Wang et al. BMC Genomics 2011, 12:289 http://www.biomedcentral.com/1471-2164/12/289 species that were derived by alternative biogenesis path- ways and only partially met classical definitions, such as small RNAs derived from genomic loci containing repeat sequences [7], snoRNA [8] and tRNA [9]. Although most of these studies focused on nuclear or cytosolic members, small ncRNAs from subcellular genomes, such as chloroplasts and mitochondria, have gradually risen into view in animals, plants and fungi [10-12]. Chloroplasts and mitochondria, widely accepted as endosymbiontic eubacteria in cells, retain separate circu- lar genomes, and their own gene expression machinery [13]. One hallmark of organellar genomes is the predo- minance of post-transcriptional control, including RNA processing and turnover, which are exerted both at the gene-specific and global levels [14-16]. In chloroplasts, a small number of non-coding RNAs have been reported previously. For example, a 218-nt-long plastid-encoded RNA is thought to be relevant to the maturation of 16S ribosomal RNA [17] that was initially discovered in tobacco and then found to be conserved in several other angiosperms, and 12 ncRNAs have been identified in tobacco chloroplast with unknown functions, some of which were predicted to form possible stem-loop struc- tures [10]. In chloroplasts, nearly all polycistronic tran- scripts are processed by endonucleases or/and exonucleases [18], which may relieve secondary struc- tures for ribosome assembly [14]. Also, 3’ end matura- tion in chloroplasts follows a prokaryotic pathway with rho-independent termination, resulting in mature ter- mini flanking stem-loop structures [19]. Nevertheless, RNA processing is still poorly understood in chloroplast, compared to that of well-studied Escherichia coli. The enzymatic machinery is only beginning to be explored, and its molecular nature remains unclear. Chinese cabbage (Brassica rapa ssp. chinensis) is one of the most widely grown leafy vegetables, and its plant is composed of numerous green leaves, which contain abundant chloroplasts. This botanical characteristic, along with its close genetic relationship to Arabidopsis, make it suitable material for the study of chloroplast development. The adaptable growth temperature for Chinese cabbage ranges from 18°C to 22°C, and its pro- duction is seriously threatened by heat stress in many regions. Heat stress causes a visible growth inhibition of shoot and root [20]. One of the phenotypes is leaf etio- lation and bleaching. In the etiolated leaf segments, a clear-cut inhibition of photosynthetic activity, chloro- phyll accumulation and chloroplast development can be observed [21], thus indicating a strong response of chloroplasts to heat stress. In recent years, a great deal of attention has been paid to the elucidation of the mechanisms of heat-sensitivity for breeding heat-resis- tant cultivars of Chinese cabbage and other important Page 2 of 14 crops. Considering the importance of small RNAs in abiotic stress resistance [22-24] and their advantage in terms of regulating the plurality of target genes rather than one single gene and existence of small RNAs in the chloroplast [10], we were inspired to search for small RNAs in chloroplast associated with heat resis- tance of Chinese cabbage. In this study, we report a novel class of chloroplast small RNAs (csRNAs) in Chinese cabbage and Arabi- dopsis. Significant numbers of csRNAs are responsive to high temperature. These novel csRNAs may play poten- tial roles in the heat response, probably by regulating chloroplast RNA processing and target gene expression. Results Leaf etiolation under heat stress To define the effects of high temperature on leaf etiola- tion, we incubated the seedlings of non-heading Chinese cabbage at 44, 45, 46, 47 and 48°C for durations of 0.5, 1 and 2 hours. As indicated in Figure 1, the seedlings exposed to 44°C for 0.5 h grew as normally as those exposed to 22°C, while the seedlings exposed to 44°C for 1 hour grew more slowly than those exposed to 22° C and were etiolated on the 22nd day after the heat treatment. This indicated that the heat treatment of 44° C for 1 hour caused morphological and physiological change in the seedlings. As the temperature rose and the heat treatment prolonged, the inhibitory effects on plant growth became more severe and leaf etiolation Figure 1 Seedlings of Chinese cabbage are etiolated under heat stress. (A) A seedling growing 20 days after treatment at 22°C for 1 h (MT). (B) A seedling growing 20 days after treatment at 44°C for 1 h. (C) A seedling growing 20 days after treatment at 46°C for 1 h (HT). (D) Green color indicates the green leaves after HT treatment while yellow color shows the etiolated leaves after HT treatment.