Wang et al. BMC Genomics 2011, 12:289 http://www.biomedcentral.com/1471-2164/12/289 representing specific positions in the chloroplast gen- ome. b) Despite wide varieties in size, the csRNA families exhibited distinct size distributions. A csRNA family is usually predominated by small RNAs of one or two determinate sizes, although it is classified as a group of small RNAs, constant in their core sequence and varying in the 5’ or 3’ extender length. c) Some extra nucleotides ahead and behind the matured RNA sequences were revealed from the putative csRNA pre- cursors (such as Pre1 and Pre2 of csR-5sr-1). Consider- ing the high abundance and end-specificity of some csRNA families such as csR-5sr-1, the csRNAs may not be the degradation products. d) csRNAs not only origi- nated from the region corresponding to the mature RNAs but also from the externally and internally tran- scribed spacer regions, indicating that their biogenesis may require un-transcribed precursors or intermediate products. Collectively, our data suggest an undiscovered biogenesis pathway of small RNAs from RNA processing that differs from the existing knowledge about the single nucleotide decay from the end of a DNA or RNA chain by exonucleases. csRNAs are highly sensitive to high temperature Comparing the csRNAs from heat-treated samples with those from the non-stressed control group, the most remarkable change was that the rRNA-derived csRNAs declined significantly in the HT dataset to only ~50% of their original abundance shown in the MT dataset. However, the reduction in the 5S rRNA-derived csRNAs may due to the decease of chloroplast rRNA/rDNA by heat stress, as reported in maize chloroplast rRNA [34] and black mustard (Brassica nigra) [35]. The rRNA transcripts could be the sources of not only mature rRNA, but also csRNA, as most chloroplast small RNA sequences were identical to the sense strand of chloro- plast DNA. Under these circumstances, csRNA biogen- esis from rRNA may be passively controlled by the quantity of total transcripts. However, other possibilities still exist: the csRNAs may decrease to a certain extent in an initiative manner compared to the changes in transcripts. It is challenging to compare the changes in transcripts, mature rRNAs and csRNAs directly. Since the relations between two of the three are still indeter- minable, it is unknown whether there is another factor involved in their processing pathways. One possible alternative model for the regulation of RNA levels by csRNA is as follows: polycistronic precursors produce both csRNAs and mature RNAs; as a result, fewer csRNAs lead to more mature rRNAs processed from the precursor, and less interference caused by the base-pair- ing competition between fragments with the same nucleotide sequences. In addition, a previous study involving 5S rDNA-derived siRNAs in DDM1 and Page 11 of 14 MET1-deficient mutants suggested a role of siRNAs in 5S rDNA chromatin organisation [36], which may hint to a similar role for the 5S rRNA-originated csRNAs in the chloroplast. Among the tRNA-derived csRNAs, the number of shorter csRNAs (15-25nt) was reduced, while the num- ber of longer csRNAs (29-32nt) was increased. Interest- ingly, the 3’ termini of the shorter and longer csRNAs mapped within the D arm and anticodon arm, respec- tively, indicating that the tRNA cleavage sites were located in the middle. tRNA halves, corresponding to the 5’ or 3’ sequences of the tRNAs, derived from mature tRNAs cleaved at their anticodon loops by an unidentified RNase, were able to inhibit translation and potentially transmit long-distance signals in plant phloem sap [27]. Processed tRNA halves were also detected in prokaryotes, fungi, yeast, Arabidopsis, and human cells, under various stresses, such as early amino acid starvation or oxidative stress [37-40]. Although we do not yet know whether those csRNAs associated with tRNA 5’ ends in our datasets are functional 5’ halves or not, there is a possibility that they are generated by a similar pathway in the chloroplast, reflecting an ancient mechanism centrally associated with stress conditions. In contrast to small RNAs from rRNA, tRNA and mRNA, the igRNA-derived csRNAs increased by 30% after heat treatment. csRNA may play roles in plant heat resistance Leaf etiolation is one of the major indicators of plant sensitivity or tolerance to high temperature. When the intracellular environment is exposed to moderate heat stress, the structure of the thylakoid membrane is chan- ged and cyclic electron flow around photo system I is increased, resulting in massive photo-oxidative stress and the concomitant release of highly cyto-toxic free radicals and reactive oxygen species [41,42]. When the subcellular structures of the chloroplast are damaged by heat stress, photosynthetic capacity is greatly reduced [41]. This change is usually concomitant with leaf etiola- tion. For the heat-resistant genotypes of Chinese cab- bage, leaf etiolation appears later or only slightly compared with the heat-sensitive genotypes (data not shown). In the HT seedlings, most of the csRNAs showed reduced abundances 1 hour after the 46°C heat treatment, the time period before the appearance of leaf etiolation. One hypothesis is that the heat-responsive csRNAs play roles in the maintenance of subcellular structures and photosynthetic capacity of the chloro- plast. The csRNAs derived from tRNAs are possibly play therole similar to the reported tRNA halves under var- ious stress conditions in prokaryotes, fungi, yeast, Arabi- dopsis and human cells [27,38-40]. When seedlings are exposed to high temperature, the biogenesis of the heat-