteins effectively accumulated higher Ca2+ levels than cells expressing each of the proteins separately. Bimolecular fluorescence complementation (BiFC) assays allowed visualization of the direct interaction between the proteins in tobacco plants and in yeast, and also showed the self-interaction of VvCAX3 but not of VvCXIP4. Subcellular localization studies showed that, despite being primarily localized to the nucleus, VvCXIP4 is able to move to other cell compartments upon a Ca2+ stimulus, becoming prone to interaction with the tonoplast-localized VvCAX3. qPCR analysis showed that both genes are more expressed in grapevine stems and leaves, followed by the roots, and that the steady-state transcript levels were higher in the pulp than in the skin of grape berries. Also, both VvCXIP4 and VvCAX3 were upregulated by Ca2+ and Na+, indicating they share common regulatory mechanisms. However, VvCXIP4 was also upregulated by Li+, Cu2+ and Mn2+, and its expression increased steadily throughout grape berry development, contrary to VvCAX3, suggesting additional physiological roles for VvCXIP4, including the regulation of VvCAXs not yet functionally characterized. The main novelty of the present study was the demonstration of physical interaction between CXIP and CAX proteins from a woody plant model by BiFC assays, demonstrating the intracellular mobilization of CXIPs in response to Ca2+.

Flavescence Dorée-Derived Leaf Yellowing in Grapevine (Vitis vinifera L.) Is Associated to a General Repression of Isoprenoid Biosynthetic Pathways.

Flavescence dorée (FD), caused by the phytoplasma Candidatus Phytoplasma vitis, is a major threat to vineyard survival in different European grape-growing areas. It has been recorded in French vineyards since the mid-1950s, and rapidly spread to other countries. In Portugal, the phytoplasma was first detected in the DOC region of 'Vinhos Verdes' in 2006, and reached the central region of the country in 2009. The infection causes strong accumulation of carbohydrates and phenolics in the mesophyll cells and a simultaneous decrease of chlorophylls, events accompanied by a down regulation of genes and proteins involved in the dark and light-dependent reactions and stabilization of the photosystem II (PSII). In the present study, to better elucidate the basis of the leaf chlorosis in infected grapevine cv. Loureiro, we studied the isoprenoid transcript-metabolite correlation in leaves from healthy and FD-infected vines. Specifically, targeted metabolome revealed that twenty-one compounds (out of thirty-two), including chlorophylls, carotenoids, quinones and tocopherols, were reduced in response to FD-infection. Thereafter, and consistently with the biochemical data, qPCR analysis highlighted a severe FD-mediated repression in key genes involved in isoprenoid biosynthetic pathways. A more diverse set of changes, on the contrary, was observed in the case of ABA metabolism. Principal component analysis (PCA) of all identified metabolites clearly separated healthy from FD-infected vines, therefore confirming that the infection strongly alters the biosynthesis of grapevine isoprenoids; additionally, forty-four genes and metabolites were identified as the components mostly explaining the variance between healthy and infected samples. Finally, transcript-metabolite network correlation analyses were exploited to display the main hubs of the infection process, which highlighted a strong role of VvCHLG, VvVTE and VvZEP genes and the chlorophylls intermediates aminolevulunic acid and porphobilinogen in response to FD infection. Overall, results indicated that the FD infection impairs the synthesis of isoprenoids, through the repression of key genes involved in the biosynthesis of chlorophylls, carotenoids, quinones and tocopherols.

Vineyard calcium sprays shift the volatile profile of young red wine produced by induced and spontaneous fermentation.

Calcium supplements have increasingly been used at pre-harvest stages for improving fruit firmness, aiming at mitigating environmental stress. However, as recent studies demonstrated that calcium modifies the polyphenolic profile of grape berries, we hypothesize in this study that it also affects wine volatile profile. In a two-year study, grapevines cv. "Vinhão" were sprayed with 2% CaCl2 throughout the fruiting season, and musts were prepared at a laboratory scale. Musts from calcium-treated fruits contained higher calcium levels and less anthocyanins. Increased calcium content did not affect the course of fermentation induced with a S. cerevisiae starter inoculum, but impacted the course of spontaneous fermentations carried out by endogenous berry microflora. Several compounds associated to varietal and fermentative aromas were largely influenced by the calcium treatment. For instance, volatile phenols decreased, together with ß-damascenone, benzaldehyde and γ-nonalactone, while several acetates and alcohols increased. Principal component analysis showed that the volatile profile of control wines produced by spontaneous fermentation substantially differed between replicates, but calcium treatment lowered replicate variability. Volatile profiles were also influenced by the vintage and fermentation type. The shift in wine volatile profile upon calcium treatment may be relevant from an oenological perspective.

