Transcription factor TCP20 regulates peach bud endodormancy by inhibiting DAM5/DAM6 and interacting with ABF2.

The dormancy-associated MADS-box (DAM) genes PpDAM5 and PpDAM6 have been shown to play important roles in bud endodormancy; however, their molecular regulatory mechanism in peach is unclear. In this study, by use of yeast one-hybrid screening, we isolated a TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR transcription factor, PpTCP20, in the peach cultivar 'Zhongyou 4' (Prunus persica var. nectarina). The protein was localized in the nucleus and was capable of forming a homodimer. Electrophoretic mobility shift assays demonstrated that PpTCP20 binds to a GCCCR element in the promoters of PpDAM5 and PpDAM6, and transient dual luciferase experiments showed that PpTCP20 inhibited the expression of PpDAM5 and PpDAM6 as the period of the release of flower bud endodormancy approached. In addition, PpTCP20 interacted with PpABF2 to form heterodimers to regulate bud endodormancy, and the content of abscisic acid decreased with the release of endodormancy. PpTCP20 also inhibited expression of PpABF2 to regulate endodormancy. Taken together, our results suggest that PpTCP20 regulates peach flower bud endodormancy by negatively regulating the expression of PpDAM5 and PpDAM6, and by interacting with PpABF2, thus revealing a novel regulatory mechanism in a perennial deciduous tree.

Introduction

Perennial deciduous fruit trees in temperate and boreal zones have seasonal growth, and bud endodormancy is an adaptive way for plants to avoid damage in winter (Busov, 2019) in a process that is regulated by internal physiological factors (Lang, 1987; Tuan ). Endodormancy is a complex mechanism that is essential for plant development and productivity (Chuine and Beaubien, 2001), and it requires a sufficient period of chilling to be completed. For example, the peach cultivar ‘Zhongyou 4’ (Prunus persica var. nectarina) requires ~650–700 h of chilling accumulation (hours below 7.2 °C) (Wang ). As a result of climate change, insufficient chilling accumulation in winter is becoming a major challenge for deciduous fruit production (Campoy ; Kuroki ). It is therefore crucial to understand the molecular mechanisms that regulate bud endodormancy in order to maintain fruit yields.

The peach DORMANCY ASSOCIATED MADS-box (DAM) genes DAM5 and DAM6 belong to the MIKC-type MADS-box transcription factor family, which includes SHORT VEGETATIVE PHASE (SVP) and AGAMOUS-LIKE 24 (AGL24) in Arabidopsis (Yamane ). Bielenberg found that there are six tandem genes (DAM1–6) in the peach mutant evergrowing (evg) that lacks dormant behavior. Subsequent studies have found that DAM5 and DAM6 are induced by short days (Li ), and that the DAM genes are essential for the growth and development of peach (Jiménez ). Yamane determined that the expression patterns of DAMs were associated with the endodormancy status. DAM-like genes have been studied in many perennial species in relation to bud dormancy, including leafy spurge (Horvath ), apricot (Sasaki ), pear (Liu ; Niu ), and apple (Mimida ), which suggests that DAMs control bud dormancy in a similar manner across perennial plants.

Abscisic acid (ABA) is known to enhance the induction of dormancy and to regulate its maintenance in grape (Zheng ). The ABA content decreases with chilling accumulation in grape (Zheng ) and in peach (Wang ), and it is the major inhibitor that releases corm dormancy in gladiolus (Wu ). It has been hypothesized that ABA can inhibit cell proliferation and shoot growth, and that dormancy can be induced by ABA biosynthesis, catabolism, and signaling (Wang ; Zheng ). ABA signaling genes belong to the basic leucine zipper (bZIP) transcription factor family (Jakoby ). ABA response elements (ABREs), such as the ABRE-binding factors ABF1, ABF2, ABF3, and ABI5, belong to the bZIP family and have important roles in ABA-dependent stress signaling (Zhang ). The AREB1/ABF2 gene is induced by the ABA content in Arabidopsis (Fujita ). In poplar, AREB/ABF is up-regulated during bud dormancy by increasing ABA content (Ruttink ), and AREB1 binds to the DAM1 promoter region and negatively regulates its activity in pear (Tuan ). These results suggest that the ABF transcription factor plays an essential role in bud dormancy.

Teosinte branched1/Cycloidea/Proliferating cell factors (TCPs) are a class of plant-specific transcriptional regulators that play an important role in development, including cell proliferation and growth (Nicolas and Cubas, 2016). The TCP transcription factor family has a highly conserved TCP domain (a non-canonical basic helix–loop–helix motif) that is involved in DNA binding and dimerization (Cubas ). Based on the characteristics of this domain, TCP family members are divided into class I (also known as PCF or TCP-P) and class II (also known as TCP-C). The class-I TCP consensus binding site II motif is GGNCCCAC, of which the core elements are GCCCR (R = A or G), and class-II TCPs bind DNA motifs of the sequence GTGGNCCC (Li ). In Arabidopsis, class-II BRC1 (or TCP12) binds to, and is positively regulated, by HB21, HB40, and HB53. These four proteins enhance the expression of NCED3, leading to the accumulation of ABA and triggering a hormonal response that promotes axillary bud dormancy (González-Grandío et al., 2013, 2017). The class-I TCP14 has the ability to regulate embryo growth potential in seeds, and the lower ABA1 expression in tcp14 mutants delays germination (Tatematsu ; Rueda-Romero ). In addition, all class-I TCPs can interact with the REPRESSOR of GA1-3 protein (Davière ), and TCP14 and TCP15 interact with DELLA proteins to regulate seed germination in Arabidopsis (Resentini ). In addition, TCP19 modulates corm dormancy release by repressing NCED expression and increasing cytokinin levels in gladiolus (Wu ). Recently, all TCP transcription factors from the peach genome have been identified and analysed in relation to fruit ripening (Guo ); however, their relationships with bud endodormancy are still poorly understood.

