Chloroplast biogenesis from proplastids requires coordinate expression of nuclear and chloroplast genes [1], and is largely regulated by developmental and environmental cues such as light. Approximately 3000 proteins in chloroplasts are encoded by the nucleus [2]. They participate in a large number of functional processes that are required for chloroplast biogenesis. These processes include import of nuclear encoded proteins through the Toc/Tic complexes, protein assembly and disassembly with chaperone proteins, thylakoid formation, pigment synthesis, plastid divisions, and retrograde signaling [3, 4]. In addition, a great number of proteins localized outside chloroplasts, such as photoreceptors, light-signaling transducers, and transcription factors, have been shown to be involved in chloroplast development [3, 4]. On the one hand, most genes belonging to these two classes are essential for chloroplast development since suppression of their expressions leads to impaired chloroplasts. On the other hand, some light signaling pathway genes, such as constitutive photomorphogenic 1 (COP1), COP10, COP11, De-etiolated 1 (DET1) and Phytochrome-interacting transcription factor 3 (PIF3), function as suppressors of light-regulated gene expression and loss-of-function mutations of these genes result in ectopic chloroplast development [5–7]. In contrast, Elongated Hypocotyl 5 (HY5) that acts downstream of multiple families of photoreceptors [8–10] has been genetically characterized as a positive regulator of photomorphogenesis under a broad spectrum of light and affects chloroplast development [4, 11]. Overexpression of HY5-ΔN77 has been shown to result in precocious development of chloroplasts in the hypocotyls [12]. Determining how these genes are coordinately expressed during chloroplast development requires a genome-wide examination of gene expression during the transition from non-colored plastids into chloroplasts.

Mutations in model and other plant species are important resources for functional genomics studies. Analyses of some plastid development mutants identify important regulatory genes of plastid development. For example, ARC6, the first gene discovered to have a global effect on plastid differentiation in higher plants, was identified from an Arabidopsis mutant arc6 [13]. The Orange (Or) gene that encodes a zinc-finger DnaJ cysteine rich domain containing protein is isolated from the orange curd cauliflower mutant and has been proven to be responsible for the conversion of leucoplasts into chromoplasts [14]. The green curd cauliflower mutant is a spontaneous mutation with an abnormal pattern of chloroplast development in curds. Compared with other mutants in which chloroplast development is impaired, the green curd mutant is unique in turning otherwise non-photosynthetic white tissue into green color with the ectopic development of chloroplasts in the inflorescence meristematic cells. The mutation in the green curd cauliflower could involve the gene(s) sufficient for chloroplast development, although there is possibility that the white curd cauliflower carries a genetic mechanism for the suppression of chloroplast development, which the green curd mutation would suppress.

In the present study, we profiled gene expression in green and white curds on the genome scale using the RNA-seq approach. We assembled 118,000 unigenes with an average length of 406 bp from cDNA libraries of green and white curds and detected 7155 differentially expressed genes with a change in expression of at least 3-fold. Among them are a large number of genes associated with chloroplast development. We also observed that BoHY5 was expressed at higher level in green curds than in white curds and that 2616 HY5-targeted genes were expressed differentially. Among these HY5-targeted genes, all the 1600 up-regulated genes were found to be HY5-targeted genes in the light in Arabidopsis, suggesting a role of BoHY5 with the ectopic chloroplast development in the green curd cauliflower mutant.

Large-scale transcriptome sequencing by next generation sequencing platforms such as the Illumina GA sequencing system has been proven to be a powerful and efficient approach for gene expression analysis at the genome level and offers several advantages over microarray technologies [29]. Since the RNA-seq approach provides digital representation of the gene abundance and the statistics are well modeled by the Poisson distribution, even a single replication has been shown to be adequate [30]. Currently, the RNA-seq approach has been widely used to investigate transcriptomes of plants and animals, especially for those having whole genome sequences [31]. A number of tools to map RNA-seq data to reference genomes and to quantify the expression of transcripts have been developed [32]. However, relatively fewer reports have shown studies on using the RNA-seq approach for organisms without reference genomes. In this report we employed the RNA-seq approach to investigate the gene expression changes in a green curd mutant in order to elucidate the genetic basis of chloroplast biogenesis and development. RNA-seq reads along with publicly available ESTs of cauliflower were assembled de novo using a novel assembly strategy as described in the Methods section. A total of 118, 000 unigenes were obtained and 7155 genes showed at least 3-fold changes in expression in green curd mutant. Among them, a large number of genes involved in photomorphogenesis including chloroplast development were revealed, demonstrating a successful use of the RNA-seq approach to profile gene expression in a species without a fully sequenced genome.

