Dual functions of PsmiR172b-PsTOE3 module in dormancy release and flowering in tree peony (Paeonia suffruticosa)

Abstract

MicroRNAs (miRNAs) are non-coding RNAs that interact with target genes and are involved in many physiological processes in plants. miR172-AP2 mainly plays a role in the regulation of flowering time and floral organ differentiation. Bud dormancy release is necessary for forcing culture of tree peony in winter, but the mechanism of dormancy regulation is unclear. In this study, we found that a miR172 family member, PsmiR172b, was downregulated during chilling-induced bud dormancy release in tree peony, exhibiting a trend opposite to that of PsTOE3. RNA ligase-mediated (RLM) 5'-RACE (rapid amplification of cDNA ends) confirmed that miR172b targeted PsTOE3, and the cleavage site was between bases 12 (T) and 13 (C) within the complementary site to miR172b. The functions of miR172b and PsTOE3 were detected by virus-induced gene silencing (VIGS) and their overexpression in tree peony buds. PsmiR172b negatively regulated bud dormancy release, but PsTOE3 promoted bud dormancy release, and the genes associated with bud dormancy release, including PsEBB1, PsEBB3, PsCYCD, and PsBG6, were upregulated. Further analysis indicated that PsTOE3 directly regulated PsEBB1 by binding to its promoter, and the specific binding site was a C-repeat (ACCGAC). Ectopic expression in Arabidopsis revealed that the PsmiR172b-PsTOE3 module displayed conservative function in regulating flowering. In conclusion, our results provided a novel insight into the functions of PsmiR172-PsTOE3 and possible molecular mechanism underlying bud dormancy release in tree peony.

Figure 1

Homology comparison of miR172s in tree peony and the other plants, and expression patterns of PsmiR172s and PsTOE3 during chilling-induced bud dormancy release. (A) Homology comparison of miR172s in tree peony and the other plants. Ps, Paeonia suffruticosa; Ath, Arabidopsis thaliana; Csi, Camellia sinensis; Osa, Oryza sativa; Vvi, Vitis vinifera. (B) Expression levels of mature PsmiR172a, PsmiR172b, and PsmiR172d during chilling-induced dormancy release in tree peony. (C) Expression levels of pre-PsmiR172a, pre-PsmiR172b, and pre-PsmiR172d during chilling-induced dormancy release in tree peony. (D and E) Expression levels of PsmiR172b and PsTOE3 after chilling and after being transferred to the greenhouse when exposed to chilling for 7 and 21 days. Blue arrows show the relative expression levels of PsmiR172b and PsTOE3 when being transferred to the greenhouse after chilling for 7 days, and black arrows show the relative expression levels of PsmiR172b and PsTOE3 when transferred to the greenhouse after chilling for 21 days. Data are the mean ± standard deviation of three replications. ^*Significant difference at P < .05, ^**significant difference at P < .01.

Figure 2

cDNA sequence of PsTOE3, phylogenetic tree, and subcellular localization of PsTOE3. (A) cDNA sequence of PsTOE3. The initiation codon and termination codon are marked by bold italics, and the putative binding sites of PsmiR172b are marked by double underline; the synonymous mutation sites (for mPsTOE3) are marked by a red color. The primer (PsTOE3-P1, -P2, -P3, and -P4) sites for RT–PCR are marked by underlines. (B) Phylogenetic tree containing PsTOE3 and 147 AtAP2/ERFs constructed using the neighbor-joining method with 1000 bootstrap replications. Different colors mark different subfamilies of the AP2 family, and PsTOE3 is marked with a blue dot. (C) Subcellular localization of PsTOE3 by fluorescent microscopy with a stimulating wavelength of 488 nm.