In this study, we show that the TCP transcription factor PpTCP20 acts as a transcriptional repressor to inhibit the expression of PpDAM5 and PpDAM6 during the period of release of bud endodormancy in peach (the transition stage). In addition, PpTCP20 can interact with PpABF2 to synergistically regulate the release of endodormancy. Taken together, our findings reveal new molecular mechanisms for peach bud endodormancy.

Materials and methods

Plant material and identification of dormancy stages of flower buds

Peach trees (Prunus persica var. nectarina cv. Zhongyou 4) were grown in the Shandong Institute of Pomology in Tai’an, Shandong Province, China. To examine the dormancy stages of the flower buds, annual shoots were collected approximately every 15 d from 15 October 2017 to 30 January 2018, as described previously (Wang ). At each sampling time, 20 of the shoots were placed in tap water under 200 μmol m−2 s−1 light for 16 h at 25 °C, with a humidity of 75%. After 25 d, the percentage of flower buds that had broken dormancy was determined. If the bud break was less than 50%, the flower buds were considered to be in the endodormancy stage (Lang, 1987).

Morphological analysis of flower buds

Microscopic observation of flower bud morphology was performed by paraffin sectioning, as described previously (Ren ). A series of flower buds (30 October 2017 to 15 January 2018) was fixed in 70% FAA (formaldehyde, ethanol, acetic acid) overnight at 4 °C. The paraffin sections were then eluted with xylene and absolute ethanol, stained with a red dye solution, and decolorized with absolute ethanol. The sections were then stained with a solid green dye solution and dehydrated with absolute ethanol. Finally, the sections were placed in xylene, sealed with a neutral gum, and observed under an optical microscope (Nikon Eclipse E100).

RNA extraction and quantitative PCR

Flower buds were sampled from 15 October 2017 to 30 January 2018. Total RNA was isolated from 0.5 g of bud tissue using a RNAprep Pure Plant Kit (Tian Gen, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was generated using HiScript Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using SYBR Premix Ex Taq (Takara) on a CFX96 real-time PCR detection system (Bio-Rad). Three biological replicates were used for each analysis. The relative expression levels were calculated using the 2−ΔΔ method (Livak and Schmittgen, 2001) using the PpUBQ gene as the internal control. The data were analysed using SPSS Statistics v. 20. The qPCR primers are listed in Supplementary Table S1 at JXB online.

Measurement of ABA

The ABA content of flower buds collected from 15 October 2017 to 30 January 2018 was determined by HPLC/ electrospray ionization tandem MS (HPLC/ESI-MS/MS), as described previously by Zhao with slight modifications. Accurately weighed samples of 0.5 g of flower buds were pulverized in liquid nitrogen, and 10 ml of isopropanol/hydrochloric acid extraction buffer was added followed by shaking at 4 °C for 30 min. Then, 20 ml of dichloromethane was added, and the mixture was shaken at 4 °C for 30 min. Following centrifugation at 13 000 rpm for 5 min at 4 °C, the lower organic phase was collected, dried under nitrogen gas, and redissolved in 400 μl of methanol (0.1% formic acid). HPLC/ESI-MS/MS was then used to measure the ABA content after passing through a 0.22-μm filter, using a 1290 HPLC system (Agilent) with a 6500 Qtrap MS/MS (AB SCIEX company).

Cloning and bioinformatic analysis of PpTCP20

The full-length open-reading frame (ORF) of PpTCP20 was amplified using the flower bud cDNA (sampled on 15 October 2017) and inserted into the expression vector, using the primer sequences listed in Supplementary Table S1. The sequences of proteins homologous to PpTCP20 in different species were obtained from PlantTFDB (Jin ). The TCP genes in Arabidopsis were obtained from TAIR (http://www.arabidopsis.org/). Sequence alignment was performed using the DNAMAN software. The neighbor-joining method in MEGA6 was used to analyse the phylogenetic tree (Tamura ).

Subcellular localization of PpTCP20 and transgenic plant material

The ORF sequence of PpTCP20 was amplified with the stop codon removed, and ligated into the PRI-GFP (35S::GFP) vector for detection of subcellular localization, as described previously (Hu ). The primers are listed in Supplementary Table S1. PpTCP20-GFP and the control GFP construct were used to infect onion epidermal cells via Agrobacterium tumefaciens strain GV3101, as described previously (Chen ). After 3 d incubation, the GFP fluorescence signals in the transformed onion cells were observed using a Zeiss LSM880 microscope, and the images were analysed using the ZEN lite software (Zeiss).

Transgenic Nicotiana benthamiana was obtained via Agrobacterium tumefaciens GV3101 with the 35S::PpTCP20 construct as described by Cao , with some modifications. Briefly, leaf sections were scratched with a blade and incubated with the Agrobacterium strain for 20 mins. The sections were transferred to selective medium containing kanamycin, and identification of transgenic plants was performed by PCR and qPCR analysis. The primers are shown in Supplementary Table S1.

