The presence and role of melatonin in plants are still under debate owing to difficulties of identification and quantification. Accordingly, although it has been frequently proposed that melatonin acts as an antioxidant in phototrophic organisms, experimental data on its physiological role are scarce. This study describes the use of a rapid and simple new method for quantification of melatonin in the marine macroalga Ulva sp., organisms routinely exposed to tide-related environmental stresses and known for their high tolerance to abiotic conditions. The method was used here to show that exposure to oxidative stress-inducing environmental conditions (elevated temperature and heavy metals) induced a rise in melatonin level in the algae. Addition of exogenous melatonin alleviated the algae from cadmium-induced stress. Interestingly, although the algae were taken from a culture growing free floating and kept under constant photoperiod and water level, they exhibited a semi-lunar rhythm of melatonin levels that correlated with predicted spring tides. The correlation can probably be interpreted as reflecting preparation for predicted low tides, when the algae are exposed to increasing temperature, desiccation, and salinity, all known to induce oxidative stress. Given the simplicity of the described method it can easily be adapted for the study of melatonin in many other phototrophic organisms. These results provide, for the first time, experimental data that support both an antioxidant role for melatonin and its semi-lunar rhythm in macroalgae.

The nhaA gene of Escherichia coli, which encodes a pH-activated Na+/H+ antiporter, has been modified; six of its eight histidine codons were mutated to arginine codons by site-directed mutagenesis, yielding the mutations H254R-H257R (a double mutant), H226R, H39R, H244R, and H319R. In addition a deletion (delta nhaA1-14) lacking the remaining two histidines, His-3 and His-5, has been constructed. By comparing the phenotypes conferred by plasmids bearing the various mutations to the phenotype of the wild type upon transformation of strains NM81 (delta nhaA) or EP432 (delta nhaA, delta nhaB) we found that none of the NhaA histidines are essential for the Na+/H+ antiporter activity of the NhaA protein. However, the replacement of His-226 by Arg markedly changes the pH dependence of the antiporter. All mutants except H226R confer to NM81 and EP432 Na+ resistance up to pH 8.5 as well as Li+ resistance. Cells bearing H226R are resistant to Li+ and to Na+ at neutral pH, but they become sensitive to Na+ above pH 7.5. Analysis of the Na+/H+ antiporter activity of membrane vesicles derived from H226R cells shows that the mutated protein is activated by pH to the same extent as the wild type. However, whereas the activation of the wild-type NhaA occurs between pH 7 and pH 8, that of H226R antiporter occurs between pH 6.5 and pH 7.5. Furthermore, while the wild-type antiporter remains almost fully active at least up to pH 8.5, H226R is reversibly inactivated above pH 7.5, reaching 10-20% of the maximal activity at pH 8.5. We suggest that His-226 is part of a pH-sensitive site that regulates the activity of NhaA.