Dormancy-associated MADS-box genes and microRNAs jointly control dormancy transition in pear (Pyrus pyrifolia white pear group) flower bud

Abstract

Bud dormancy in perennial plants is indispensable to survival over winter and to regrowth and development in the following year. However, the molecular pathways of endo-dormancy induction, maintenance, and release are still unclear, especially in fruit crops. To identify genes with roles in regulating endo-dormancy, 30 MIKC(C)-type MADS-box genes were identified in the pear genome and characterized. The 30 genes were analysed to determine their phylogenetic relationships with homologous genes, genome locations, gene structure, tissue-specific transcript profiles, and transcriptional patterns during flower bud dormancy in 'Suli' pear (Pyrus pyrifolia white pear group). The roles in regulating bud dormancy varied among the MIKC gene family members. Yeast one-hybrid and transient assays showed that PpCBF enhanced PpDAM1 and PpDAM3 transcriptional activity during the induction of dormancy, probably by binding to the C-repeat/DRE binding site, while DAM proteins inhibited the transcriptional activity of PpFT2 during dormancy release. In the small RNA-seq analysis, 185 conserved, 24 less-conserved, and 32 pear-specific miRNAs with distinct expression patterns during bud dormancy were identified. Joint analyses of miRNAs and MIKC genes together with degradome data showed that miR6390 targeted PpDAM transcripts and degraded them to release PpFT2. Our data show that cross-talk among PpCBF, PpDAM, PpFT2, and miR6390 played important roles in regulating endo-dormancy. A model for the molecular mechanism of dormancy transition is proposed: short-term chilling in autumn activates the accumulation of CBF, which directly promotes DAM expression; DAM subsequently inhibits FT expression to induce endo-dormancy, and miR6390 degrades DAM genes to release endo-dormancy.

Keywords: Dormancy; MIKCC-type MADS-box genes; PpCBF; PpFT2; microRNA; transient assays; yeast one-hybrid..

Fig. 1.

Bud break percentage of ‘Suli’ pear after 21 d of forcing conditions. Dormant shoots of field-grown ‘Suli’ pear trees were collected from 15 November 2010 to 15 February 2011, and kept in water in a phytotron at day/night temperatures of 25±1/18±1 °C, with a 12h photoperiod of white light (320 μmol photon m^–2 s^–1), and 75% humidity. Percentage bud break was assessed after 21 d using 12 shoots per sampling period. Error bars show the standard deviation of three biological replicates. Means with the same letter among stages are not significantly different (P ≤ 0.05).

Fig. 2.

Phylogenetic tree of the MIKC gene family in pear. The phylogenetic tree was constructed based on a multiple sequence alignment of predicted full-length MIKC protein sequences of Pyrus pyrifolia (Pp), Actinidia deliciosa (Ad), Arabidopsis (At), Brassica juncea (Bj), Coffea arabica (Ca), Citrus sinensis (Cs), Citrus trifoliata (Ct), Dendrobium crumenatum (Dc), Malus domestica (Md), Oryza sativa (Os), Platanus acerifolia (Pa), Panax ginseng (Pg), Prunus mume (Pm), Solanum lycopersicum (Sl), and Vitis vinifera (Vv). Numbers at nodes are percentage bootstrap values based on Neighbor–Joining analysis. The groups were marked with bold bars outside of the tree. The MIKC proteins identified in the pear genome were marked with black dots.

Fig. 3.

Transcript profiles of pear MIKC genes. Transcript analyses were performed by qRT-PCR. (A) Transcript profiles of pear MIKC genes in different pear organs/tissues. (B) Transcript profiles of pear MIKC genes during bud dormancy transition.

Fig. 4.

Interaction between PpCBF and PpDAM promoter as determined by Y1H assay. (A) Upstream regions of PpDAM A- and C-type promoters showing location of C-repeat/DRE transcription factor binding site. (B) Y1H assays showing interaction between PpCBF and PpDAM promoters. (C) The promoter of PpDAM with mutated C-repeat/DRE site was synthesized artificially and was inserted into pAbAi plasmid for Y1H assays. The pAbAi vector ligated to the promoter of PpDAM with non-mutated C-repeat/DRE site as a positive control. Y1H assays showed interaction between PpCBF and promoters of PpDAM with mutated C-repeat/DRE site and non-mutated C-repeat/DRE site. (This figure is available in colour at JXB online.)

Fig. 5.

Dual luciferase transient expression assays to probe functions of promoters and transcription factors. Interaction between PpDAM promoters and PpCBF in tobacco leaves. The activity of firefly and renilla luciferase in tobacco leaves was detected 3 d after infiltration. Error bars show standard error (SE) of three independent experiments with at least four replicate reactions. Means with the same letter among different injections are not significantly different (P ≤ 0.05).

Fig. 6.

Expression levels of dormancy-associated genes in pear flower buds during different bud dormancy stages. Error bars show the standard deviation of three biological replicates. Means with the same letter among stages are not significantly different (P ≤ 0.05).

Fig. 7.

Interaction between PpDAM and promoter of PpFT2 as determined by Y1H assay. (A) Upstream regions of PpFT2 A-, B- and C-type promoters. (B) Y1H assays showing interaction between PpDAM and PpFT2 promoters. Note that PpDAMs associated with the –312 to +131bp fragment of ProFT2. (This figure is available in colour at JXB online.)

Fig. 8.

Dual luciferase transient expression assays to probe functions of promoters and transcription factors. The activity of firefly and renilla luciferase in tobacco leaves was detected 3 d after infiltration. Error bars show standard error (SE) of three independent experiments with at least four replicate reactions. (A) Interaction between PpFT2 promoter and PpDAM1 in tobacco leaves. (B) Interaction between PpFT2 promoter and PpDAM2 in tobacco leaves. (C) Interaction between PpFT2 promoter and PpDAM3 in tobacco leaves. Means with the same letter among different injections are not significantly different (P ≤ 0.05).

Fig. 9.

Expression profiles of conserved and less-conserved miRNAs in pear flowering buds during bud dormancy. (A) Expression profiles of conserved miRNAs. (B) Expression profiles of less-conserved miRNAs. (C) Expression profiles of pear-specific miRNAs. Detailed list of miRNAs used in this figure can be found in Supplementary Tables S2 and S4 in Supplementary File 3 at JXB online.

Fig. 10.

qRT-PCR validations of the expression levels of miRNAs in pear flower buds during bud dormancy. Error bars show the standard deviation of three biological replicates. Means with the same letter among stages are not significantly different (P ≤ 0.05).

Fig. 11.

The secondary structures of miR6390 precursor and its target genes PpDAMs. (A) Predicted secondary structure of pre-miR6390. (B) The pairing between miR6390 and its target sites within PpDAMs is illustrated. (C) Target plot (t-plot) for miR6390 targets confirmed by degradome sequencing and the cleavage site was confirmed by the 5′-RACE nested PCR. (This figure is available in colour at JXB online.)

Fig. 12.

Proposed model of genetic factors that may affect dormancy transition in pear.Solid arrows/bars indicate genes, hormones, metabolites, or environmental conditions that have been proven to induce/inhibit targets; dashed arrows/bars indicate those that have been proposed but not yet confirmed in induction/inhibition of targets in this study. Short-term chilling in autumn activates the accumulation of CBF, which directly promotes DAM expression; DAM subsequently inhibits FT2 expression to induce endo-dormancy and miR6390 degrades DAM genes to release endo-dormancy. Short-term cold also induced ABA accumulation that might enhance the endo-dormancy by activating the ABI gene.