Vineyard calcium sprays induce changes in grape berry skin, firmness, cell wall composition and expression of cell wall-related genes.

Having a central role in cell wall pectin cross-linking, calcium has been increasingly used as supplement to promote fruit firmness and extended shelf-life. However, the molecular rearrangements associated to increased fruit robustness are still a matter of debate. In this study, mechanical, histochemical and molecular assays were conducted to understand the mechanisms underlying the effects of Ca in fruit physical properties. In a two-year field trial, grapevines were sprayed with exogenous CaCl2 throughout the fruiting season. Results showed an increase in berry Ca concentration at harvest, associated to increased fruit consistency and skin resistance. Scanning electron microscopy showed that fruits from Ca-treated plants had smoother skin surfaces than control fruits, and that microcracks encircling the lenticels were less prominent. Histochemistry assays suggested higher deposition of pectin-like material in skin cell walls in grapes from Ca-treated vines, but no evident modifications in cellulose content were observed. Accordingly, the expression of cellulose synthase family gene CesA3 was not affected by exogenous Ca, while polygalacturonase-encoding genes PG1 and PG2 were downregulated, together with EXP6 belonging to expansin family, and CER9 and CYP15 involved in cuticle biosynthesis. These results suggested that Ca acts by inhibiting pectin degradation and cell wall loosening, while remodeling cuticle structure.

Chitosan Application in Vineyards (Vitis vinifera L. cv. Tinto Cão) Induces Accumulation of Anthocyanins and Other Phenolics in Berries, Mediated by Modifications in the Transcription of Secondary Metabolism Genes.

Despite the numerous beneficial properties and uses of chitosan in agriculture, the molecular mechanisms behind its elicitation potential are still unclear. This study aimed at understanding the effect of chitosan application in the levels of phenolic compounds of Vitis vinifera L. red grapes berry skin (cv. Tinto Cão) during veraison. Grapevines were treated with chitosan (0.01% in 0.01% acetic acid) while control grapevines were sprayed with 0.01% acetic acid. Results showed that several monomeric anthocyanins increased significantly in berry skins after treatment with chitosan. Additionally, Catechin, Rutin and Querecetin-3-O-galactoside were also recorded in higher amount upon chitosan treatment. Besides modulating the phenolic content, chitosan treatment also induced modifications in several target genes encoding key enzymes and transporters involved in secondary metabolic pathways. For instance, the genes PAL, CHS, F3H, ANR, UFGT, ABCC1, GST, MATE1 were upregulated in leaves and berry skins at veraison cessation in response to chitosan treatment. Overall, the results demonstrated that chitosan has a stimulatory effect on the accumulation of phenolic compounds, including anthocyanins, mediated by modifications in the transcription of key genes involved in their biosynthesis and transport in grape berries.

Exogenous calcium deflects grape berry metabolism towards the production of more stilbenoids and less anthocyanins.

Calcium supplements have increasingly been used at pre- and post-harvest stages for improving fruit firmness, but elevated calcium levels in grape cells were shown to reduce total anthocyanin content. In this study, we hypothesized that exogenous calcium influences specific polyphenolic compounds, and performed targeted UPLC-MS analysis in fruits collected from vines cv. "Vinhão" sprayed with 2% (w/v) CaCl2 throughout the fruiting season, in two consecutive vintages, and in grape cell cultures elicited with calcium. Results showed that anthocyanin content is reduced upon calcium treatment, while stilbenoid synthesis is generally stimulated, in line with UFGT and STS expression patterns. The main metabolites involved in this response were malvidin-3-O-glucoside, E-piceid, E-ε-viniferin and E-resveratrol. The accumulation of phenolic acids, catechin and some quercetin derivatives was also favored by calcium, while other flavonols and flavan-3-ols were affected according to the vintage and berry developmental stage. In cell cultures, the entire flavonoid pathway was repressed.

Genome Wide Identification, Molecular Characterization, and Gene Expression Analyses of Grapevine NHX Antiporters Suggest Their Involvement in Growth, Ripening, Seed Dormancy, and Stress Response.