Yeast one-hybrid assays

The PpDAM6 promoter fragment containing three tandem repeat sequences of the site II motifs (–801 to –772 relative to the translational start site) was ligated into the pAbAi vector to generate the bait plasmid PpDAM6-pAbAi. This plasmid was inserted into the yeast strain Y1H Gold to detect the minimum concentration of aureobasidin A (AbA) that completely inhibited the growth of the bait yeast strains. The yeast one-hybrid (Y1H) screening assay was performed following the Matchmaker® Gold Yeast One-Hybrid Library Screening System (Clontech).

To determine the interaction between PpTCP20 and PpDAM6-pAbAi, the ORF of PpTCP20 was cloned into the pGADT7 vector to generate a AD-PpTCP20 fusion plasmid using the primers listed in Supplementary Table S1. The AD-PpTCP20 construct was transformed into the yeast strain Y1H Gold (containing pAbAi-PpDAM6) and incubated on a SD/–Leu plate. After 3 d, the yeast culture clones (OD600= 0.002) were spotted on SD/–Leu AbA200 and cultured at 28 ℃ for 3–5 d. The empty pGADT7 vector was used as a negative control.

Yeast two-hybrid assays

The yeast two-hybrid (Y2H) interaction was verified using the Matchmaker Yeast Two-Hybrid System (Clontech) according to the manufacturer’s manual. The ORF of PpTCP20 was recombined into the pGBKT7 vector for verification of self-activation, and the ORFs of PpTCP20 and PpABF2 were cloned into the pGADT7 vector using the primers listed in Supplementary Table S1. The two recombinant plasmids were then co-transformed into yeast strain Y2H Gold and cultured on selective medium (SD/–Trp/–Leu) at 30 °C for ~3 d. After the yeast cells had grown, the putative transformants were transferred to selective medium (SD/–Leu/–Trp/–His/–Ade) with X-α-gal.

Electrophoretic mobility shift assays

The ORF of PpTCP20 was cloned into the pGEX4T-1 vector to generate the PpTCP20-GST vector. PpTCP20-GST was transferred to Escherichia coli Transetta (DE3) for expression of the PpTCP20-GST fusion protein. The recombinant protein PpTCP20-GST was obtained and purified using a Pierce™ GST Spin Purification Kit (ThermoFisher Scientific) according to the manufacturer’s instructions.

Electrophoretic mobility shift assays (EMSAs) were performed using a LightShift™ Chemiluminescent EMSA Kit (ThermoFisher Scientific) according to the manufactuer’s instructions, as described previously (An ). The primers were used for 3′ biotin labeling (Sangon, Shanghai, China). Briefly, a biotinylated probe was incubated at 24 °C in binding buffer with or without the PpTCP20-GST fusion protein for 20 min. Unlabeled probes were used for labeled-probe competition. A mutant probe was generated in which the core motif sequence was substituted. The probe sequences are listed in Supplementary Table S2. Free and bound DNA were separated using an acrylamide gel.

Dual luciferase assays

Dual luciferase (dual-LUC) assays were performed as previous described (An ). The full-length PpTCP20 cDNA was inserted into the pGreenII 0029 62‐SK vector to generate the effector construct. The PpDAM5, PpDAM6, and PpABF2 promoter fragments were individually cloned into pGreenII 0800‐LUC vectors to generate the reporter constructs. All recombinant constructs were individually transformed into A. tumefaciens strain GV3101. Nicotiana benthamiana leaves were infected with the mixed Agrobacterium strain. Detection of fluorescence was performed using an in vivo imaging system (IVIS Lumina II, Xenogen, Alameda, CA, USA). The primers are listed in Supplementary Table S1.

Firefly luciferase complementation assays

Agrobacterium-mediated firefly luciferase complementation (LCI) assays were performed as previously described by Wang . The coding sequences of PpTCP20 and PpABF2 were fused to 35S::nLuc and 35S::cLuc, respectively. TCP20-nLuc and ABF2-cLuc were transformed into A. tumefaciens strain GV3101 and injected into N. benthamiana leaves. The leaves were then grown for 2 d in a humid environment, after which they were sprayed with 100 mM luciferin. After 6 min in the dark, the luciferase activity was monitored using a live imaging system (IVIS Lumina II, Xenogen, Alameda, CA, USA). Each assay was performed with three biological replicates.

Results

Identification of the dormancy stage and morphological changes in the flower bud

In order examine the molecular regulatory mechanism of PpDAM6, the dormancy stages of peach flower buds were first identified. From 15 October to 15 November, no bud-break was observed on the shoots. Thereafter, the buds started to break, and on 30 December the bud-break was 59% (Fig. 1A), at which time there was 1057 h of chilling accumulation (Supplementary Table S3). Zhongyou 4 requires ~700 h of chilling, and 725 h had been accumulated by 15 December. Therefore, we determined that the flower buds were in the endodormancy stage from 15 October to 15 December and in the endodormancy release period (transition stage) from 15 November to 15 December. In addition, the period 15 December 2017 to 30 January 2018 was considered as ecodormancy during which bud-burst was inhibited by the unfavorable environmental conditions (winter cold).

Fig. 1.

