The role of sulfated metabolites in the wound response of siphonous green algae

The giant unicellular macroalgae exhibit an extraordinary wound repair mechanism. The rapid assimilation of cellular contents into an insoluble wound plug prevents detrimental cytoplasmic loss and limits the intrusion of extracellular components which could otherwise prove fatal. Using chemical and biochemical investigations in conjunction with ultra performance liquid chromatography mass spectrometry (UPLC-MS) and MS/MSanalysis, we demonstrate that biopolymerization reactions are adopted by the organism to assist in the assembly of the wound plug. We found that wounding of the cell initiates biopolymerisation by the rapid hydrolysis of a novel secondary metabolite, 6,7-dihydroxycoumarin-3-sulfate (DHCS) via cellular sulfatases to produce 3,6,7-trihydroxycoumarin (THC). Our results further substantiate the hypothesis that, upon generation, THC immediately undergoes oxidation mediated by an oxidative burst to produce o- and p-quinones. These may subsequently form multiple covalent bonds with thiol or amino functionalities present in a number of proteins, proteoglycans or other small molecules located within the wounded region. The resulting matrix of crosslinked proteins reinforces the structural integrity of the wound plug. Preliminary results suggest that DHCS may also function as a metallophore. Hydroxycoumarin analogues and deuterium labeled amino acid incubations were used to probe the biosynthetic pathway for DHCS assembly. We propose that C3-hydroxyl group introduction proceeds via an α-hydroxy-2-hydroxycaffeic acid intermediate. Subsequent spontaneous ring cyclization followed by sulfate transfer mediated by a sulfotransferase yields DHCS. Following secondary metabolite profile screening, we used a combination of analytical techniques to identify new sulfated metabolites in selected species of the Dasycladales and the seagrass Zostera marina.