Plant NHX antiporters are critical for cellular pH, Na+, and K+ homeostasis and salt tolerance. Even though their genomic and functional studies have been conducted in many species, the grapevine NHX family has not been described yet. Our work highlights the presence of six VvNHX genes whose phylogenetic analysis revealed their classification in two distinct groups: group I vacuolar (VvNHX1-5) and group II endosomal (VvNHX6). Several cis-acting regulatory elements related to tissue-specific expression, transcription factor binding, abiotic/biotic stresses response, and light regulation elements were identified in their promoter. Expression profile analyses of VvNHX genes showed variable transcription within organs and tissues with diverse patterns according to biochemical, environmental, and biotic treatments. All VvNHXs are involved in berry growth, except VvNHX5 that seems to be rather implicated in seed maturation. VvNHX4 would be more involved in floral development, while VvNHX2 and 3 display redundant roles. QPCR expression analyses of VvNHX1 showed its induction by NaCl and KNO3 treatments, whereas VvNHX6 was induced by ABA application and strongly repressed by PEG treatment. VvNHX1 plays a crucial role in a bunch of grape developmental steps and adaptation responses through mechanisms of phyto-hormonal signaling. Overall, VvNHX family members could be valuable candidate genes for grapevine improvement.

Transgenic expression of Hyp-1 gene from Hypericum perforatum L. alters expression of defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens.

MAIN CONCLUSION: Phenolic oxidative coupling protein (Hyp-1) isolated from Hypericum perforatum L. was characterized as a defense gene involved in H. perforatum recalcitrance to Agrobacterium tumefaciens-mediated transformation Hypericum perforatum L. is a reservoir of high-value secondary metabolites of increasing interest to researchers and to the pharmaceutical industry. However, improving their production via genetic manipulation is a challenging task, as H. perforatum is recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, phenolic oxidative coupling protein (Hyp-1), a pathogenesis-related (PR) class 10 family gene, was selected from a subtractive cDNA library from A. tumefaciens-treated H. perforatum suspension cells. The role of Hyp-1 in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum and Lactuca sativa overexpressing Hyp-1, and in Catharanthus roseus silenced for its homologous Hyp-1 gene, CrIPR. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in Hyp-1 transgenic plants. However, silencing of CrIPR induced CrPR-5 expression and decreased expression efficiency of Agrobacterium. The expression of core genes involved in several defense pathways was also analyzed in Hyp-1 transgenic tobacco plants. Overexpression of Hyp-1 led to an ample down-regulation of key genes involved in auxin signaling, microRNA-based gene silencing, detoxification of reactive oxygen species, phenylpropanoid pathway and PRs. Moreover, Hyp-1 was detected in the nucleus, plasma membrane and the cytoplasm of epidermal cells by confocal microscopy. Overall, our findings suggest Hyp-1 modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

Resveratrol-Loaded Lipid Nanocarriers Are Internalized By Endocytosis in Yeast.

Different positive pharmacological effects have been attributed to the natural product resveratrol (RSV), including antioxidant, antiaging, and cancer chemopreventive properties. However, its low bioavailability and rapid metabolic degradation has led to the suspicion that many of the biological activities of this compound observed in vitro may not be attainable in humans. To improve its bioavailability and pharmacokinetic profile, attempts have been made to encapsulate RSV into lipid-based nanocarrier systems. Here, the dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) liposomal system (1:2) loaded with RSV revealed appropriate characteristics for drug release purposes: reduced size for cellular uptake (157 ± 23 nm), stability up to 80 days, positive surface charge (ζ ≈ +40 mV), and a controlled biphasic release of RSV from the lipid nanocarriers over a period of almost 50 h at pH 5.0 and 7.4. Moreover, the encapsulation efficiency of the nanocarrier ranged from 70% to 92% and its RSV loading capacity from 9% to 14%, when [RSV] was between 100 and 200 µM. The partition coefficient ( Kp) of RSV between lipid and aqueous phase was log Kp = 3.37 ± 0.10, suggesting moderate to high lipophilicity of this natural compound and reinforcing the lipid nanocarriers' suitability for RSV incorporation. The thermodynamic parameters of RSV partitioning in the lipid nanocarriers at 37 °C (Δ H = 43.76 ± 5.68 kJ mol-1; Δ S = 0.20 ± 0.005 kJ mol-1; and Δ G = -18.46 ± 3.48 kJ mol-1) reflected the spontaneity of the process and the establishment of hydrophobic interactions. The cellular uptake mechanism of the RSV-loaded nanocarriers labeled with the lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was studied in the eukaryotic model system Saccharomyces cerevisiae. Thirty minutes after incubation, yeast cells readily internalized nanocarriers and the spots of blue fluorescence of DPH clustered around the central vacuole in lipid droplets colocalized with the green fluorescence of the lipophilic endocytosis probe FM1-43. Subsequent studies with the endocytosis defective yeast deletion mutant ( end3Δ) and with the endocytosis inhibitor methyl-ß-cyclodextrin supported the involvement of an endocytic pathway. This novel nanotechnology approach opens good perspectives for medical applications.

