Acetic acid acts as a signal molecule, strongly enhancing xanthone biosynthesis in Hypericum perforatum root cultures. This activity is specific, as demonstrated by the comparison with other short-chain monocarboxylic acids. We have recently demonstrated that Hypericum perforatum root cultures constitutively produce xanthones at higher levels than the root of the plant and that they respond to chitosan (CHIT) elicitation with a noteworthy increase in xanthone production. In the present study, CHIT was administered to H. perforatum root cultures using three different elicitation protocols, and the increase in xanthone production was evaluated. The best results (550 % xanthone increase) were obtained by subjecting the roots to a single elicitation with 200 mg l(-1) CHIT and maintaining the elicitor in the culture medium for 7 days. To discriminate the effect of CHIT from that of the solvent, control experiments were performed by administering AcOH alone at the same concentration used for CHIT solubilization. Unexpectedly, AcOH caused an increase in xanthone production comparable to that observed in response to CHIT. Feeding experiments with (13)C-labeled AcOH demonstrated that this compound was not incorporated into the xanthone skeleton. Other short-chain monocarboxylic acids (i.e., propionic and butyric acid) have little or no effect on the production of xanthones. These results indicate that AcOH acts as a specific signal molecule, able to greatly enhance xanthone biosynthesis in H. perforatum root cultures.

Botrytis cinerea, responsible for grey mold diseases, is a pathogen with a broad host range, affecting many important agricultural crops, in pre and post harvesting of fruits and vegetables. Commercial fungicides used to control this pathogen are often subjected to photolysis, volatilization, degradation, leaching, and runoff during application. In this context, the use of a delivery system, based on poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) represents an innovative approach to develop new pesticide formulations to successfully fight B. cinerea infections. In order to study NPs uptake, B. cinerea conidia and mycelium were treated with PLGA NPs loaded with the high fluorescent probe coumarin 6 (Cu6-PLGA NPs) and analyzed under ApoTome fluorescence microscopy. The observations revealed that 50 nm Cu6-PLGA NPs penetrated into B. cinerea conidia and hyphae, as early as 10 min after administration. Pterostilbene, a natural compound, and fluopyram, a synthetic antifungal, were entrapped in PLGA NPs, added to B. cinerea conidia and mycelium, and their antifungal activity was tested. The results revealed that the compounds loaded in NPs exhibited a higher activity against B. cinerea. These results lay the foundations for the use of PLGA NPs as a new strategy in plant pest management.

Water-soluble chitosan oligosaccharides (COS) affect xanthone and volatile organic compound content, as well as antifungal activity against human pathogenic fungi of extracts obtained from Hypericum perforatum root cultures. Several studies have demonstrated the elicitor power of chitosan on xanthone biosynthesis in root cultures of H. perforatum. One of the major limitations to the use of chitosan, both for basic and applied research, is the need to use acidified water for solubilization. To overcome this problem, the elicitor effect of water-soluble COS on the biosynthesis of both xanthones and volatile organic compounds (VOCs) was evaluated in the present study. The analysis of xanthones and VOCs was performed by HPLC and GC-MS headspace analysis. The obtained results showed that COS are very effective in enhancing xanthone biosynthesis. With 400 mg L COS, a xanthone content of about 30 mg g DW was obtained. The antifungal activity of extracts obtained with 400 mg L COS was the highest, with MIC of 32 µg mL against Candida albicans and 32-64 µg mL against dermatophytes, depending on the microorganism. Histochemical investigations suggested the accumulation of isoprenoids in the secretory ducts of H. perforatum roots. The presence of monoterpenes and sesquiterpenes was confirmed by the headspace analysis. Other volatile hydrocarbons have been identified. The biosynthesis of most VOCs showed significant changes in response to COS, suggesting their involvement in plant-fungus interactions.

Xanthones are a class of secondary metabolites produced by plant organisms. They are characterized by a wide structural variety and numerous biological activities that make them valuable metabolites for use in the pharmaceutical field. This review shows the current knowledge of the xanthone biosynthetic pathway with a focus on the precursors and the enzymes involved, as well as on the cellular and organ localization of xanthones in plants. Xanthone biosynthesis in plants involves the shikimate and the acetate pathways which originate in plastids and endoplasmic reticulum, respectively. The pathway continues following three alternative routes, two phenylalanine-dependent and one phenylalanine-independent. All three routes lead to the biosynthesis of 2,3′,4,6-tetrahydroxybenzophenone, which is the central intermediate. Unlike plants, the xanthone core in fungi and lichens is wholly derived from polyketide. Although organs and tissues synthesizing and accumulating xanthones are known in plants, no information is yet available on their subcellular and cellular localization in fungi and lichens. This review highlights the studies published to date on xanthone biosynthesis and trafficking in plant organisms, from which it emerges that the mechanisms underlying their synthesis need to be further investigated in order to exploit them for application purposes.

To date, most endocytosis studies in plant cells have focused on clathrin-dependent endocytosis, while limited evidence is available on clathrin-independent pathways. Since dynamin a is a key protein both in clathrin-mediated endocytosis and in clathrin-independent endocytic processes, this study investigated its role in the uptake of poly-(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs). The experiments were performed on cultured cells and roots of Arabidopsis thaliana. Dynasore was used to inhibit the activity of dynamin-like proteins to investigate whether PLGA NPs enter plant cells through a dynamin-like-dependent or dynamin-like-independent endocytic pathway. Observations were performed by confocal microscopy using a fluorescent probe, coumarin 6, loaded in PLGA NPs. The results showed that both cells and roots of A. thaliana rapidly take up PLGA NPs. Dynasore was administered at different concentrations and exposure times in order to identify the effective ones for inhibitory activity. Treatments with dynasore did not prevent the NPs uptake, as revealed by the presence of fluorescence emission detected in the cytoplasm. At the highest concentration and the longest exposure time to dynasore, the fluorescence of NPs was not visible due to cell death. Thus, the results suggest that, because the NPs’ uptake is unaffected by dynasore exposure, NPs can enter cells and roots by following a dynamin-like-independent endocytic pathway.