MicroRNAs and their targets involved in unreduced pollen formation induced by heat stress in Camellia nitidissima

Heat stress is often employed as a physical mutagenic agent to produce unreduced gametes because of its operational advantages and consistency of treatment. MicroRNAs (miRNAs) are critical for the control of gene expression through post-transcription in plants exposed to abiotic stresses. Here, the effect of heat stress on flower buds and pollen in Camellia nitidissima was investigated, and it was demonstrated that heat treatment significantly stimulated the formation of unreduced pollen. The highest frequency of unreduced pollen, 48.67 +/- 2.10%, was obtained in the treatment at 42 degrees C for 4 hrs. Integrated analysis was conducted to identify and characterize the miRNAs and their targets involved in unreduced pollen formation induced by heat stress, through small RNA, degradome, and transcriptome sequencing. In all 268 known and 234 novel miRNAs were identified from heated anthers (AnT), untreated anthers (AnC), heated petals (PeT), and untreated petals (PeC) samples, of which 42 miRNAs were specifically regulated in the comparison of AnT vs AnC. The targets of miRNAs were predicted by degradome analysis, and the expression profiles of these targets were characterized based on transcriptome sequencing. A total of 150 negatively regulated DE miRNA-DEG pairs were found using the 42 specifically regulated DE miRNAs in AnT vs AnC as screening criteria. Among them, ten candidate miRNA-target pairs that were involved in unreduced pollen formation induced by heat stress were screened based on the GO annotation of the targets. One pair (Cni-miR535a-MUS81), two pairs (Cni-miR168b-TUBA1 and Cni-miR6478-TUBB), seven pairs (Cni-miR8674-STL1, Cni-miR159f-CSLG2, Cni-miR535b-SBT1.6, Cni-miRn37-TBL6, Cni-miRn112-BGAL2, Cni-miRn61-RHD3, and Cni-miR168b-TUBA1), and one pair (Cni-miR156k-GRV2) were associated with cross-overs, microtubule, cell wall, and vacuole, respectively. For the first time, our findings indicate the potential roles of miRNAs in unreduced pollen formation. It is also a fundamental resource for further study on miRNA-mediated regulation of unreduced pollen formation induced by heat stress in C. nitidissima.