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.