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.

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.

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.

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.

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.