Identification and Validation of the miR156 Family Involved in Drought Responses and Tolerance in Tea Plants ( Camellia sinensis (L.) O. Kuntze).

The microRNA156 (miR156) family, one of the first miRNA families discovered in plants, plays various important roles in plant growth and resistance to various abiotic stresses. Previously, miR156s were shown to respond to drought stress, but miR156s in tea plants ( Camellia sinensis (L.) O. Kuntze) have not been comprehensively identified and analyzed. Herein, we identify 47 mature sequences and 28 precursor sequences in tea plants. Our evolutionary analysis and multiple sequence alignment revealed that csn-miR156s were highly conserved during evolution and that the rates of the csn-miR156 members' evolution were different. The precursor sequences formed typical and stable stem-loop structures. The prediction of cis -acting elements in the CsMIR156s promoter region showed that the CsMIR156s had diverse cis -acting elements; of these, 12 CsMIR156s were found to be drought-responsive elements. The results of reverse transcription quantitative PCR (RT-qPCR) testing showed that csn-miR156 family members respond to drought and demonstrate different expression patterns under the conditions of drought stress. This suggests that csn-miR156 family members may be significantly involved in the response of tea plants to drought stress. Csn-miR156f-2-5p knockdown significantly reduced the Fv/Fm value and chlorophyll content and led to the accumulation of more-reactive oxygen species and proline compared with the control. The results of target gene prediction showed that csn-miR156f-2-5p targeted SQUAMOSA promoter binding protein-like ( SPL ) genes. Further analyses showed that CsSPL14 was targeted by csn-miR156f-2-5p, as confirmed through RT-qPCR, 5' RLM-RACE, and antisense oligonucleotide validation. Our results demonstrate that csn-miR156f-2-5p and CsSPL14 are involved in drought response and represent a new strategy for increasing drought tolerance via the breeding of tea plants.