Abstract
MicroRNAs are non-coding small RNA molecules that are involved in the post-transcriptional regulation of the genes that control various developmental processes in plants, including zygotic embryogenesis (ZE). miRNAs are also believed to regulate somatic embryogenesis (SE), a counterpart of the ZE that is induced in vitro in plant somatic cells. However, the roles of specific miRNAs in the regulation of the genes involved in SE, in particular those encoding transcription factors (TFs) with an essential function during SE including LEAFY COTYLEDON2 (LEC2), remain mostly unknown. The aim of the study was to reveal the function of miR165/166 and miR160 in the LEC2-controlled pathway of SE that is induced in in vitro cultured Arabidopsis explants.In ZE, miR165/166 controls the PHABULOSA/PHAVOLUTA (PHB/PHV) genes, which are the positive regulators of LEC2, while miR160 targets the AUXIN RESPONSE FACTORS (ARF10, ARF16, ARF17) that control the auxin signaling pathway, which plays key role in LEC2-mediated SE. We found that a deregulated expression/function of miR165/166 and miR160 resulted in a significant accumulation of auxin in the cultured explants and the spontaneous formation of somatic embryos. Our results show that miR165/166 might contribute to SE induction via targeting PHB, a positive regulator of LEC2 that controls embryogenic induction via activation of auxin biosynthesis pathway (Wójcikowska et al., 2013). Similar to miR165/166, miR160 was indicated to control SE induction through auxin-related pathways and the negative impact of miR160 on ARF10/ARF16/ARF17 was shown in an embryogenic culture. Altogether, the results suggest that the miR165/166- and miR160-node contribute to the LEC2-mediated auxin-related pathway of embryogenic transition that is induced in the somatic cells of Arabidopsis. A model summarizing the suggested regulatory interactions between the miR165/166-PHB and miR160-ARF10/ARF16/ARF17 nodes that control SE induction in Arabidopsis was proposed.
Highlights
The unique developmental plasticity of plant cells has been widely documented by the successful formulation of in vitro culture protocols that enable the efficient clonal propagation of numerous plant species
The candidate miR160, miR165/166 Involved in Somatic Embryogenesis targets of miR165/miR166 (PHB/PHV) and miR160 (ARF10/ARF16/ARF17) were evaluated in somatic embryogenesis (SE) culture of Arabidopsis with a disturbed expression/function of the studied genes
We found that accumulation of the PHB transcripts in the gainof-function phb1-d mutant and a STTM165/166 line led to the miR160, miR165/166 Involved in Somatic Embryogenesis significantly increased LEAFY COTYLEDON2 (LEC2) transcription (Figure 3A)
Summary
The unique developmental plasticity of plant cells has been widely documented by the successful formulation of in vitro culture protocols that enable the efficient clonal propagation of numerous plant species (reviewed Misra and Saema, 2016). In numerous plants including Arabidopsis thaliana (Arabidopsis), zygotic embryos at precisely defined developmental stages provide the most efficient tissue to induce SE (Elithi and Stasolla, 2011; Wójcikowska and Gaj, 2016). In Arabidopsis, the culture of zygotic embryos at a late cotyledonary stage of development on an auxin medium has been recommended to induce SE for molecular studies on plant cell totipotency (Gaj, 2004). Using this model SE system has resulted in the remarkable progress in deciphering the genetic mechanisms that govern SE induction that has been achieved in recent years (reviewed in Wójcikowska and Gaj, 2016)
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