Determination of the dormancy stages of peach flower buds. (A) Bud-break percentage of the Zhongyou 4 variety from 15 October 2017 to 30 January 2018. Annual shoots were collected from peach trees in the field on the dates indicated and placed in water for 25 d before assessment. Shoots with <50% bud-break were considered as dormant, and the values are means of 20 shoots. (B) Morphology of flower buds over time: (a) 30 October 2017, (b) 15 November 2017, (c) 30 November 2017, (d) 15 December 2017, (e)30 December 2017, and (f) 15 January 2018). Scale bars in (c, f) are 200 μm. (C) Expression of PpDAM6 during bud dormancy. Data are means (±SD) of the three biological replicates, and expression is relative to that ofPpUBQ. Different letters indicate significant differences between means as determined by ANOVA followed by Duncan’s multiple range test (P<0.05). (This figure is available in colour at JXB online.)

We examined the morphological characteristics of the flower buds using paraffin sections. As shown in Fig. 1B, from 30 October to 15 November, the stigma grew quickly, and there was no obvious change in internal morphology during the whole endodormancy stage. The expression of PpDAM6 was determined every ~15 d from 15 October 2017 to 30 January 2018. The transcript levels began to increase on 15 October, peaked around 15 November during the endodormancy period, and then decreased markedly in the transition stage (Fig. 1C).

PpTCP20 binds to the PpDAM6 promoter

Bioinformatic analysis revealed that the site-II motifs to which the TCP transcription factor can bind (–801 to –772 relative to the translational start site) were found in the promoter of PpDAM6, (Supplementary Fig. S1). The bait fragment sequences of the PpDAM6-pAbAi vectors were used to search the TCP transcription factors in peach (Fig. 2A; Supplementary Fig. S2).

Fig. 2.

Yeast one-hybrid (Y1H) screening for binding to the site-II motif elements of PpDAM6. (A) Schematic diagram of the bait fragment. The site-II motifs of the PpDAM6 promoter fragment were used to screen interactive proteins as the bait sequence. The core sequence in the site-II motif element of the PpDAM6 promoter is highlighted in bold (GGGCCC). (B) Determination of the minimum inhibitory concentration of aureobasidin A (AbA) in the SD/–Ura medium. It was found that 200 ng ml−1 of AbA was the appropriate concentration for the bait yeast strain Y1H Gold (PpDAM6-pAbAi). (C) PpTCP20 binds to the site-II motif element of PpDAM6. Plasmid AD-PpTCP20 and the empty control pGADT7 were transferred to the bait Y1H Gold (PpDAM6-pAbAi) and selected on SD/–Leu/AbA200 agar plates. A transformant from the combination of Y1H Gold (PpDAM6-pAbAi/AD-PpTCP20) was able to grow on the medium, but the PpDAM6-pAbAi/pGADT7 combination was not able to grow and was used as a control. (This figure is available in colour at JXB online.)

The Y1H Gold (PpDAM6-pAbAi) culture was grown on SD/–Ura with different concentrations of AbA and indicated that 200 ng ml−1 was an appropriate concentration for Y1H screening (Fig. 2B). Prupe.3G308700 and three other genes were thus identified. After comparison with the TCP transcription factor in Arabidopsis, we designated Prupe.3G308700 as PpTCP20 (Supplementary Fig. S3). Next, the ORF of PpTCP20 was ligated into pGADT7 to construct AD-PpTCP20 and this was verified with the bait protein in Y1H Gold (PpDAM6-pAbAi). As shown in Fig. 2C, the combination of Y1H Gold (PpDAM6-pAbAi/AD-PpTCP20) was able to grow on SD/–Leu/AbA200, which indicated that PpTCP20 bound to PpDAM6 in yeast cells.

PpTCP20 encodes a TCP protein and is located in the nucleus

PpTCP20 was 4358 bp in length, and the clone of the coding sequence (CDS) was 933 bp (Supplementary Fig. S4A). The clone of the PpTCP20 CDS from the flower bud of Zhongyou 4 was three bases longer than the sequence, it encoded 311 amino acids (Supplementary Fig. S4B), and belonged to the class I family (Supplementary Fig. S3). The homologous sequences showed that PpTCP20 contained a conserved TCP domain–basic helix–loop–helix (bHLH) motif (Fig. 3A), which allows protein–protein interactions and DNA binding (Martín-Trillo and Cubas, 2010). The phylogenetic tree showed that PpTCP20 is closely related to Prunus mume and has the most distant relationship with Arabidopsis (Supplementary Fig. S5), with only 57.54% identity between PpTCP20 and AtTCP20.

Fig. 3.

PpTCP20 is localized in the nucleus of onion epidermal cells. (A) Sequence alignment of the PpTCP20 amino acids in Arabidopsis thaliana (AT3G27010, AtTCP20), Malus domestica (MDP0000915616, MdTCP20), Pyrus bretschneideri (Pbr041545, PbTCP20), Prunus mume (XP_008231091, PmTCP20), and Prunus persica (Prupe.3G308700, PpTCP20). (B) PpTCP20 is localized in the nucleus of onion epidermal cells. The PpTCP20-GFP vector (green fluorescent protein) was used for transient expression, and GFP was used as a blank control. The epidermal tissues were stained with DAPI prior to imaging. The experiment was repeated at least three times with similar results. The scale bars in the merged images are 50 μm. (C) Homodimerization of PpTCP20. The full-length CDS sequence of PpTCP20 was ligated into the pGBKT7 and pGADT7 vectors to generate the activation domain (AD) and binding domain (BD) fusions. The yeast two-hybrid (Y2H) Gold yeast strain was co-transformed with the bait and prey and cultured on SD/–Leu/–Trp (DDO) medium.The growth of yeast cells on SD/–Leu/–Trp/–His/–Ade/X-α-Gal (QDO/X) medium indicated an interaction. Yeast cells transformed with AD + BD-TCP20 were included as negative controls. (This figure is available in colour at JXB online.)

