Actin organization during eucalyptus root hair development and its response to fungal hypaphorine.

The fungus Pisolithus microcarpus establishes an ectomycorrhiza with Eucalyptus globulus. This symbiosis involves a fungal synthesis and secretion of hypaphorine, an indolic compound. Previous studies have shown that hypaphorine induces an alteration in the actin cytoskeleton of elongating root hairs and inhibits hair elongation. Using an alternative approved method, we analyzed the effects of hypaphorine on the E. globulus root hair cyto-architecture and actin configuration in more detail and provide new results. One mM hypaphorine stops root hair elongation within 20 min, and changes the hair cyto-architecture. Semi-quantitative analysis of the actin cytoskeleton before and after treatment with hypaphorine shows that hypaphorine induces a shift from fine F-actin to F-actin bundles in the sub-apex of the hair, which occurs first in the mid-plane of the cell. This creates a sub-apical cell centre free of filamentous actin, an actin configuration that differs from that during developmental growth arrest. The mechanism of action of hypaphorine is discussed.

Metabolomics meets functional assays: coupling LC-MS and microfluidic cell-based receptor-ligand analyses.

INTRODUCTION: Metabolomics has become a valuable tool in many research areas. However, generating metabolomics-based biochemical profiles without any related bioactivity is only of indirect value in understanding a biological process. Therefore, metabolomics research could greatly benefit from tools that directly determine the bioactivity of the detected compounds. OBJECTIVE: We aimed to combine LC-MS metabolomics with a cell based receptor assay. This combination could increase the understanding of biological processes and may provide novel opportunities for functional metabolomics. METHODS: We developed a flow through biosensor with human cells expressing both the TRPV1, a calcium ion channel which responds to capsaicin, and the fluorescent intracellular calcium ion reporter, YC3.6. We have analysed three contrasting Capsicum varieties. Two were selected with contrasting degrees of spiciness for characterization by HPLC coupled to high mass resolution MS. Subsequently, the biosensor was then used to link individual pepper compounds with TRPV1 activity. RESULTS: Among the compounds in the crude pepper fruit extracts, we confirmed capsaicin and also identified both nordihydrocapsaicin and dihydrocapsaicin as true agonists of the TRPV1 receptor. Furthermore, the biosensor was able to detect receptor activity in extracts of both Capsicum fruits as well as a commercial product. Sensitivity of the biosensor to this commercial product was similar to the sensory threshold of a human sensory panel. CONCLUSION: Our results demonstrate that the TRPV1 biosensor is suitable for detecting bioactive metabolites. Novel opportunities may lie in the development of a continuous functional assay, where the biosensor is directly coupled to the LC-MS.