Gene expression analysis revealed Hbr-miR396b as a key piece participating in reaction wood formation of Hevea brasiliensis (rubber tree)

Rubber tree (Hevea brasiliensis) is a perennial tree, which is important for wood-related industrial applications. However, the molecular basis of wood formation in H. brasiliensis is largely unknown. In this study, we induced the formation of reaction wood by artificial bending three 3−year−old rubber trees for 7 or 100 days, and collected xylem samples from tension wood, opposite wood, and normal wood, and subjected them to small RNA sequencing to identify the microRNAs (miRNAs) participated in rubber tree wood formation. We identified 25 known microRNAs (miRNAs) belonging to 21 miRNA families in 7-day wood and 24 known miRNAs belonging to 20 miRNA families in 100-day wood. Three known miRNAs, hbr-miR396b, hbr-miR6168, and hbr-miR6485, that accumulated in 100-day wood targeted gene members of the three transcription factor families C3H, SQUAMOSA BINDING PROTEIN (SBP), and AUXIN RESPONSE FACTOR (ARF). Gene Ontology analysis showed genes co-expressed with transcription factor genes targeted by miRNAs were associated with cellulose metabolism, cellulose biosynthesis, and cellulose synthase in 100-day but not 7-day wood, likely reflecting the longer duration of artificial bending treatment. We also constructed a co-expression network that includes miRNAs, their target transcription factor genes, and the genes co-expressed with these genes, revealing that cellulose biosynthesis was regulated by 3 known and 13 novel miRNAs, which may all participate in reaction wood formation. In summary, this study identified potential key miRNA−transcription factor−mRNA networks involved in reaction wood formation of rubber tree and laid a theoretical foundation for further elucidating the mechanism of wood formation in this species.