To identify the subcellular localization of PpTCP20, the PpTCP20-GFP fusion protein was transiently expressed in onion epidermal cells. Confocal microscopy revealed that PpTCP20-GFP was only localized in the nucleus, while the empty control vector GFP was expressed throughout the cells (Fig. 3B). This indicated that PpTCP20 is a nuclear protein, consistent with its function as a transcription factor, which is typically in the cell nucleus. The TCP protein is capable of forming homodimers or heterodimers to regulate downstream gene expression (Parapunova ). Our Y2H assays indicated that PpTCP20 had no autoactivation activity and could form homodimers (Fig. 3C).

PpTCP20 binds to the site-II motifs and inhibits expression of PpDAM6

To further demonstrate that PpTCP20 could bind to the site-II motifs, the PpTCP20-GST fusion protein was used for EMSAs. The results showed a clearly shifted band when PpTCP20-GST was incubated with labeled probes containing site-II motifs (Fig. 4B). In addition, the core sequence of the site-II motifs, GGGCCC, was replaced with AAATTT (Fig. 4A) and the shifted band disappeared, indicating that GGGCCC is essential for the binding of PpTCP20 to the site-II motifs. Because GCCCR is known to be a binding target of the PpTCP20 protein, we examined other GCCCR motifs in the promoter of PpDAM6 to which PpTCP20 might bind (Supplementary Table S2). EMSAs showed that PpTCP20 could not bind to the other three site-II motifs (Supplementary Fig. S6), indicating that PpTCP20 directly binds only to the motifs of the PpDAM6 promoter.

Fig. 4.

PpTCP20 inhibits expression of PpDAM6. (A) The probes of the nucleotide sequence to which PpTCP20 may be bound. The predicted site-II motif is underlined (CACGTG), and the mutation site (Mut) is that for which the 5´-GGGCCC-3´ motif is replaced by 5´-AAATTT-3´. (B) Electrophoretic mobility shift assay (EMSA) showing that the PpTCP20-GST fusion protein binds to the site-II motifs of the PpDAM6 promoter. The PpTCP20 fusion protein was incubated with a labeled or a mutated probe DNA fragment. The unlabeled probe fragment was used as a competitor. The free and bound probes were separated on an acrylamide gel: –, absent; +, present. (C) Effector and reporter vector construction diagrams for the dual luciferase assays. (D) PpTCP20 inhibited the expression of PpDAM6 in a transient expression assay. In PpDAM6pro(Mut)-Luc the 5´-GGGCCC-3´ motif was replaced by 5´-AAATTT-3´. The graph shows a quantitative analysis of luminescence intensity, and the value for PpTCP20-62SK + PpDAM6pro-Luc was set to 1. Data are means (±SD) of the three biological replicates. Different letters indicate significant differences between means as determined by ANOVA followed by Duncan’s multiple range test (P<0.05). (This figure is available in colour at JXB online.)

To determine whether PpTCP20 could regulate the expression of PpDAM6, dual-LUC assays were performed in tobacco leaves. Compared with the control, PpTCP20-SK co-expressed with Luc-PpDAM6pro showed significantly reduced luminescence intensity. In contrast, 35S::PpTCP20 failed to induce the Luc expression activity of Luc-PpDAM6pro (Fig. 4C, D). These results indicated that the PpTCP20 protein may inhibit the expression of PpDAM6.

PpTCP20 binds to GCCR motifs and inhibits PpDAM5 expression

PpDAM5 and PpDAM6 have been identified as candidate genes for bud endodormancy (Yamane ), and the expression of PpDAM5 was similar to that of PpDAM6. The PpDAM5 transcript levels also decreased rapidly in the transition stage (Fig. 5A). Three GCCCR motifs were found in the promoter of PpDAM5 (Supplementary Fig. S7) and EMSAs showed that PpTCP20 could bind to the promoter of PpDAM5 (Supplementary Fig. S6). When the core CCC sequence of the GCCCR motif was replaced with TTT, a clearly shifted band disappeared, indicating that PpTCP20 can also bind to PpDAM5 (Fig. 5B). Dual-LUC assays also showed that the PpTCP20 protein may inhibit the expression of PpDAM5 (Fig. 5C).

Fig. 5.

PpTCP20 inhibits the expression of PpDAM5. (A) Relative expression of PpDAM5 during peach flower bud dormancy. Data are means (±SD) of the three biological replicates, and expression is relative to that of PpUBQ. Different letters indicate significant differences between means as determined by ANOVA followed by Duncan’s multiple range test (P<0.05). (B) Electrophoretic mobility shift assay (EMSA) showing that the PpTCP20-GST fusion protein binds to the PpDAM5 promoter. The PpTCP20 fusion protein was incubated with a labeled or a mutated (Mut) probe DNA fragment. In 5mT2 the 5´-GGG-3´ motif was replaced with 5´-TTT-3´. The unlabeled probe fragment was used as a competitor. The free and bound probes were separated on an acrylamide gel: –, absent; +, present. (C) PpTCP20 inhibited the expression of PpDAM5 in a transient expression assay. In PpDAM5pro(Mut)-Luc the 5´-GGG5-3´ motif was replaced by 5´-5TTT-3´. The graph shows a quantitative analysis of luminescence intensity, and the value for 62SK + Luc was set to 1. Data are means (±SD) of the three biological replicates. Different letters indicate significant differences between means as determined by ANOVA followed by Duncan’s multiple range test (P<0.05). (This figure is available in colour at JXB online.)

