Phenolic Compounds and Capsaicinoids in Three Capsicum annuum Varieties: From Analytical Characterization to In Silico Hypotheses on Biological Activity.

The affinity of specific phenolic compounds (PCs) and capsaicinoids (CAPs) present in three Capsicum annuum varieties (Friariello, Cayenne and Dzuljunska Sipka) to the transient receptor potential vanilloid member 1 (TRPV1) was investigated by integrating an analytic approach for the simultaneous extraction and analysis through high-performance liquid chromatography coupled with ion trap mass spectrometry (HPLC/ITMS) and UV detection (HPLC-UV) of PCs and CAPs and structural bioinformatics based on the protein modelling and molecular simulations of protein-ligand docking. Overall, a total of 35 compounds were identified in the different samples and CAPs were quantified. The highest content of total polyphenols was recorded in the pungent Dzuljunska Sipka variety (8.91 ± 0.05 gGAE/Kg DW) while the lowest was found in the non-pungent variety Friariello (3.58 ± 0.02 gGAE/Kg DW). Protein modelling generated for the first time a complete model of the homotetrameric human TRPV1, and it was used for docking simulations with the compounds detected via the analytic approach, as well as with other compounds, as an inhibitor reference. The simulations indicate that different capsaicinoids can interact with the receptor, providing details on the molecular interaction, with similar predicted binding energy values. These results offer new insights into the interaction of capsaicinoids with TRPV1 and their possible actions.

Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations.

Plasma membrane vesicles isolated by two-phase partitioning from the storage root of Beta vulgaris show atypically high water permeability that is equivalent only to those reported for active aquaporins in tonoplast or animal red cells (Pf=542 microm s(-1)). The values were determined from the shrinking kinetics measured by stopped-flow light scattering. This high Pf was only partially inhibited by mercury (HgCl2) but showed low activation energy (Ea) consistent with water permeation through water channels. To study short-term regulation of water transport that could be the result of channel gating, the effects of pH, divalent cations, and protection against dephosphorylation were tested. The high Pf observed at pH 8.3 was dramatically reduced by medium acidification. Moreover, intra-vesicular acidification (corresponding to the cytoplasmic face of the membrane) shut down the aquaporins. De-phosphorylation was discounted as a regulatory mechanism in this preparation. On the other hand, among divalent cations, only calcium showed a clear effect on aquaporin activity, with two distinct ranges of sensitivity to free Ca2+ concentration (pCa 8 and pCa 4). Since the normal cytoplasmic free Ca2+ sits between these ranges it allows for the possibility of changes in Ca2+ to finely up- or down-regulate water channel activity. The calcium effect is predominantly on the cytoplasmic face, and inhibition corresponds to an increase in the activation energy for water transport. In conclusion, these findings establish both cytoplasmic pH and Ca2+ as important regulatory factors involved in aquaporin gating.

Tonoplast vesicles of Beta vulgaris storage root show functional aquaporins regulated by protons.

BACKGROUND INFORMATION: Water is crucial for plant development and growth, and its transport pathways inside a plant are an ongoing topic for study. Plants express a large number of membrane intrinsic proteins whose role is now being re-evaluated by considering not only the control of the overall plant water balance but also in adaptation to environmental challenges that may affect their physiology. In particular, we focused our work on water movements across the root cell TP (tonoplast), the delimiting membrane of the vacuole. This major organelle plays a central role in osmoregulation. RESULTS: An enriched fraction of TP vesicles from Beta vulgaris (red beet) storage roots obtained by a conventional method was used to characterize its water permeability properties by means of the stopped-flow technique. The preparation showed high water permeability (485 microm x s(-1)), consistent with values reported in the literature. The water permeability was strongly blocked by HgCl(2) (reduced to 16%) and its energy activation was low. These observations allow us to postulate the presence of functional water channels in this preparation. Moreover, Western-blot analysis demonstrated the presence of a tonoplast intrinsic protein. With the purpose of studying the regulation of water channels, TP vesicles were exposed to different acidic pH media. When the pH of a medium was low (pH 5.6), the water permeability exhibited a 42% inhibition. CONCLUSIONS: Our findings prove that although almost all water channels present in the TP vesicles of B. vulgaris root are sensitive to HgCl(2), not all are inhibited by pH. This interesting selectivity to acidification of the medium could play a role in adapting the water balance in the cell-to-cell pathway.