Identification of miRNA858 long-loop precursors in seed plants

Abstract

MicroRNAs (miRNAs) are a class of nonprotein-coding short transcripts that provide a layer of post-transcriptional regulation essential to many plant biological processes. MiR858, which targets the transcripts of MYB transcription factors, can affect a range of secondary metabolic processes. Although miR858 and its 187-nt precursor have been well studied in Arabidopsis (Arabidopsis thaliana), a systematic investigation of miR858 precursors and their functions across plant species is lacking due to a problem in identifying the transcripts that generate this subclass. By re-evaluating the transcript of miR858 and relaxing the length cut-off for identifying hairpins, we found in kiwifruit (Actinidia chinensis) that miR858 has long-loop hairpins (1,100 to 2,100 nt), whose intervening sequences between miRNA generating complementary sites were longer than all previously reported miRNA hairpins. Importantly, these precursors of miR858 containing long-loop hairpins (termed MIR858L) are widespread in seed plants including Arabidopsis, varying between 350 and 5,500 nt. Moreover, we showed that MIR858L has a greater impact on proanthocyanidin and flavonol levels in both Arabidopsis and kiwifruit. We suggest that an active MIR858L-MYB regulatory module appeared in the transition of early land plants to large upright flowering plants, making a key contribution to plant secondary metabolism.

Figure 1.

The distribution of mature miR858s in seed plants. Small RNA sequencing data for 32 representative land plants were screened for mature miR858. Mature miR858s were found in all examined seed plants, but not in lycophytes and bryophytes. Solid circles indicate where mature miR858 was identified in small RNA sequencing data of the corresponding species. In contrast, a hollow circle shows that mature miR858 was not found.

Figure 2.

A functional long-loop precursor ach-MIR858Lc identified in kiwifruit. A) An irregular loop is marked. ach-miR858c and ach-miR858c* are highlighted. B) The schematic diagram of the miRNA dual-luciferase assay for miR858. A artificial target (reverse complementary sequence of miR858) was inserted into the pGreen_dualluc_3′UTR_sensor vector, located behind firefly luciferase coding sequence. C) Dual-luciferase assay showing ach-MIR858Lc has a significant inhibitory effect on the artificial target. EV pSAK277 is used as control, and the REN/LUC ratio of EV is set as 1. D) Transient assays in N. benthamiana leaves. The data in (C) and (D) are plotted as means ± SE. n = 3 or 4 biological replicates.

Figure 3.

A comparison of the secondary structure and function between short hairpin ath-MIR858a and long hairpin ath-MIR858L in Arabidopsis. A) The short and long precursors show a typical hairpin structure or a long-loop hairpin-like structure, respectively. The 3′ stem in ath-MIR858a and ath-MIR858L are termed SR1 and SR2 (Stem Region). miR858 generation region formed a stem within SR1 in ath-MIR858a. In the long-loop precursor, SR2 forms the stem, and SR1 becomes part of the loop in ath-MIR858L. B) Dual-luciferase assay showed that ath-MIR858L had a stronger inhibitory effect on the artificial target than ath-MIR858a. EV pSAK277 was used as control, and the REN/LUC ratio of EV was set as 1. C) Transient assays in N. benthamiana leaves. The expression of miR858 produced by ath-MIR858a was set as 1. D) Dual-luciferase assay of SR1^mut (ath-MIR858L-SR1^mut) and SR2^mut (ath-MIR858L-SR2^mut). EV pSAK277 was used as control, and the REN/LUC ratio of EV was set as 1. E) Levels of ath-miR858a when SR1^mut and SR2^mut are over-expressed. The expression of ath-miR858a produced by SR1^mut was set as 1. The data in (B), (C), (D), and (E) were plotted as means ± SE. n = 3 or 4 biological replicates.

Figure 4.

MIR858L over-expression reduces proanthocyanidin and flavonol content in both kiwifruit and Arabidopsis. A) The expression of ach-MIR858Lc transcript and mature ach-miR858c in kiwifruit wild-type and over-expression plants. B) The expression of ach-MIR858a/L transcripts and mature ath-miR858a in Arabidopsis wild-type and over-expression plants. C) The seed color of Arabidopsis unstained and DMACA-stained. D) HPLC detection of proanthocyanidin and flavonol in kiwifruit leaves. E) HPLC detection of Arabidopsis proanthocyanidin and flavonol in silique and leaves, respectively. The data of (A), (B), (D), and (E) were plotted as means ± SE (n = 3 biological replicates). L refers to different transgenic lines. Statistical analysis was performed using the two-tailed Student's t-test. n.s. represents not significant. n.d. represents not detected.

Figure 5.

Long-loop MIR858Ls are present widely in seed plants, while the long-loop does not influence the mode of processing and repressive action. A) The long-loop precursors of miR858 present in seed plants, but not in lycophytes and bryophytes. The solid line is the 300-nt recommended as a maximum length for plant miRNA hairpin precursors, according to Axtell and Meyers (2018). The bar on the right of phylogenetic tree shows the average length of putative hairpins in each species. B) The length of MIRs in Arabidopsis and kiwifruit, with MIR858Ls being the longest MIRs in Arabidopsis and kiwifruit. E) The expression of mature miR858 in control and after silencing of DCL1 in transient N. benthamiana assays. The expression of mature miR858 produced in control groups was set as 1. D) The loop-deleted hairpin MIR858L^Del created by retaining 15 nt in the terminal loop, while the remaining loop nucleotides were deleted. EV pSAK277 was used as control, and the REN/LUC ratio of EV was set as 1. The data in (C) and (D) were plotted as means ± SE. n = 3 or 4 biological replicates.

Figure 6.

The origin, evolution, and development of miR858. A) Phylogenetic analysis of TT2-like genes (R2R3 MYBs) and the 5′ and 3′ stem (reverse complementary sequence) of MIR858Ls. Phylogenetic analysis suggests that early MIR858L stems likely originated from TT2-like genes, and developed stems were divided into two individual. Circles represent the 5′ stems of MIR858Ls, while stars represent the 3′ stems of MIR858Ls. The phylogenetic tree was drawn with ignore branch lengths. B) TEs are prevalent in the loops of MIR858Ls. C) The classification of TEs found in loops. D) The percentage of precursors containing TEs increases when the length of loops increases. E) Proto MIR858L originates from the ancient TT2-like gene (MYB) through the inverted duplication. Subsequently, evolution of the stem sequence and insertion of TEs promote the development of MIR858L, to regulate S5 and S7 MYB transcription factors in seed plants.