Biology · Methods in Biology

In a forward genetic screen for heat stress response in Arabidopsis, researchers identified an uncharacterized gene 'x' with sequence homology to the alpha-subunit of a heterotrimeric G-protein. To validate whether the protein localizes to the membrane, they cloned it in fusion with GFP at its N-terminus under the CaMV promoter. Upon expression in tobacco protoplasts, they did not observe membrane localization of the GFP-signal. Based on this, they made a few assumptions: A. N-terminally tagging of protein 'X' with GFP may block membrane localization of protein X. B. Tagging of protein 'X' with GFP may alter the conformation of protein X because of its bigger size. C. Tobacco protoplasts are a heterologous system for the expression of gene 'x', and thus, the protein X does not localize to the membrane. D. Gene 'x' is not getting transcribed because of the wrong promoter choice. Which one of the following options represents all correct assumptions?