Functional Analysis of Four Members of the PsbP Family in Photosystem II in Nicotiana tabacum using Differential RNA Interference

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Abstract
Gene redundancy is frequently found in higher plants and complicates genetic analysis. In this study, a method referred to as ‘differential RNA interference (dRNAi)’ was used to investigate the psbP gene family in Nicotiana tabacum. PsbP is a membrane-extrinsic subunit of PSII and plays important roles in the water splitting reaction. N. tabacum has four psbP isogenes and the function of each isogene has not yet been characterized in vivo. To obtain transgenic tobacco plants with various amounts and compositions of PsbP members, the psbP isogenes were differentially silenced by RNA interference (RNAi) using the 3′-untranslated region (UTR) as a silencing trigger (dRNAi). In addition, the extra psbP genes without the 3′-UTR were complementarily transformed into the above silenced plants, which accumulated PsbP originating from the exogenous gene while differential silencing of the endogenous target was maintained. By using dRNAi and subsequent complementation (substitution) in dRNAi, we clearly demonstrated that, regardless of the of PsbP members that were accumulated, PSII activity was linearly correlated with the total amount of PsbP. Therefore, we concluded that the protein functions of the PsbP members in N. tabacum are equivalent in vivo, whereas full expression of the four isogenes is required for optimum PSII activity. These results demonstrate that the use of dRNAi and subsequent complementation/substitution in dRNAi would provide a new experimental approach for studying the function of multigene families in plants.