Figure 3

Prediction and validation of the target relationship between PsTOE3 and PsmiR172b. (A) Secondary structure of PsmiR172b precursor. Red represents the sequence of mature PsmiR172b and its complementary sequence is marked in green. (B) Identification of the PsmiR172b target site with RLM 5′-RACE. The cleaved site is marked with an arrow. The numbers above the arrow are the numbers of cleavage sites in independent clones. (C) Cotransformation of tobacco leaves with PsmiR172b:GUS and PsTOE3:GUS, PsmiR172b:GUS, and mPsTOE3:GUS, respectively. GUS staining was observed histochemically. (D) Quantitative analysis of GUS enzyme activities by fluorospectrophotometer in leaves inoculated with different recombinant vectors. 1, 35S::GUS; 2, 35S::PsmiR172b:GUS; 3, 35S::PsTOE3:GUS; 4, 35S::mPsTOE3:GUS; 5, 35S:: PsmiR172b:GUS + 35S::PsTOE3:GUS; 6, 35S::PsmiR172b:GUS + 35S::mPsTOE3:GUS. Data are the mean ± standard error of the mean of 10 plants. Lowercase letters on the columns indicate significant difference at the P < .05 level. (E) Change trends of PsTOE3 transcripts during chilling-induced dormancy release using RT–PCR. Primer pair P1 and P2 was used to amplify the fragment containing the target region, and P3 and P4 were used to obtain the fragment after the target region. The primer sites are showed in Fig. 2A. (F) Protein levels of PsTOE3 during chilling-induced dormancy release by western blot; ACTIN protein was the internal control.

Figure 4

Morphological changes and expression levels of genes associated with bud dormancy release after silencing of PsmiR172b by VIGS in tree peony. (A) Morphological changes after silencing of PsmiR172d by VIGS for 28 days. Buds infected with TRV1/TRV2 were used as control. Bar = 1.0 cm. (B) Relative expression levels of PsmiR172b in buds after silencing of PsmiR172b for 7 days. (C) Relative growth of height and width of TRV2-STTM172b buds after transforming for 14 and 28 days. (D) Relative expression levels of genes associated with peony dormancy release in TRV2-STTM172b buds after transforming for 7 days. Data are the mean ± standard deviation of six replications for relative expression. ^*Significant difference at P < .05, ^**significant difference at P < .01.

Figure 5

Morphological changes and expression levels of genes associated with bud dormancy release after overexpression of PsTOE3 in tree peony. (A) Morphological changes in PsTOE3 overexpression buds after transformation for 28 days. Buds with empty pBI121 vector were used as control. Scale bar = 1.0 cm. (B) Relative expression levels of PsTOE3 in PsTOE3-OE buds using qRT–PCR and the amount of PsTOE3 protein in three PsTOE3-OE buds (#2, #4, and #6) using western blot after transformation for 7 days. (C) Relative growth in height and width in PsTOE3-OE buds after transformation for 14 and 28 days. (D) Relative expression levels of genes associated with peony dormancy release using qRT–PCR in PsTOE3-OE buds after transformation for 7 days. Data are the mean ± standard deviation of six replications for relative expression. ^*Significant difference at P < .05, ^**significant difference at P < .01.

Figure 6

Transcript levels of the genes associated with dormancy release as revealed by qRT–PCR and PsTOE3 directly regulated the expression of PsEBB1. (A) Expression levels of the genes associated with dormancy release, including PsCYCD, PsEBB1, PsEBB3, and PsBG6 during chilling-induced dormancy release using qRT–PCR. (B) Schematic diagram of PsEBB1 promoter and the result of the yeast one-hybrid assay. The C-repeat is marked with blue arrows. (F) Full length of PsEBB1 promoter; F1 (−2068 to −629), a fragment without C-repeat as a negative control; F2 (−628 to −1), a fragment with C-repeat. (C) Interaction of PsTOE3 protein and the C-repeat promoter of PsEBB1 by EMSA. The C-repeat (ACCGAC) and mutation C-repeat (AGCGAC) were synthesized with four replications, and the mutation C-repeat was used as negative control. (D) Regulation of PsTOE3 protein to PsEBB1 using the dual luciferase assay after infiltration for 3 days. (E) Relative LUC/REN activities after infiltration for 3 days. Data are the mean ± standard deviation of six replications for relative expression. ^*Significant difference at P < .05, ^**significant difference at P < .01.

Figure 7

Hypothetical model of the roles of PsmiR172b-PsTOE3 during chilling-induced bud dormancy release in tree peony. PsmiR172b responds to chilling treatment and is downregulated, and the expression of its target gene, PsTOE3, is induced during the same process. Both PsmiR172b silencing and PsTOE3 overexpression lead to the upregulation of PsEBB1, PsEBB3, PsCYCD, and PsBG6; PsTOE3 can directly bind to the promoter of PsEBB1 and activate its expression, indirectly accelerate cell proliferation and finally lead to bud dormancy release and budbreak. Red arrows represent positive regulation, black bars represent negative regulation. Dotted red arrows indicate indirectly positive regulation.