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 . 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 . Currently, the RNA-seq approach has been widely used to investigate transcriptomes of plants and animals, especially for those having whole genome sequences . A number of tools to map RNA-seq data to reference genomes and to quantify the expression of transcripts have been developed . 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 . 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 . 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 . 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 . 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 . 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.