Responses of the Azolla filiculoides Stras.–Anabaena azollae Lam. association to elevated sodium chloride concentrations: Amino acids as indicators for salt stress and tipping point

► The Azolla filiculoides–Anabaena azollae has a salt resistance limit of 90mM NaCl. ► The Az. filiculoides–An. azollae association appeared to acclimate to salt stress. ► Az. filiculoides needs a functional root system to survive salt stress. ► Constitutively high glutamine concentration may contribute to salt tolerance in Azolla. ► Proline and glutamine/glutamate ratios in Azolla can be used to predict the tipping point. The water fern genus Azolla has been found in marine sediments, showing that ancestral species grew in marine ecosystems during the Eocene. Modern Azolla species, however, only live in freshwater. Besides aiming to elucidate experimentally the conditions that prevailed over the Arctic Ocean and adjacent Nordic seas during the Eocene, studies on tolerance of these plants to salt stress have applied potential, as Azolla may be used as a fertilizer, due to its symbiosis with N2-fixing Anabaena cyanobacteria, and grown on salt-infiltrated agricultural fields. Here, the response of a temperate Azolla filiculoides–Anabaena azollae association to a wide salinity gradient (0–210mM NaCl) was studied by measuring growth, nutrient content, nitrogenase activity and the accumulation of free amino acids in the association. The association was able to grow at salt concentrations up to 90mM NaCl and appeared to acclimate in this range, but only after a period of 75 days. At concentrations exceeding 120mM NaCl, however, roots were shed and impairment of water and nutrient uptake (including dinitrogen fixation) resulted in die off. Increased Na concentrations in the plants grown at external salt concentrations of 30mM NaCl were to some extent reverted by decreased K concentrations, suggesting that K in the plants was replaced by Na as plant growth was not affected. Absolute nutrient concentrations in plant tissue were not correlated with plant growth and therefore not suitable as reliable indicators for salt stress. However, significant increases in the free amino acids proline and glutamate and significant decreases in asparagine, glutamine and gamma-amino-butyric-acid were found with increasing salinity. The constitutively high glutamine concentrations in the plants grown up to 90mM NaCl may have contributed to the salt tolerance of the plants by providing osmotic adjustment. Proline concentrations and glutamine/glutamate ratios proved to be strong linear indicators for the level of salt stress and can therefore be used to predict the tipping point conc