MiR393 contributes to the embryogenic transition induced in vitro in Arabidopsis via the modification of the tissue sensitivity to auxin treatment.

Anna M Wójcik,Małgorzata D Gaj

doi:10.1007/s00425-016-2505-7

Abstract

Main conclusionmiR393 was found to control embryogenic transition in somatic cells in Arabidopsis via control of theTIR1andAFB2auxin receptors genes of the F-box family.miR393 molecules are believed to regulate the expression of the auxin receptors of the TAAR clade. Considering the central role of auxin in the induction of somatic embryogenesis (SE) in plant explants cultured in vitro, the involvement of miR393 in the embryogenic transition of somatic cells has been hypothesised. To verify this assumption, the reporter, overexpressor and mutant lines in genes encoded MIR393 and TIR1/AFB proteins of the F-box family were analysed during SE in Arabidopsis. Expression profiling of MIR393a and MIR393b, mature miR393 and the target genes (TIR1, AFB1, AFB2, AFB3) were investigated in explants undergoing SE. In addition, the embryogenic potential of various genotypes with a modified activity of the MIR393 and TIR1/AFB targets was evaluated. The distinct increase in the accumulation of miR393 that was coupled with a notable down-regulation of TIR1 and AFB2 targets was observed at the early phase of SE induction. Relevant to this observation, the GUS/GFP monitored expression of MIR393, TIR1 and AFB2 transcripts was localised in explant tissue undergoing SE induction. The results suggest the miR393-mediated regulation of TIR1 and AFB2 during embryogenic transition induced in Arabidopsis and a modification of the explant sensitivity to auxin treatment is proposed as underlying this regulatory pathway.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-016-2505-7) contains supplementary material, which is available to authorized users.

Highlights

Non-coding RNAs encoded by MIRNA genes, the so-called microRNAs, regulate gene expression by targeting mRNAs for degradation or translational repression (Axtell 2013)
Two mutants that were impaired in the production of functional DCL proteins were analysed in terms of their embryogenic potential in vitro—dcl1-6 (Schauer et al A survey of MIRNA gene transcripts in the embryogenic culture of Col-0 explants indicated a distinct up-regulation of MIR393a and MIR393b transcripts, which were accumulated up to 53 and 27 fold, respectively (Supplementary Fig. S1)
In explants that were induced towards somatic embryogenesis (SE) and treated with 2,4-D for 5 days, both of the MIR393 genes were found to be active in an apical part of the immature zygotic embryos (IZE) and an especially strong GUS signal was observed in the explant parts that are involved in embryogenic transition, i.e. in the adaxial parts of the cotyledons and in the vicinity of the shoot apical meristem (SAM) (Fig. 2d, e)

Summary

Introduction

Non-coding RNAs encoded by MIRNA genes, the so-called microRNAs (miRNAs), regulate gene expression by targeting mRNAs for degradation or translational repression (Axtell 2013). The biogenesis of miRNA requires activity of multiple enzyme complexes and among them the RNase-III-like enzyme DICER-. LIKE1 (DCL1) was indicated as playing a central role in the biogenesis of most of the miRNAs identified in plants (Reinhart et al 2002). DCL1 interacts with other doublestranded RNA (dsRNA) binding proteins, HYPONASTIC LEAVES1 (HYL1) and the zinc-finger protein SERRATE (SE), to promote the efficient and accurate biogenesis of miRNA (Cuperus et al 2010). In contrast to DCL1, which is involved in the biogenesis of MIRNA-encoded RNAs, the activity of DCL2-4 is related to the biogenesis of viral short interfering RNA (siRNA) and endogenous siRNA such as retrotransposon siRNA and trans-acting small interfering RNA (Xie et al 2004). DCL3 was revealed to cognate dsRNAs and produce the siRNAs that work in RNA-directed DNA methylation (Chapman and Carrington 2007)

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