Calcium- and hormone-driven regulation of secondary metabolism and cell wall enzymes in grape berry cells.

The efficacy of calcium sprays for improving fleshy fruit resistance to abiotic/biotic stress and enhancement of fruit shelf life has increasingly been explored. However, because calcium is a powerful secondary messenger in many signaling pathways, including those driven by abscisic acid (ABA) and jasmonates, it may interfere with the biosynthesis of specialized metabolites highly important for fruit and wine quality, such as phenolic compounds. In this study, a combination of biochemical and molecular biology approaches were applied to grape cell cultures and detached grape berries, in order to investigate the effect of calcium in the modulation of enzymes involved in the biosynthesis of phenolic compounds and in cell wall organization. Concentrations up to 10 mM CaCl2 did not affect cell growth, size or viability, but triggered modifications in total phenolics content, particularly in anthocyanin levels in grape cell suspensions. The effects of calcium applied alone or in combination with ABA or methyl jasmonate (MeJA) were visible in several branches of specialized metabolic pathways, confirming that the calcium-hormone interplay regulates the expression of phenylalanine ammonia lyase (PAL), stilbene synthase (STS), dihydroflavonol reductase (DFR) and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT). The activity of PAL and UFGT enzymes was also specifically modulated by calcium, ABA and MeJA. These results closely correlated to the modifications observed in the expression of VvAM1 and VvABCC1 encoding vacuolar anthocyanin transporters. Modulation of the expression and activity of pectin methyl esterases (PME) and polygalacturonases (PG) by calcium was also evident, confirming an important role of calcium in cell wall organization via the regulation of enzyme activity, besides its well-known role in the formation of cross links between pectin molecules. Overall, this study uncovers important biochemical mechanisms induced by calcium and stress hormones on grape berries, and highlights the need to consider the consequences of calcium treatments and stress for fruit quality.

The grapevine VvCAX3 is a cation/H+ exchanger involved in vacuolar Ca2+ homeostasis.

MAIN CONCLUSION: The grapevine VvCAX3 mediates calcium transport in the vacuole and is mostly expressed in green grape berries and upregulated by Ca 2+ , Na + and methyl jasmonate. Calcium is an essential plant nutrient with important regulatory and structural roles in the berries of grapevine (Vitis vinifera L.). On the other hand, the proton-cation exchanger CAX proteins have been shown to impact Ca2+ homeostasis with important consequences for fruit integrity and resistance to biotic/abiotic stress. Here, the CAX gene found in transcriptomic databases as having one of the highest expressions in grapevine tissues, VvCAX3, was cloned and functionally characterized. Heterologous expression in yeast showed that a truncated version of VvCAX3 lacking its NNR autoinhibitory domain (sCAX3) restored the ability of the yeast strain to grow in 100-200 mM Ca2+, demonstrating a role in Ca2+ transport. The truncated VvCAX3 was further shown to be involved in the transport of Na+, Li+, Mn2+ and Cu2+ in yeast cells. Subcellular localization studies using fluorescently tagged proteins confirmed VvCAX3 as a tonoplast transporter. VvCAX3 is expressed in grapevine stems, leaves, roots, and berries, especially at pea size, decreasing gradually throughout development, in parallel with the pattern of calcium accumulation in the fruit. The transcript abundance of VvCAX3 was shown to be regulated by methyl jasmonate (MeJA), Ca2+, and Na+ in grape cell suspensions, and the VvCAX3 promotor contains several predicted cis-acting elements related to developmental and stress response processes. As a whole, the results obtained add new insights on the mechanisms involved in calcium homeostasis and intracellular compartmentation in grapevine, and indicate that VvCAX3 may be an interesting target towards the development of strategies for enhancement of grape berry properties.