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. 2022 Aug 10;13:971482. doi: 10.3389/fpls.2022.971482

PpMYB52 negatively regulates peach bud break through the gibberellin pathway and through interactions with PpMIEL1

Yuzheng Zhang 1,2,3, Qiuping Tan 1,2,3, Ning Wang 1,2,3, Xiangguang Meng 1,2,3, Huajie He 1,2,3, Binbin Wen 1,2,3, Wei Xiao 1,2,3, Xiude Chen 1,2,3, Dongmei Li 1,2,3, Xiling Fu 1,2,3,*, Ling Li 1,2,3,*
PMCID: PMC9413399  PMID: 36035719

Abstract

Bud dormancy, which enables damage from cold temperatures to be avoided during winter and early spring, is an important adaptive mechanism of deciduous fruit trees to cope with seasonal environmental changes and temperate climates. Understanding the regulatory mechanism of bud break in fruit trees is highly important for the artificial control of bud break and the prevention of spring frost damage. However, the molecular mechanism underlying the involvement of MYB TFs during the bud break of peach is still unclear. In this study, we isolated and identified the PpMYB52 (Prupe.5G240000.1) gene from peach; this gene is downregulated in the process of bud break, upregulated in response to ABA and downregulated in response to GA. Overexpression of PpMYB52 suppresses the germination of transgenic tomato seeds. In addition, Y2H, Bimolecular fluorescence complementation (BiFC) assays verified that PpMYB52 interacts with a RING-type E3 ubiquitin ligase, PpMIEL1, which is upregulated during bud break may positively regulate peach bud break by ubiquitination-mediated degradation of PpMYB52. Our findings are the first to characterize the molecular mechanisms underlying the involvement of MYB TFs in peach bud break, increasing awareness of dormancy-related molecules to avoid bud damage in perennial deciduous fruit trees.

Keywords: peach, bud break, PpMYB52, PpMIEL1, ABA, GA

Introduction

Bud dormancy, which enables buds to avoid damage from cold temperatures during winter and early spring, is an important adaptive mechanism of deciduous fruit trees to cope with seasonal environmental changes and temperate climates (Yamane, 2014; Yordanov et al., 2014). The stages of bud dormancy is divided into paradormancy, endodormancy, and ecodormancy (Lang et al., 1987). Endodormancy is a complex mechanism regulated by multiple internal and external physiological factors and cannot released until certain chilling requirements are met (Singh et al., 2018). Ecodormancy is a growth stagnation caused by natural environmental, such as low temperature and drought (Horvath et al., 2003). With increasing global warming, fruit trees have been budding and blossoming earlier (Atauri et al., 2010). Trees that have broken bud are more vulnerable to late spring frost damage than trees yet to break bud (Jones and Cregg, 2006). Delaying bud break is one way to avoid spring frost damage (Chayani et al., 2015). Therefore, understanding the regulatory mechanism of bud break in fruit trees is highly important for the artificial control of bud break and the prevention of spring frost damage.

Bud dormancy is regulated by temperature, plant hormones, genes and other factors. ABA is generally considered to be a key hormone involved in dormancy induction and maintenance (Horvath et al., 2008; Gillespie and Volaire, 2017). And GA is considered to be a key hormone regulating bud dormancy release. The dormancy-related MADS-Box (DAM) Genes are Identified to be key factors controlling dormancy and dormancy release in many species, including leafy spurge (Horvath et al., 2010), pear (Niu et al., 2016), apple (Mimida et al., 2015), and peach (Hisayo et al., 2011). In addition to DAM, EARLY BUD BREAK 1 (PpEBB1) can promote bud break by regulating hormone metabolism, the cell cycle, and cell wall modifications (Zhao X. et al., 2020). PpTCP20 can regulates peach flower bud endodormancy by negatively regulating the expression of PpDAM5 and PpDAM6, and by interacting with PpABF2 (Wang et al., 2020). Moreover, Gibberellin receptor GID1 gene might play a role in dormancy release in peach vegetative bud (Hollender et al., 2016).

