LaMIR166a-mediated auxin biosynthesis and signalling affect somatic embryogenesis in Larix leptolepis

Somatic embryogenesis (SE) involves complex molecular signalling pathways. Understanding molecular mechanism of SE in Larix leptolepis ( L. leptolepis ) can aid research on genetic improvement of gymnosperms. Previously, we obtained five LaMIR166a (miR166a precursor) -overexpression embryonic cell lines in the gymnosperm Larix leptolepis . The proliferation rates of pro-embryogenic masses in transgenic and wild-type lines were calculated. Overexpression of the miR166a precursor LaMIR166a led to slower proliferation. When pro-embryogenic masses were transferred to maturation medium, the relative expression of LaMIR166a and miR166a in the LaMIR166a -overexpression lines was higher than in the wild-type during SE, while LaHDZ31–34 expression levels also increased without negative control by miR166, suggesting that regulation of HD-ZIP III by miR166a exits stage-specific characteristics. The key indole-3-acetic acid (IAA) biosynthetic gene Nitrilase of L. leptolepis ( LaNIT ) was identified and the effects of miR166a on auxin biosynthesis and signalling genes were studied. During SE, LaNIT, Auxin response factor1 ( LaARF1 ) and LaARF2 mRNA levels and IAA contents were markedly higher in LaMIR166a -overexpression lines, which revealed lower deformity rate of embryos, indicating endogenous IAA synthesis is required for somatic embryo maturation in L. leptolepis . Additionally, the IAA biosynthesis and signalling genes showed similar expression patterns to LaHDZ31–34 , suggesting HD-ZIP III genes have a positive regulatory effect on LaNIT . Our results suggest miR166a and LaHDZ31–34 have important roles in auxin biosynthesis and signalling during SE, which might determine if the somatic embryo normally developed to mature in L. leptolepis .