PpTCP20 is involved in peach bud endodormancy

The PpTCP20 transcript levels were lowest on 15 November and then increased up to 15 December during the transition stage (Fig. 6A), which was the opposite to what was observed for PpDAM6 (Fig. 1C). This indicated that PpTCP20 may be involved in regulating peach bud endodormancy release.

Fig. 6.

PpTCP20 is involved in peach bud endodormancy. (A) Relative expression of PpTCP20 during the peach flower bud dormancy. Data are means (±SD) of the three biological replicates, and expression is relative to that of PpUBQ. Different letters indicate significant differences between means as determined by ANOVA followed by Duncan’s multiple range test (P<0.05). (B) RT-PCR and qRT-PCR analyses of three PpTCP20-overexpressing transgenic lines of tobacco. NtACTIN is the reference gene. WT, wild-type. Data are means (±SD) of three biological replicates. (C) Comparison of seed germination for WT and the three transgenic lines after 5 d on MS medium. (D) Phenotypes of the WT and transgenic lines after 65 d. The circles indicate the positions of the flowers. (This figure is available in colour at JXB online.)

Due to the difficulty in obtaining transgenic peach plants, the 35S::PpTCP20 fusion plasmid was heterologously transformed into tobacco. Three positive transgenic lines (PpTCP20-L1, PpTCP20-L2, and PpTCP20-L4) were identified (Fig. 6B). These PpTCP20‐overexpressing lines exhibited enhanced germination compared with the wild-type (Fig. 6C; Supplementary Fig. S8A). In particular, the overexpressing lines showed an early flowering phenotype (Fig. 6C; Supplementary Fig. S8B).

Changes in ABA content and PpABF2 expression during bud dormancy

ABA metabolism has been reported as being involved in dormancy release (Zheng ). The ABA content of peach flower buds was highest on 15 October, gradually decreased as endodormancy was released, and then remained at a low, constant level in the ecodormancy period (Supplementary Fig. S9). Previous studies have shown that the AREB1/ABF2 gene together with the ABA signal play an important role in the dormancy release process of grape and pear (Zheng ; Tuan ). PpABF2 was found to have the highest similarity to the ARB1/ABF2 protein in Arabidopsis (Supplementary Fig. S10), and germination is known to be significantly inhibited by ABF2 overexpression in Arabidopsis. PpABF2 has high expression during the endodormancy period in peach (Sun ) and this is recognized as key regulator for bud dormancy. The expression level of PpABF2 was the lowest on 15 November (Fig. 7A). The expression peaked on 15 December as chilling accumulation increased, indicating that PpABF2 has an essential role in bud endodormancy release. Moreover, PpABF2 had an expression pattern similar to that of PpTCP20 (Fig. 6A), indicating that they may interact with each other.

Fig. 7.

Interaction between PpTCP20 and PpABF2. (A) Relative expression of PpABF2 in peach flower buds during different dormancy stages. Data are means (±SD) of the three biological replicates, and expression is relative to that of PpUBQ. Different letters indicate significant differences between means as determined by ANOVA followed by Duncan’s multiple range test (P<0.05). (B) PpTCP20 interacts with PpABF2 in yeast two-hybrid (Y2H) assays. PpTCP20 and PpABF2 were fused to pGBKT7 and pGADT7, respectively. The Y2H-Gold yeast strain was co-transformed with the bait and prey to SD/–Leu/–Trp medium (DDO). The growth of yeast cells on SD/–Leu/–Trp/–His/–Ade/X-α-Gal medium (QDO/X) indicated an interaction. Yeast cells transformed with AD + BD-TCP20 were included as negative controls. (C) In vivo interaction of PpTCP20 and PpABF2 in tobacco leaves. The coding regions of PpTCP20 and PpABF2 were fused to pCAMBIA1300-nLUC and pCAMBIA1300-cLUC, respectively, and used to infect the leaves. Agrobacterium strains expressing nLuc and cLuc were used as negative controls. (This figure is available in colour at JXB online.)

PpTCP20 interacts with PpABF2 and directly binds to the PpABF2 promoter

To test whether PpTCP20 can interact with PpABF2, PpTCP20 and PpABF2 were fused to pGBKT7 and pGADT7 to generate BD-PpTCP20 and AD-PpABF2, respectively, for Y2H assays. The results indicated that PpTCP20 was able to interact with the PpABF2 protein (Fig. 7B). A LCI assay was used to further verify the interaction, with the ORFs of PpTCP20 and PpABF2 being fused to pCAMBIA1300-nLUC and pCAMBIA1300-cLUC, respectively. PpTCP20-nLUC and PpABF2-cLUC co-expression resulted in strong luciferase activity compared to the control (Fig. 7C).

The TCP-binding site (TBS) motif (GGTCCCAC, –800 to –792 relative to the translational start site) to which AtTCP20 can bind was also found in the promoter of PpABF2 (Supplementary Fig. S11; Wu ). EMSAs verified that PpTCP20 could bind to the GCCCR motif of PpABF2 promoter (Supplementary Fig. S12A). In addition, dual-LUC assays showed PpTCP20 inhibited the expression of PpABF2. Thus, as a result of the expression of PpTCP20 and PpABF2 (Figs 6A, 7A), PpTCP20 may inhibit the expression of PpABF2 during the deep bud endodormancy period in peach.

