Two vacuolar green fluorescent proteins (GFP) were stably inserted in Nicotiana tabacum and Nicotiana benthamiana genome, with unexpected difficulties, and compared with A. thaliana cv. Wassilewskaja transgenic plants expressing the same constructs. GFP fluorescence was strong in all tissues of A. thaliana but it was barely visible in Nicotiana. Confocal microscopy analysis revealed a variable distribution of the marker in those cells where GFP fluorescence was visible. The role of light dependent proteases was the variable pointing out more inter-species diversity. GFPs degradation was much higher in Nicotiana spp. than in A. thaliana. The version of GFP used appeared not to be a good vacuolar marker for Nicotiana differentiated tissues, although it can efficiently label vacuoles in protoplasts or calli. Nevertheless the sensitivity of the reporter protein can be used as an indicator of hidden characteristics of the plant vacuoles, revealing differences otherwise invisible. One of the markers in our system, GFP-Chi, evidenced a clear morphological difference in the vacuolar system of guard cells of the three species.