Nitrate-ammonium (N-A) and ammonium-nitrate (A-N) transition experiments were performed with tobacco (Nicotiana tabacum, cv Gatersleben) plants to study whether the increased expression of ammonium assimilation-related genes found under short-term boron (B) deficiency is maintained when the nitrogen source changes. Asparagine synthetase (AS), glutamate dehydrogenase (GDH) and glutamine synthetase (GS) gene overexpressions in roots under B deprivation were detected in both nitrogen transitions, although GS and, especially, AS up-regulations were observed earlier in the ammonium-nitrate transition. Transcript levels were inversely correlated with hexose contents after 24 h of B deprivation. Our results suggest that tobacco roots respond to short-term B deficiency by increasing the expression of AS, GDH and GS genes irrespectively of the nitrogen source. A hypothetical model to explain these results is proposed and discussed. Thus, the combined action of these enzymes would avoid the accumulation of ammonia and help maintain the activity of the tricarboxylic acid cycle when tobacco roots are affected by B deficiency, the plant organ that first senses this stress. Therefore, under B deficiency, they would act as an ammonium detoxifying mechanism, being a complement to the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway. We propose that the protective role of these enzymes could be extended to changing nitrogen-supply conditions. © 2014 Japanese Society of Soil Science and Plant Nutrition.