In vitro transcription is an essential tool to study the molecular mechanisms of transcription. For over a decade, we have developed an in vitro transcription system from tobacco (Nicotiana tabacum)-cultured cells (BY-2), and this system supported the basic activities of the three RNA polymerases (Pol I, Pol II, and Pol III). However, it was not suitable to study photosynthetic genes, because BY-2 cells have lost their photosynthetic activity. Therefore, Arabidopsis (Arabidopsis thaliana) in vitro transcription systems were developed from green and etiolated suspension cells. Sufficient in vitro Pol II activity was detected after the minor modification of the nuclear soluble extracts preparation method; removal of vacuoles from protoplasts and L-ascorbic acid supplementation in the extraction buffer were particularly effective. Surprisingly, all four Arabidopsis Rubisco small subunit (rbcS-1A, rbcS-1B, rbcS-2B, and rbcS-3B) gene members were in vitro transcribed from the naked DNA templates without any light-dependent manner. However, clear light-inducible transcriptions were observed using chromatin template of rbcS-1A gene, which was prepared with a human nucleosome assembly protein 1 (hNAP1) and HeLa histones. This suggested that a key determinant of light-dependency through the rbcS gene transcription was a higher order of DNA structure (i.e. chromatin).In plants, the molecular mechanisms of lightdependent gene regulation remain largely unknown. Generally, transcription is the major regulation step during gene expression in the eukaryotic genome. In plants, most genes are mainly regulated at the level of transcription, and the expressions of many genes depend on surrounding light conditions relating to photosynthesis, photomorphogenesis, phototropism, circadian rhythms, gametogenesis, etc. Therefore, light-dependent transcription has been studied intensively. Three distinct classes of photoreceptors have been discovered: phytochromes, blue-light receptors (cryptochromes, phototropins, and ZTL / FKF1 / LKP2 family), and photoreceptors for UV-B (Banerjee and Batschauer, 2005). These photoreceptors affect downstream gene expression mediated by the specific binding of transcription factors to cis-regulatory elements in promoters. Light regulatory elements are essential cisregulatory elements found in photo-responsible genes, such as the conserved modular array 5 (CMA5) on the Rubisco small subunit (rbcS) genes (López-Ochoa et al., 2007). Our knowledge of transacting factors that associate with light regulatory elements is quite limited, mainly because plant materials are inconvenient to use for biochemical research. Therefore, to understand transcription regulation comprehensively, we need to develop a plant-specific in vitro transcription system.The eukaryotic (human cell) in vitro transcription system was reported by Weil et al. (1979) and Manley et al. (1980). Subsequently, Dignam et al. (1983) established finely tuned systems. Nowadays, in vitro transcription systems are indispensable for molecular biology;...