In eukaryotes, small non-protein-coding RNAs have emerged as key guidance molecules that fulfill important, vital functions in many cellular processes, such as transcription, translation, splicing, DNA replication and RNA processing . It is thought that small RNAs are produced by Dicer-like proteins (DCLs) from their precursors, which can be stem-loop RNA transcripts or long double-stranded RNAs (dsRNAs). The small RNAs directly interact with proteins from the Piwi/Argonaute (AGO) family to form the core of the RNA induced silencing complex (RISC) [2, 3]. However, data collected from animals suggest a correlation between small RNA diversity and morphological complexity [4, 5]. Recent progress in the understanding of the non-canonical mechanisms of small RNA biogenesis has been achieved in mammalian systems . Massive amounts of data produced by next generation sequencing technologies also revealed subclasses of small non-coding RNA species that were derived by alternative biogenesis pathways and only partially met classical definitions, such as small RNAs derived from genomic loci containing repeat sequences , snoRNA  and tRNA . 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 circular genomes, and their own gene expression machinery . One hallmark of organellar genomes is the predominance 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  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 structures . In chloroplasts, nearly all polycistronic transcripts are processed by endonucleases or/and exonucleases , which may relieve secondary structures for ribosome assembly . Also, 3' end maturation in chloroplasts follows a prokaryotic pathway with rho-independent termination, resulting in mature termini flanking stem-loop structures . 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 production is seriously threatened by heat stress in many regions. Heat stress causes a visible growth inhibition of shoot and root . One of the phenotypes is leaf etiolation and bleaching. In the etiolated leaf segments, a clear-cut inhibition of photosynthetic activity, chlorophyll accumulation and chloroplast development can be observed , 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-resistant cultivars of Chinese cabbage and other important 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 , we were inspired to search for small RNAs in chloroplast associated with heat resistance of Chinese cabbage.
In this study, we report a novel class of chloroplast small RNAs (csRNAs) in Chinese cabbage and Arabidopsis. Significant numbers of csRNAs are responsive to high temperature. These novel csRNAs may play potential roles in the heat response, probably by regulating chloroplast RNA processing and target gene expression.