Small RNA sequencing reveals various microRNAs involved in piperine biosynthesis in black pepper (Piper nigrum L.)

Black pepper (Piper nigrum L.), an important and long-cultivated spice crop, is native to South India and grown in the tropics. Piperine is the main pungent and bioactive alkaloid in the berries of black pepper, but the molecular mechanism for piperine biosynthesis has not been determined. MicroRNAs (miRNAs), as classical endogenous noncoding small RNAs, play important roles in regulating secondary metabolism in many species, but less is known regarding black pepper or piperine biosynthesis. To dissect the functions of miRNAs in secondary metabolism especially in piperine biosynthesis, a total of 109 known miRNAs, 153 novel miRNAs and 1,757 individual targets were identified from different tissues of black pepper by small RNA sequencing. qRT-PCR and 5'-RLM-RACE experiments were conducted to validate the reliability of sequencing data and predicted targets. We found 4 miRNAs along with their targets including miR166-4CL, miR396-PER, miR397-CCR and novel_miR43-AOC2/3 modules involved in piperine biosynthesis. Finally, a regulatory network of miRNAs in piperine biosynthesis in black pepper was proposed.