MYBs constitute the largest transcription factor (TF) family in plants, and MYB TFs contain a highly conserved DNA-binding domain, the MYB domain, which typically contains one to four incomplete amino acid sequence repeats (R). Each R sequence consists of approximately 52 amino acids, forming three helices (Dubos et al., 2010). In addition, there are regularly spaced tryptophan (W) residues in each R sequence; these residues forms a hydrophobic core with a helix-turn-helix (HTH) structure between the second and third helices (Kanei-Ishii et al., 1990; Dubos et al., 2010).

The MYB TF family comprises proteins with a wide range of functions involved in the regulation of almost all biological processes in plants (Stracke et al., 2001; Dubos et al., 2010). A large number of studies have shown that plant MYB TFs are widely involved in the response to abiotic stress, such as drought (Huang et al., 2018; Alexander et al., 2019; Fang et al., 2019), heat (Li J. et al., 2021; Wu et al., 2021), cold (Gong et al., 2018), and salt (Zhao K. et al., 2020), and the regulation of the biosynthesis of various secondary metabolites, such as glucosinolates (Dubos et al., 2010), flavonoids (Kortstee et al., 2011; Anwar et al., 2019; Li Y. et al., 2020) and terpenoids (Matías-Hernández et al., 2017). In addition, MYB TFs are involved in the regulation of plant cell morphology and pattern developing (Lai et al., 2005; Guimil and Dunand, 2006) and the regulation of multiple growth and development processes (Byrne et al., 2000; Wang et al., 2009; Zhuang et al., 2021). In peach, the synthesis of anthocyanins and proanthocyanidins and the formation of trichomes on the surface of fruit are regulated by MYB TFs (Vendramin et al., 2014; Zhou et al., 2015; Rahim et al., 2019). In recent years, MYB TFs have been found to play an important role in regulating seed dormancy and germination. In Arabidopsis, AtMYB96 inhibits seed germination by regulating the expression of abscisic acid (ABA)-insensitive 4 (ABI4) (Lee and Seo, 2015). AtMYB7 can interact with AtbZIP60 to coregulate seed germination (Xian et al., 2016). Moreover, the MYB TF family protein RSM1 can interact with HY5/HYH to regulate seed germination (Yang et al., 2018). In wheat, dormancy release was shown to occur earlier after the TaMyB10-A1 gene was mutated (Mares and Himi, 2021). In maize, ZmMYB59 plays a negative regulatory role in germination (Zhai et al., 2020). Studying tomato, Xu et al. (2018) found that overexpression of SlMYB102 can improve the seed germination rate. In Paeonia suffruticosa, PsMYB1 functions in response to low temperature to regulate bud dormancy release and germination (Zhang et al., 2015). However, there are few reports on MYB TFs regulating bud break in perennial woody plants, and the molecular mechanism underlying the involvement of MYB TFs in bud break remains unclear. In this study, we found that PpMYB52, which encodes a MYB TF in peach, was downregulated in the process of bud break, which may negatively regulate peach bud break.

To further explore the molecular mechanism of PpMYB52 in peach bud break, a PpMYB52-interacting protein was identified from the peach dormancy-associated SSHcDNA library via a yeast two-hybrid (Y2H) assay, namely, RING-type E3 ligase MYB30-ligase 1 (PpMIEL1), a RING-type E3 ubiquitin ligase, which plays an important role in ubiquitin-mediated degradation of target proteins (Chen et al., 2021). RING-type E3 ubiquitin ligases compose a class of E3 ligases containing a RING-finger domain, and RING-type E3 ligases play an important role in abscisic acid signal transduction (Stone et al., 2006; Zhang et al., 2007); anthocyanin biosynthesis (An et al., 2017a,2020); and the response to abiotic stress, such as cold (An et al., 2020, 2021), drought (Bae et al., 2011) and salt (Du et al., 2021). In Arabidopsis, the ubiquitin ligase AtMIEL1 mediates the degradation of AtMYB30 and weakens plant defense (Marino et al., 2013). In addition, AtMIEL1 also interacts with AtMYB96 in Arabidopsis stems, and AtMIEL1 can ubiquitinate AtMYB96 and negatively regulate ABA sensitivity and cuticle wax biosynthesis (Lee and Seo, 2016; Lee et al., 2017). In apple, MdMIEL1 can negatively regulate anthocyanin accumulation through ubiquitination-mediated degradation of MdMYB1 proteins (An et al., 2017a) and interact with MdMYB308L to promote the ubiquitination-based degradation of MdMYB308L, which negatively regulates cold resistance and anthocyanin accumulation (An et al., 2020). However, there are few reports about MIEL1 in peach, and the molecular mechanism of PpMIEL1 in bud break is still unclear.

