Transcriptional Expression Characteristics and Subcellular Localization of ADP-Glucose Pyrophosphorylase in the Oil Plant Perilla frutescens

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Abstract
Three ADP-glucose pyrophosphorylase clones were isolated from the cotyledon cDNA library of the oil plant, Perilla frutescens, and their intracellular localization investigated. Two of three cDNAs (PfagpS1 and PfagpS2) were homologous to the catalytic small subunit of AGPases found in other plants, while the third clone (PfagpL) was highly similar to the large subunit type. Transcripts for PfagpS1 and PfagpS2 were observed in both photosynthetic and non-photosynthetic tissue, showing the highest expression in the stem, while PfagpL transcripts were abundantly expressed in stem and cotyledon. To evaluate the subcellular localization of PfagpS2 and PfagpL as well as the maize BT2, N-terminus-GFP DNA fusion were constructed and transformed into tobacco plants. Immunoblot analysis showed that the expressed PfagpS2- and PfagpL-GFP fusions were targeted to the plastid in the heterologous tobacco system whereas the BT2-GFP remained intact, suggesting a cytoplasmic location. These intracellular assignments were confirmed by direct confocal microscopic examination. GFP signals were localized to the cytoplasm as well as in the nucleus in BT2-GFP plants, and to the plastids in PfagpS2- and PfagpL-GFP plants. Our results indicate that Perilla cotyledons contain multiple AGPase subunits, of which at least two isoforms and very likely the third, are plastidial in nature.