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

Abstract

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.

MiRNA expression profiling and zeatin dynamic changes in a new model system ofin vivoindirect regeneration of tomato

“…Additionally, REV function is necessary for polar auxin transport in the shoot[83]. Li et al[51,84,85] demonstrated that over-expression of LaMIR166a and down-regulated of LaHDZIP31-34 genes results in different IAA levels in pro-embryogenic masses of L. leptolepis . The authors speculated that LaMIR166 targeted HD-ZIP III genes likely regulate auxin biosynthesis and response genes.…”

miRNA expression profiling and zeatin dynamic changes in a new model system of in vivo indirect regeneration of tomato

“…Additionally, REV function is necessary for polar auxin transport in the shoot [79]. Li et al [45,80,81] demonstrated that over-expression of La-miR166a and down-regulated of LaHD-ZIP31-34 genes results in different IAA levels in pro-embryogenic masses of L. leptolepis. The authors speculated that La-miR166 targeted HD-ZIP III genes likely regulate auxin biosynthesis and response genes.…”

Metabolome and Transcriptome Association Analysis Reveals Regulation of Flavonoid Biosynthesis by Overexpression of LaMIR166a in Larix kaempferi (Lamb.) Carr

“…Wild embryogenic cultures were obtained from L. kaempferi immature zygotic embryos on the induction medium, LaMIR166a was cloned into a psuper1300+ binary vector and successfully transformed into embryogenic suspensor masses (ESMs), and five transgenic embryogenic cell lines were obtained, which increased lateral root number and biomass at the germination stage [19][20][21]. Based on our previous studies, we selected three of these soft and fresh cell clusters (named a-3, a-4, and a-5), which were grown for 20 days on the proliferation medium at the same developmental stage; they were cultured in triplicate and taken from different culture masses.…”

“…Previous studies have found that LaMIR166a may indirectly regulate the development of SAM by affecting WOX expression [19]. LaMIR166a plays an important role in auxin biosynthesis and signal transmission during somatic embryo maturation and germination [20,21]. However, the role of miR166 and its regulatory mechanisms in the proembryo stage are not clear.…”