In this study, we found that peach PpMYB52, which is upregulated by ABA and downregulated by gibberellins (GAs), negatively regulates peach bud break. In addition, we identified a RING-type E3 ubiquitin ligase, PpMIEL1, which interacts with PpMYB52, and its expression was continuously upregulated during bud break. We hypothesized that PpMIEL1 may positively regulate peach bud break by the ubiquitination and degradation of PpMYB52.

Materials and methods

Plant materials and treatments

Peach (Prunus persica var. nectarina cv. Zhongyou 4) trees were grown at the Shandong Agricultural University Horticultural Experiment Station in Tai’an, Shandong Province. Flower bud samples were collected approximately every 15 days from 15 October 2021 to 15 March 2022.Phloem samples were taken from the middle and lower part of annual branches. Two ring cuts of the branches with an interval of 5cm and two longitudinal cuts on both sides of the branches were cut with a knife. All cuts were down to the xylem. The phloem was gently raised with the knife edge and then quickly placed in liquid nitrogen.

Eighteen annual shoots with a length of 40–50 cm were removed from Zhongyou 4 described above, and were divided into three groups, namely, those treated with deionized water + 0.5% Triton 100, 1 mM GA3 + 0.5% Triton 100 and 1 mM ABA + 0.5% Triton 100, ABA and GA3 were dissolved in 8 mL ethanol, thendeionized water and 2.5 ml Triton 100 were added to 500 ml, the same volume of ethanol and Triton 100 were added to the thendeionized water, and the solution was evenly sprayed onto the branches, on 30 January. The shoots of each treatment group were placed in tap water under 200 μmol m–2 s–1 light for 16 h at 25°C and 8 h of darkness at 23°C and were then collected at 0, 1, 5, 9, and 14 days after treatment. All the flower buds were removed, immediately placed in liquid nitrogen and then stored at -80°C for subsequent experiments.

To determine the flower bud break rate of cultivars throughout the dormancy period, at each sampling time, 25 shoots were placed in tap water under 200 μmol m–2 s–1 light for 16 h at 25°C and 8 h of darkness at 23°C with a relative humidity of 75%. After 25 days, 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).

RNA isolation and quantitative PCR

Total RNA was extracted from 0.1 g of peach bud tissue and tomato leaves using an RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was then generated using HiScript qRT SuperMix for qPCR (+ gDNA-wiper) (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Quantitative PCR (qPCR) was performed on a CFX96 real-time PCR detection system (Bio-Rad) together with SYBR premix Ex Taq (Takara). Three biological replicates were included for each analysis. The relative expression levels were calculated using the 2–Δ Δ CT method (Livak and Schmittgen, 2001), with the PpUBQ gene used as an internal control in peach and the SlActin gene used as an internal control in tomato. The primers used are shown in Supplementary Table 1. The data were analyzed using SPSS Statistics v20 to analyze the significance of the differences among data, with a significance level of p < 0.05 under Duncan’s test.

