PMC:4307275 / 1821-5430 JSONTXT

{"project":"2_test","denotations":[{"id":"25561231-21454800-50799887","span":{"begin":391,"end":392},"obj":"21454800"},{"id":"25561231-20168296-50799888","span":{"begin":393,"end":394},"obj":"20168296"},{"id":"25561231-20168296-50799889","span":{"begin":547,"end":548},"obj":"20168296"},{"id":"25561231-23075219-50799890","span":{"begin":549,"end":550},"obj":"23075219"},{"id":"25561231-18667720-50799891","span":{"begin":704,"end":705},"obj":"18667720"},{"id":"25561231-15937229-50799892","span":{"begin":706,"end":707},"obj":"15937229"},{"id":"25561231-22789293-50799893","span":{"begin":938,"end":939},"obj":"22789293"},{"id":"25561231-12136113-50799894","span":{"begin":967,"end":968},"obj":"12136113"},{"id":"25561231-11746085-50799895","span":{"begin":969,"end":970},"obj":"11746085"},{"id":"25561231-11746085-50799896","span":{"begin":1005,"end":1006},"obj":"11746085"},{"id":"25561231-11746085-50799897","span":{"begin":1609,"end":1610},"obj":"11746085"},{"id":"25561231-21843626-50799898","span":{"begin":2312,"end":2314},"obj":"21843626"},{"id":"25561231-22275386-50799899","span":{"begin":2888,"end":2890},"obj":"22275386"},{"id":"25561231-3943125-50799900","span":{"begin":3171,"end":3173},"obj":"3943125"},{"id":"25561231-3554144-50799901","span":{"begin":3289,"end":3291},"obj":"3554144"},{"id":"25561231-3556162-50799902","span":{"begin":3292,"end":3294},"obj":"3556162"},{"id":"25561231-2041747-50799903","span":{"begin":3295,"end":3297},"obj":"2041747"},{"id":"T98575","span":{"begin":391,"end":392},"obj":"21454800"},{"id":"T55168","span":{"begin":393,"end":394},"obj":"20168296"},{"id":"T73652","span":{"begin":547,"end":548},"obj":"20168296"},{"id":"T81243","span":{"begin":549,"end":550},"obj":"23075219"},{"id":"T52692","span":{"begin":704,"end":705},"obj":"18667720"},{"id":"T57502","span":{"begin":706,"end":707},"obj":"15937229"},{"id":"T70341","span":{"begin":938,"end":939},"obj":"22789293"},{"id":"T68632","span":{"begin":967,"end":968},"obj":"12136113"},{"id":"T38074","span":{"begin":969,"end":970},"obj":"11746085"},{"id":"T56943","span":{"begin":1005,"end":1006},"obj":"11746085"},{"id":"T94795","span":{"begin":1609,"end":1610},"obj":"11746085"},{"id":"T83347","span":{"begin":2312,"end":2314},"obj":"21843626"},{"id":"T80635","span":{"begin":2888,"end":2890},"obj":"22275386"},{"id":"T88485","span":{"begin":3171,"end":3173},"obj":"3943125"},{"id":"T58912","span":{"begin":3289,"end":3291},"obj":"3554144"},{"id":"T29108","span":{"begin":3292,"end":3294},"obj":"3556162"},{"id":"T95387","span":{"begin":3295,"end":3297},"obj":"2041747"}],"text":"1. Introduction\nA protoplast is a naked plant cell bounded only by the cell membrane that is obtained by enzymatic treatment to remove the cell wall. The structural property of protoplasts facilitates the study of transient expression systems. Recently, protoplasts have been reported to retain their tissue- and cell-specific features within the time frame of a transient expression assay [1,2]. This property is combined with the advantage of a fluorescence-activated cell sorting (FACS) that sorts protoplasts only having a fluorescent signal [2,3] to facilitate molecular biological studies on the expression profiling of local tissues, such as the Arabidopsis root quiescent center and sperm cells [4,5]. Those studies have highlighted the potential of single cell-based FACS using protoplasts. Also, single cell-based flow cytometric analysis (FCA) has been successfully applied to in vivo analysis of protein-protein interactions [6,7], and promoter activity [8,9], etc.\nHagenbeek and Rock (2001) [9] reported the usefulness of FCA for the promoter analysis system using protoplasts, and described the advantages and disadvantages of FCA, in detail. As a major disadvantage, they showed that the fluorescence intensity of GFP was unusually detected to be as low as the threshold level of background signals, and subsequently, it caused the transfection ratio determined to be as low as 4%, which represented the population size of protoplasts expressing GFP-fluorescent signals on FCA. Since the average transfection efficiency determined on hemocytometer analysis has been reported to be ca. 40%–60% [9], a large number of transfected protoplasts might supposedly have been missed on FCA. It could be a bottleneck on application of plant protoplast system to various FCA or FACS. To overcome these limitations of FCA using protoplasts, higher DNA quantities (10–130 μg) were used, but the transfection ratio, (amounting to 6%), was not markedly improved. Thus, the low fluorescence intensity of FCA using plant protoplasts remains a challenging limitation.\nAs chloroplasts are an isolated space bounded by an envelope membrane and a single plant cell comprises many (tens to hundreds) of chloroplasts [10], they have been reported to be an excellent reservoir for producing various recombinant proteins [11]. To localize the fluorescent protein (FP) in the chloroplast, the transit peptide originated from the small subunit of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) of rice [12], used to localize various recombinant proteins into chloroplasts, was selected. Chloroplasts have been reported to have a limited set of protein degradation pathways in the stroma compared with that of the cytoplasm; it has been an advantage of chloroplasts that recombinant proteins localized within them could be kept more stable than those expressed in the cytoplasm [13,14].\nAdditionally, a Kozak (Kz) sequence, a defined consensus sequence adjacent to the ATG initiation codon in eukaryotic mRNAs, was considered for the improvement of the translation efficiency, which has been reported to mediate efficient initiation of translation by ribosomes [15]. The Kz sequences have been identified in various eukaryotes including vertebrates, yeasts, protozoa, and plants [16,17,18].\nBased on the above advantages, the chloroplast and Kz sequence in this study were adopted for the localization space of FPs and the expression efficiency, respectively. The useful strategy of FCA using rice protoplasts was developed for increasing the fluorescence intensity of FPs analyzed by flow cytometry."}