MicroRNA Zma-miR528 Versatile Regulation on Target mRNAs during Maize Somatic Embryogenesis

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. While several targets have been described for this miRNA, the regulation has not been experimentally confirmed for the SE process. Here, we explored the accumulation of zma-miR528 and several predicted targets during embryogenic callus induction, proliferation, and plantlet regeneration using the maize cultivar VS-535. We confirmed the cleavage site for all tested zma-miR528 targets; however, PLC1 showed very low levels of processing. The abundance of zma-miR528 slightly decreased in one month-induced callus compared to the immature embryo (IE) explant tissue. However, it displayed a significant increase in four-month sub-cultured callus, coincident with proliferation establishment. In callus-regenerated plantlets, zma-miR528 greatly decreased to levels below those observed in the initial explant. Three of the target transcripts (MATE, bHLH, and SOD1a) showed an inverse correlation with the miRNA abundance in total RNA samples at all stages. Using polysome fractionation, zma-miR528 was detected in the polysome fraction and exhibited an inverse distribution with the PLC1 target, which was not observed at total RNA. Accordingly, we conclude that zma-miR528 regulates multiple target mRNAs during the SE process by promoting their degradation, translation inhibition or both.

Keywords: maize; miRNA-target regulation; somatic embryogenesis; zma-miR528.

Figure 1

Target prediction for zma-miR528. (a) Circos plot between zma-miR528a and predicted targets showing their chromosomal location. The yellow-filled box represents the MIR528a gene location. Gray dark boxes refer to targets selected and analyzed in this study. For Circos plot construction, we used zma-MIR528a location only to illustrate the miRNA-target pairing, as both genes produce the same mature miRNA targeting the same transcripts. Pale gray boxes denote other putative targets not approached in our investigation. (*) Experimentally validated targets for zma-miR528 in a previous report: ZmLAC3 (Zm00001d052243_T001); ZmLAC5 (Zm00001d042901_T001). (b) List of the selected potential targets showing the recognition site pairing for zma-miR528 and their reported function in plant pathways according to several reports [19,23,24,25,26,27,28,29,30,31].

Figure 2

Experimentally validated cleavage sites for zma-miR528 targets. Validation for cleavage was obtained after 5′-RLM-RACE mapping, as described in the methods. The presence of cleavage products is shown in Supplementary Material, Figure S1. Arrows designate the cleavage sites, and the above numbers indicate the proportion of clones showing the same site. The upper strand represents targets with the complementary site (blue-filled fragment), and the bottom strand shows the pairing with the miRNA sequence. Mismatches are shown in red. (:) pairing, (.) non-canonical pairing.

Figure 3

Zma-miR528 and mRNA target accumulation levels in VS-535 SE. (a) Different tissue samples obtained from maize SE stages used for miRNA and target profiling. Immature embryos (IE) at 15–18 days after pollination (DAP) used as initial explant for SE initiation. One month after induction, friable embryogenic callus (C1) was selected for sampling. Embryogenic calli were subcultured monthly, and proliferation was established after four months (C4). By gradual hormone reduction and photoperiod exposure, plantlet (PL) regeneration was achieved. (b) zma-miR528 and selected target levels were analyzed by RT-qPCR in total RNA from the samples mentioned above. Fold change represents abundance relative to IE and normalized either by U6 snRNA internal control (microRNA) or 18S rRNA (transcripts). The results were obtained from three independent biological replicates (n = 3) with three technical replicates for each one (n = 9). Data were analyzed by performing a one-way ANOVA with multiple comparisons by the Tukey post hoc test. Boxes that do not share at least an identical letter differ significantly (p < 0.005) from each other.

Figure 4

Inverse correlation between zma-miR528 and some of the selected targets during VS-535 SE stages. The heatmap shows the log2-fold-change (Log2FC) of each transcript in samples. Numbers on the right side represent Pearson’s coefficient (R) and the significance value (p) for the correlation analysis between the miRNA and each relative target abundance. * p < 0.05, ns: no significance. IE: immature embryo. C1: one-month embryogenic callus. C4: four-month embryogenic callus. PL: regenerated plantlet.

Figure 5

Distribution of zma-miR528 and targets in polysomal fractions. Upper panels show profiles for the immature embryo, one-month callus, four-month callus, and germinated embryo axis. Each profile is composed of 16 minor fractions further pooled into broader fractions according to the absorbance profile and rRNA observation on depicted agarose gels: F1 (free RNA and ribonucleoproteins complexes), F2 (monosomes), F3 (light polysomes), and F4 (heavy polysomes). Lower panels display the distribution of miR528 and some targets in each fraction. The percentage level was calculated for each transcript using the lower Ct value as a normalizer. Error bars indicate ± SD of two biological replicates with three technical replicates for each one (n = 6). A254 nm: absorbance value at 254 nm wavelength.

Figure 6

Mechanisms and impact of zma-miR528 target regulation during maize SE. (a) Zma-miR528 and its targets experience accumulation patterning during the induction, proliferation, and plantlet regeneration in maize SE. Upregulation: green upward arrow; downregulation: red downward arrow. IE: immature embryo (explant); proEC: pro embryogenic callus mass; EC: embryogenic callus; RS: regenerative spots; PL: regenerated plantlet. (b) Proposed model for zma-miR528 regulatory mechanisms exerted on targets. Mature miRNA arises from either of two MIR528 genes present in maize. miRNA-RISC assembles either in the cytoplasm or on membrane-bound polysomes, where zma-miR528 directs target recognition to promote target cleavage or translational repression. Mechanism selection could depend on miRNA-target kinetic parameters or the presence/absence of certain accessory proteins in the RISC complex. However, either mechanism could be linked to the production of miRNA-mediated cleavage products, as suggested in the literature [38]. According to the target function, zma-miR528 could be involved in embryogenic calli proliferation by maintaining metabolic and physiologic processes required for SE.