Vector construction and genetic transformation

The full-length open reading frames (ORFs) of PpMYB52 and PpMIEl1 were amplified using flower bud cDNA via 2 × Phanta Max Master Mix (P515, Vazyme) according to the manufacturer’s instructions. The primers used were designed using CE Design v1.04 (Vazyme) and are shown in Supplementary Table 1. The vectors were constructed using a ClonExpress Ultra One Step Cloning Kit (C115, Vazyme) according to the manufacturer’s instructions. Full-length PpMYB52 was inserted into the pRI101 vector under the control of the CaMV35S promoter. Subsequently, the obtained 35S:PpMYB52 vector was transformed into Agrobacterium tumefaciens strain GV3101 according to the freeze–thaw method, which was then used to infect Micro Tom tomato explants (Jian et al., 2019). DNA was extracted for PCR for transgene detection. Three independent homozygous T3 transgenic lines (OE-1, OE-2, and OE-3) were selected for subsequent experiments.

Yeast two-hybrid assays

A peach dormancy-associated SSHcDNA library was constructed from peach buds collected from dormancy until bud break. The full-length ORF, 1–300 bp ORF and 301–777 bp ORF of PpMYB52 were inserted into a pGBKT7 bait vector for verification of self-activation, the 1–300 bp region of PpMYB52 was used to screen interacting genes whose products contain an Myb_DNA-bind_6 domain, as the full-length PpMYB52 protein had self-activation activity (Figure 1A and Supplementary Figure 1). And the full-length ORF of PpMIEl1 was cloned into a pGADT7 vector via the primers listed in Supplementary Table 1. The two recombinant plasmids were cotransformed into yeast Y2H Gold strains, and the transformants were cultured on selective media (SD/-Trp/-Leu) at 30°C for ∼3 days. After the yeast cells had grown, the putative transformants (OD600 = 0.002) were transferred to selective media (SD/-Ade/-His/-Leu/-Trp/x-α-Gal).

FIGURE 1.

FIGURE 1

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PpMYB52 interaction with PpMIEL1 and the expression pattern of PpMIEL1. (A) Analysis results of conserved domains according to the NCBI. (B) PpMYB52 interacts with PpMIEL1 in yeast. Because of the autoactivation of the full-length PpMYB52 protein, we used a 1–300 bp region to perform the experiment. Yeast cells transformed with BD-PpMYB52 (1–300 bp) + AD were included as negative controls. (C) PpMYB52 and PpMIEL1 BiFC verification. (D) Expression level of PpMIEL1 during the various stages of dormancy and bud break from 15 October 2021 to 15 March 2022. (E) Expression of PpMIEL1 in different tissues of Zhongyou 4 peach.

Bimolecular fluorescence complementation assays

The full-length ORF of PpMYB52 was cloned into a pSPYNE vector, yielding a PpMYB52-NYFP plasmid, and the full-length ORFs of PpMIEl1 were inserted into a pSPYCE vector, yielding a PpMIEl1-CYFP plasmid. All the recombinant plasmids were individually transformed into A. tumefaciens strain GV3101. Equal concentrations of A. tumefaciens strain GV3101 containing the plasmids of interest were transiently coexpressed in onion epidermal cells. After incubation at 25°C in the dark for 48 h, fluorescent and differential interference contrast (DIC) images were observed with a laser-scanning confocal microscope (Zeiss LSM880), and the images were analyzed using Zen Lite software (Zeiss) and Adobe Photoshop 7.0. The yellow fluorescent protein (YFP) was visualized by excitation with an argon laser at 514 nm.

Subcellular localization of PpMYB52

The ORF sequence of PpMYB52 without the stop codon was amplified and ligated into a pRI101-GFP (35S:GFP) vector for detection of subcellular localization (Hu et al., 2016). The primers used are listed in Supplementary Table 1. PpMYB52-GFP and a control GFP construct were infiltrated into 4-week-old tobacco (Nicotiana benthamiana) leaves via A. tumefaciens strain GV3101. After 3 days of incubation, the GFP fluorescence signals in the transformed onion cells were observed using a Zeiss LSM880 microscope, images were collected, and the images were analyzed using Zen Lite software (Zeiss).

