Abstract
Maize somatic embryogenesis (SE) requires the induction of embryogenic callus and establishment of proliferation before plant regeneration. The molecular mechanisms underlying callus embryogenic potential are not well understood. Here we explored the role of small RNAs (sRNAs) and the accumulation of their target transcripts in maize SE at the dedifferentiation step using VS-535 zygotic embryos collected at distinct developmental stages and displaying contrasting in vitro embryogenic potential and morphology. MicroRNAs (miRNAs), trans-acting siRNAs (tasiRNAs), heterochromatic siRNAs (hc-siRNAs) populations and their RNA targets were analyzed by high-throughput sequencing. Abundances of specific miRNAs, tasiRNAs and targets were validated by qRT-PCR. Unique accumulation patterns were found for immature embryo at 15 Days After Pollination (DAP) and for the callus induction from this explant, as compared to 23 DAP and mature embryos. miR156, miR164, miR166, tasiARFs and the 24 nt hc-siRNAs displayed the most strikingly different patterns between explants and during dedifferentiation. According to their role in auxin responses and developmental cues, we conclude that sRNA-target regulation operating within the 15 DAP immature embryo explant provides key molecular hints as to why this stage is relevant for callus induction with successful proliferation and plant regeneration.
Highlights
Plant zygotic embryogenesis is determined by complex gene regulatory networks to achieve appropriate spatiotemporal cell division and differentiation[1,2,3]
Both miRNA and small interfering RNAs (siRNAs) duplexes are stabilized through HUA ENHANCER 1 (HEN1)-dependent methylation at their 3′ ends19. small RNAs (sRNAs) loading on specific ARGONAUTE (AGO) proteins yields a functional RNA-Induced Silencing Complex (RISC), with AGO specificity further diversifying sRNA roles based on mature size, 3′ end nucleotide identity, and the specific silencing mechanism used on targets[15,19]
Our findings support a role of differential sRNA-mediated regulation during maize callus induction depending on the explant developmental stage
Summary
Plant zygotic embryogenesis is determined by complex gene regulatory networks to achieve appropriate spatiotemporal cell division and differentiation[1,2,3]. An alternative gene expression program, promoted by high phytohormone concentrations, allows the accomplishment of embryogenesis and plant regeneration from somatic cells. This process is known as Somatic Embryogenesis (SE)[4,5]. The double-stranded RNA (dsRNA) portion of a pri-miRNA is recognized by a DICER-LIKE (DCL) enzyme, DCL1 in plants, to generate the miRNA www.nature.com/scientificreports/. Among the ten different AGO proteins present in Arabidopsis, AtAGO1 loads miRNAs and phasiRNAs, AtAGO10 loads miR165/166, AtAGO7 loads a unique miRNA (miR390) involved in tasiRNA biogenesis from TAS3, AtAGO2 functions in antiviral defense, and the AGO4 clade (AGO4, AGO6, AGO9) functions in silencing of transposons and repeated sequences through a specific RNA-directed DNA methylation mechanism, RdDM20,21. The genomes of many plant species encode additional AGO family members
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