Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation.

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Abstract
In this paper we demonstrate that RNA sequences present upstream and downstream of a reporter gene coding region play an important role in determining the amount of protein from an mRNA. A translational enhancer, .OMEGA., derived from tobacco mosaic virus, when present at the 5''-end of .beta.-glucuronidase mRNA increased the efficiency of translation 16-fold to 18-fold in electroporated tobacco or carrot protoplasts, and threefold to 11-fold in maize or rice protoplasts. The presence of .OMEGA. did not alter the half-life of the mRNA in vivo. We also demonstrate for the first time that a minimum polyadenylated tail length of 25 adenylate residues is sufficient to substantially increase the expression and half-life of the reporter mRNA in plants. When in vitro-produced mRNAs were synthesized such that extra sequence was added to the 3''-end of the poly(A) tail, however, the final level of expression was decreased up to 80%. .OMEGA., the translational enhancer, and a poly(A) tail function independently of each other; their combined effect of translation, when both are present in an mRNA, is the multiplication of their individual effects. Histochemical analysis for the presence of .beta.-glucuronidase in tobacco established that virtually all viable cells receive mRNA during electroporation. Video image analysis of tobacco protoplasts electroporated with luciferase mRNA demonstrated that there is a wide range in the level of expression of this marker. Carrier RNA, when present during electroporation, had only a modest effect on increasing mRNA uptake. Reporter mRNA expression in electroporated protoplasts was directly proportional to the input mRNA up to at least 30 .mu.g/ml.