Germination of transgenic tomato seeds

Seeds from three homozygous T3 transgenic tomato lines overexpressing PpMYB52 were surface disinfected with 75% ethanol for 60 s followed by 50% sodium hypochlorite for 15 min and then washed with sterile water five times (5 min each time). Finally, the seeds were cultured on Murashige and Skoog (MS) solid media, and the germination was recorded every 12 h.

Results

The transcript level of PpMYB52 is negatively correlated with peach flower bud break

To evaluated the relative expression of PpMYB52 from dormancy to bud break, we first identified the dormancy stages of Zhongyou 4 flower buds. The endodormancy stage lasted from 15 October to 30 December, and the flower buds broke in the field a few days after sampling on March 15 (Figure 2B). The expression of PpMYB52 was maintained at a high level in the endodormancy stage, continued to decrease in the process of bud break (the period from endodormancy to bud break, which is also known as ecodormancy), and decreased to the lowest level before bud break (Figure 2B). And the temperature during the whole dormancy process is shown in Figure 2A. Tissue-specific analysis showed that the PpMYB52 gene was highly expressed in peach buds during endodormancy but was expressed at low levels in the flower buds before bud break, and in the flowers, petals, leaves and phloem (Figure 2C). Subcellular localization revealed that the PpMYB52 gene was localized in the nucleus (Figure 2D).

FIGURE 2.

FIGURE 2

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Definition of the dormancy stages of Zhongyou 4 flower buds and expression patterns of PpMYB52. (A) Daily maximum temperature and daily minimum temperature from October 2021 to March 2022. (B) Definition of the dormancy stages of Zhongyou 4 flower buds and expression level of PpMYB52 from 15 October 2021 to 15 March 2022. At each sampling time, 25 of the shoots were placed in tap water for 25 days. If less than 50% of the buds broke dormancy, the flower buds were considered to be in the endodormancy stage; the values are the means of 25 shoots. (C) Expression of PpMYB52 in different tissues of Zhongyou 4 peach. (D) Subcellular localization of PpMYB52. PpMYB52 was fused to YFP for transient transformation into 4-week-old tobacco (N. benthamiana) leaves. 4’,6-Diamidino-2-phenylindole (DAPI) was used as a nuclear marker. The values represent the means ± SD of three replicates, and the different letters above the bars represent significant differences; P < 0.05.

PpMYB52 transcription is regulated by gibberellin and abscisic acid

GA and ABA have been recognized as key internal factors of dormancy and bud break. To determine the response of PpMYB52 to GA and ABA, we treated the shoots of Zhongyou 4 with 1 mM GA3 and 1 mM ABA, and deionized water was used as a blank control. The results showed that, compared with that in peach flower buds treated with deionized water, the expression of PpMYB52 in peach flower buds treated with 1 mM GA3 was downregulated (Figure 3A). In contrast, the expression of PpMYB52 in peach flower buds treated with 1 mM ABA was upregulated (Figure 3B), and the upregulation was more obvious with the extension of treatment time.

FIGURE 3.

FIGURE 3

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Expression of PpMYB52 in the flower buds of Zhongyou 4 under GA3 and ABA pretreatments. (A) Expression of PpMYB52 in the flower buds of Zhongyou 4 under 1 mM GA3 + 0.5% Triton 100 treatment. (B) Expression of PpMYB52 in the flower buds of Zhongyou 4 under 1 mM ABA + 0.5% Triton 100 treatment. Deionized water + 0.5% Triton 100 was used as a blank control (CK). The values represent the means ± SD of three replicates, and the different letters above the bars represent significant differences; P < 0.05.