Discussion

PpDAM5 and PpDAM6 are negative regulators of bud endodormancy in peach

In previous studies, PpDAM5 and PpDAM6 have been found to be closely related to the required degree of cooling during bud dormancy and to flowering time in peach (Fan ; Yamane ), and the expression levels of PpDAM5 and PpDAM6 are negatively correlated with terminal bud-break (Jiménez ; Leida ). Some studies have found that down-regulation of DAM genes is accompanied by release of endodormancy in peach (Leida ), Japanese apricot (Prunus mume; Sasaki ), and pear (Niu ). Sasaki found that ectopic overexpression of apricot PmDAM6 in poplar resulted in a phenotype with growth inhibition under favorable conditions. In this study, we found that PpDAM5 and PpDAM6 were down-regulated in the peach variety ‘Zhongyou 4’ in response to low temperature during the flower-bud endodormancy release period (transition stage) (Figs 1C, 5A), which was consistent with previous studies showing that these genes have essential roles in flower bud endodormancy in peach (Li ; Yamane ; Leida ). The molecular mechanisms by which DAM genes regulate dormancy have been previously reported. For example, the DAM-like gene SVP causes inhibition of FT expression and affects flowering in Arabidopsis (Lee ). DAM1 in pear can bind to NCED3 to regulate bud dormancy (Tuan ), and the poplar transcription factor SVL is closely related to Arabidopsis SVP and can also bind to NCED3 (Singh ). Similarly, DAM can inhibit the expression of FT in leafy spurge (Hao ) and pear (Niu ) to regulate bud dormancy. Thus, we consider PpDAM5 and PpDAM6 to be negative regulators in the control of bud dormancy release in peach.

The CCCAC motif sequence is essential for PpTCP20 binding

TCPs have been identified as the DNA-binding proteins that can recognize specific motifs (Martín-Trillo and Cubas, 2010). There are 24 TCP transcription factors in Arabidopsis, divided into classes I and II (Danisman ). Arabidopsis TCP20 belongs to the class-I subfamily and can bind to site-II motifs, the core elements of which are GCCCR (R = A or G) sequences (Li ; Hervé ). Using sequence analysis, we identified the site-II motif element GGGCCCAA in the peach PpDAM6 promoter (Supplementary Fig. S1), which was similar to the Arabidopsis GGNCCCAC sequence to which AtTCP20 is targeted (Danisman ). PpTCP20 could bind to the site-II motifs of PpDAM6 (Figs 2, 4B), and EMSAs also showed that it could bind to the GCCCR motifs in the promoter of PpDAM5 (Fig. 5B). However, there were other GCCCR sequences in the promoters of PpDAM5 and PpDAM6 that had no binding affinity with PpTCP20 (Supplementary Fig. S6). A previous study has suggested that TCP20 can bind to the TBS (GGTCCCAC) motif sequence (Wu ), and we found that PpTCP20 could bind to this sequence in the promoter of PpABF2 (Supplementary Fig. S12A). Our results clearly cannot fully explain the target sequence of PpTCP20 in peach, but they suggest that the CCCAC motif is the core sequence element (Supplementary Fig. S13).

PpTCP20 regulates the release of flower bud endodormancy

Previous studies have reported that TCP20 regulates plant growth (Li ; Hervé ), nitrate foraging by roots (Guan ), and petal elongation (Wang ). It has also been shown to regulate bud activation potential and flowering in Arabidopsis (Wu ). Other studies have also found that TCP transcription factors play important roles in dormancy. TCP19 in gladiolus promotes corm dormancy release by influencing the ABA content (Wu ). TCP18/BRC1 negatively regulates axillary bud growth in Arabidopsis (Aguilar-Martínez ; Niwa ; Seale ). In addition, poplar TCP18 is a direct target gene of the SVL protein and a negative regulator of bud-break (Singh ). These studies indicate that TCP transcription factors have a function in dormancy. In our study, we found that PpTCP20‐overexpressing lines exhibited enhanced germination and showed an early flowering phenotype in tobacco compared to the wild-type (Fig. 6C, D), and a previous study had indicated that seed germination involves the breaking of dormancy (Koo ). Similarly, Arabidopsis TCP20 is involved in growth and cell division and contributes to the control of cell expansion (Hervé ). In particular, expression of PpTCP20 increased during the transition stage in peach (Fig. 6A). These results confirmed that PpTCP20 may play a key role in peach bud endodormancy.

TCP proteins can act as transcriptional activators or repressors in plants, depending on the promoter sequence and the regulatory partner (Hervé ). In Arabidopsis, TCP20 binds to the LOX2 promoter, thereby inhibiting its expression (Danisman ). TCPs can also regulate the expression of the MADS-domain transcription factors SEPALLATA3 and APETALA1 (Danisman ). Here, dual luciferase assays indicated that PpTCP20 inhibited the expression of PpDAM5 and PpDAM6 (Figs 4D, 5C). Previous studies have suggested that CBFs play important roles in regulating dormancy. The ectopic expression of peach CBF (PpCBF1) in apples alters the expression of DAMs and other dormancy-related genes and inhibits bud-break (Wisniewski ), and the CBF transcription factor is the upstream gene that activates the transcription of DAM1 in pear by binding the C-repeat/DRE element motif (Saito ; Niu ). CBF can bind to DAM in poplar, pear, and Chinese pear to regulate bud dormancy (Horvath, 2009; Saito ; Niu ). Our results indicate another mechanism for PpTCP20 to regulate PpDAM genes. Similar to the bHLH protein, the TCP transcription factor can bind to DNA to form homo- and heterodimers (Manassero ), and we confirmed that PpTCP20 is capable of forming homodimers (Fig. 4C), thus inhibiting the expression of PpDAM5 and PpDAM6 to regulate flower bud endodormancy.