Chloroplast biogenesis and development proceed with the coordinated action of many processes [3, 4]. Both environmental signals and plastidic/nuclear factors affect these processes. Light regulation of chloroplast development has been well-documented [3, 4, 33]. The light signaling pathways are composed of phytochromes, transcription factors and numerous intermediates which control photomorphogenesis including chloroplast development. The COP/DET/FUS proteins are suggested to have a function in suppressing chloroplast development in non-photosynthetic tissues [4]. Loss of function mutation of these regulators, such as cop1 and det1, has been shown to result in ectopic chloroplast development, leading to greening in Arabidopsis roots [5, 6]. The fact that the transcripts of COP1 and DET1 remained unchanged and a large number of light-responsive genes were altered in green curds of cauliflower suggests that other regulatory genes in the hierarchy of photomorphogenic regulation are responsible for chloroplast development in the green curd.

In the light signaling cascade, HY5 plays an important role in light signaling and chloroplast development. HY5 receives upstream signals and activates a large number of genes by directly binding to the G-box in the promoters of these genes [9, 26, 27]. Here, we observed higher level of HY5 transcript in the green curd mutant. Furthermore, 2616 cauliflower homologs of HY5-targeted genes were differentially expressed in green curds. Noticeably, among the 2616 genes, 1600 were up-regulated genes in green curd cauliflower. The fact that all 1600 up-regulated genes were the HY5-targeted genes in the light suggests an important role of elevated expression of BoHY5 in mediating chloroplast development in green curd cauliflower mutant. Furthermore, it is known that COP1 negatively controls HY5 activity [12]. Although COP1 was expressed at the same level between green curds and white curds, we found that CIP1 was significantly reduced in green curds (Figure 4c). Arabidopsis CIP1 is associated with the cytoskeleton and has been hypothesized to affect partitioning of COP1 in the nucleus and cytoplasm [34]. It is possible that COP1 activity in the nucleus might be affected by low level of CIP1, causing ectopic chloroplast development in green curds. Thus, BoHY5 and/or the other genes at the high hierarchy in the signal transduction cascade could be responsible or work in concert to regulate chloroplast biogenesis and development in otherwise white tissue to give rise to the striking green curd mutant phenotype.

Ultimately, the development of chloroplasts requires the coordinated action of a number of processes, including the biosynthesis of photosynthetic complexes, transportation of nuclear encoded proteins into chloroplasts, processing of the imported proteins, and assembly of the photosynthetic apparatus [3, 4]. Indeed, many genes involved in photosynthetic pigment biosynthesis along with pigment-binding proteins such as chlorophyll a/b binding proteins were discovered to be upregulated in our genome-wide profiling of green curd cauliflower. The majority of chloroplast proteins are nucleus-encoded and enter the chloroplasts via the Toc/Tic translocon complexes [1]. The increased expression of Toc genes in the green curd mutant supports an enhanced activity of chloroplast-targeted protein import. The imported proteins are folded and processed to form functional proteins. Molecular chaperones HSP70 and Cpn60 have long been known to be involved in this process [3, 20]. A recent study shows that a protein disulfide isomerase is also required for protein folding [21]. Consistent with the increased activity of protein import, genes associated with protein folding and assembling were expressed highly in the green curd mutant for chloroplast development.

In the present study, we compared gene expression on a genome-wide scale by using RNA-seq in a species without a reference genome. This study identified a great number of genes associated with chloroplast development and suggested the potential role of elevated expression of BoHY5 and/or other regulatory genes in the high hierarchy of light signaling pathways for the ectopic chloroplast development in green curd cauliflower. Our results indicate that RNA-seq as a powerful tool in a genomic era could accelerate the functional identification of genes and aid in dissecting the genetic basis of naturally-occurring variations in crops.