ABA and the ABA-signaling gene PpABF2 are involved in bud endodormancy

ABA is generally considered to be a key hormone in regulating bud dormancy (Cooke ; Wang ). It accumulates during seed development and the content decreases with the release of dormancy (Kermode, 2005). In grape, the ABA concentration decreases during the release of bud endodormancy, demonstrating that it inhibits bud-break (Zheng ). In hybrid aspen under low temperature conditions, ABA levels decrease and inhibit expression of SVL, thereby promoting bud-break (Singh ). In our study, the ABA content gradually decreased during the process of peach flower bud endodormancy (15 November to 15 December), and reached a minimum level during the ecodormancy stage (Supplementary Fig. S9), which was similar to the findings of a previous study in peach (Wang ). These results provide evidence that the ABA content plays a key role in the release of endodormancy in peach.

ABA signaling genes, such as ABFs, are known to play a role in bud endodormancy (Sun ). In pear, AREB1/ABF2 binds to DAM1 and negatively regulates its activity during bud endodormancy (Tuan ). We found that ABF2 showed high expression during the transition stage (Fig. 7), which indicated that ABF2 played an important role in bud endodormancy. The ABF protein can form a complex with other transcription factors and regulate the expression of downstream genes. For example, SnRK2s can phosphorylate AREB/ABFs to participate in ABA signaling in plants (Wang ), and the up-regulation of ABF2 that we observed was consistent with endodormancy release in pear (Tuan ). The formation of a complex of AREB1(ABF2) and NAC2 in peanut regulates the downstream gene NCED1 (Liu ). AREB1 in pear may also directly or indirectly regulate DAM1 expression in combination with the NAC transcription factor (Tuan ). Similarly, we found that ABF2 could interact with the TCP20 protein (Fig. 7B, C), which indicated that PpABF2 and PpTCP20 synergistic regulated bud endodormancy in peach. The conformation of transcription factor binding sites has been shown to be affected by combinatorial regulation in yeast (Bilu and Barkai, 2005). Similarly, there is a need to understand the diversity of TCP binding sites and the combined regulation of TCP proteins and their chaperones (Hervé ). However, as we were unable to obtain an PpABF2 fusion protein in peach, further studies are needed in order to determine whether the heterodimer between PpTCP20 and PpABF2 regulates the expression of PpDAM5 and PpDAM6. The PpTCP20 transcription factor inhibited the transcription of PpABF2 by binding the TBS motif (Supplementary Fig. S12). As a result of the expression of PpTCP20 and PpABF2 (Figs 6A, 7A), we propose that PpTCP20 inhibited PpABF2 in peach during the deep endodormancy stage (from 15 October to 30 October). Our results show that PpTCP20 may have different functions during different stages of peach endodormancy.

In summary, our study demonstrates that PpTCP20 plays an important role in flower bud endodormancy in peach by inhibiting the expression of PpDAM5 and PpDAM6. Specifically, PpTCP20 can also interact with PpABF2 to synergistically regulate the release of bud endodormancy (Fig. 8). Our findings characterize the molecular mechanisms relating to PpTCP20 in peach and provide new insights into bud dormancy in perennial deciduous fruit trees.

Fig. 8.

A proposed model of the role of PpTCP20 in peach bud endodormancy. Long-term chilling accumulation in winter directly activates the accumulation of PpTCP20 and PpABF2. PpTCP20 inhibits expression of PpDAM5 and PpDAM6 to release endodormancy. In addition, PpTCP20 interacts with PpABF2 to form heterodimers to jointly regulate the release of bud endodormancy. During the deep endodormancy period, PpTCP20 inhibits PpABF2 expression, which might enhance the endodormancy. Induction of targets is represented by solid arrows and inhibition is represented by blocked lines. The dashed arrow represents potential induction. (This figure is available in colour at JXB online.)

Supplementary data

Supplementary data are available at JXB online.

Fig. S1. The promoter sequence of PpDAM6.

Fig. S2. The PpDAM6-pAbAi bait sequence for the Y1H assay.

Fig. S3. Phylogenetic analyses of PpTCP20 and Arabidopsis TCP proteins.

Fig. S4. The sequence and gene structure of PpTCP20.

Fig. S5. Phylogenetic analysis of TCP20 proteins from different species.

Fig. S6. EMSA showing that PpTCP20 can bind to the PpDAM5 promoter.

Fig. S7. The promoter sequence of PpDAM5.

Fig. S8. Phenotypes of transgenic Nicotiana benthamiana expressing PpTCP20.

Fig. S9. The ABA content of peach flower buds during dormancy.

Fig. S10. Phylogenetic analyses of PpABF2 and AtAREB1.

Fig. S11. The promoter sequence of PpABF2.

Fig. S12. EMSA and transient expression assays showing that PpTCP20 inhibits expression of PpABF2.

Fig. S13. PpTCP20 may bind to the CCCAC motif sequence in peach.

Table S1. Primers used for amplification.

Table S2. Sequences of labeled probes used for EMSAs.

Table S3. Daily temperature data from 15 October to 30 December 2017.

Click here for additional data file.