Overexpression of PpMYB52 suppresses germination and vegetative growth of transgenic tomato

To elucidate the function of PpMYB52 in bud break, a 35S:PpMYB52 fusion plasmid was heterologously transformed into tomato; tomato was used instead of peach because transgenic peach plants are difficult to obtain. We obtained three independent transgenic lines (OE-1, OE-2, and OE-3), which were identified via PCR and qRT–PCR (Figures 4A,B). The plant height of PpMYB52 overexpression transgenic tomato lines was lower than that of the wild-type plants (Figures 4C,D). The flowering time of PpMYB52 overexpression transgenic tomato lines was longer than that of the wild-type plants (Figures 4C,G). To determine the function of PpMYB52 in bud break, the seeds of the three homozygous T3 PpMYB52-overexpressing transgenic line and Micro Tom wild-type tomato plants were sown onto MS media, and the germination was observed. The results showed that it took a longer time for the transgenic lines to achieve the same germination as that of the wild-type tomato and the transgenic lines took longer to reach 50% germination than the wild type tomato (Figures 4E,F), which indicated that the overexpression of PpMYB52 inhibited the germination of transgenic tomato seeds.

FIGURE 4.

FIGURE 4

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Overexpression of PpMYB52 in tomato suppresses seed germination and reduces plant height. (A) Reverse transcription polymerase chain reaction (RT-PCR) analysis results of PpMYB52-overexpressing transgenic lines. (B) qRT-PCR analysis results of PpMYB52 transgenic tomato lines under normal conditions. SlActin was used as a reference gene. (C) Phenotypes of PpMYB52-overexpressing transgenic tomato and Micro Tom wild-type tomato. (D) Plant height of PpMYB52-overexpressing transgenic tomato and Micro Tom wild-type tomato. Each group comprised 20 plants, and the results are the averages of 20 plants. (E,F) Determination of the germination of seeds of Micro Tom wild-type tomato and PpMYB52-overexpressing transgenic tomato in MS media. (G) Flowering time of PpMYB52-overexpressing transgenic tomato and Micro Tom wild-type tomato. The time is the number of days between planting and the first flower opening. Each group comprised 20 plants, and the results are the averages of 20 plants. The values represent the means ± SD of three replicates, and the different letters above the bars represent significant differences; P < 0.05.

Overexpression of PpMYB52 inhibits the expression of key genes involved in gibberellin synthesis and promote the expression of key genes involved in gibberellin deactivated of transgenic tomato

GAs are generally considered to be key hormones involved in regulating bud break. To determine the effect of PpMYB52 on GA synthesis, we measured the expression of genes encoding key enzymes in GA synthesis in both transgenic lines and wild-type plants. The results showed that the expression of GA biosynthesis genes copalyl diphosphate synthase (SLCPS), ent-kaurene synthase (SLKS), ent-kaurenoic acid oxidase1 (SLKAO1), ent-kaurenoic acid oxidase2 (SLKAO2) and gibberellin 20-oxidase-1(SLGA20ox1) in the PpMYB52-overexpressing transgenic tomato were lower than that in Micro Tom wild-type tomato (Figures 5A–D,F). The expression of GA deactivated genes gibberellin 2 -oxidase-1 (SLGA2ox1) and gibberellin 2 -oxidase-2(SLGA2ox2) in the PpMYB52-overexpressing transgenic tomato were higher than that in Micro Tom wild-type tomato (Figures 5K,L). However, the expression of SLKO, SLGA20ox2, SLGA20ox3, SLGA20ox4, SLGA3ox1, SLGA2ox4, and SLGA2ox5 showed no significant difference between transgentic tomato and wild-type tomato (Figures 5E,G–J,M,N). Taken together, these results suggest that overexpression of PpMYB52 inhibits GA synthesis and promote GA deactivation.

FIGURE 5.

FIGURE 5

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Expression of GA biosynthesis genes and GA deactivated genes in PpMYB52-overexpressing transgenic tomato and Micro Tom wild-type tomato. (A) Expression of SLCPS. (B) Expression of SLKS. (C) Expression of SLKAO1. (D) Expression of SLKAO2. (E) Expression of SLKO. (F) Expression of SLGA20ox1. (G) Expression of SLGA20ox2. (H) Expression of SLGA20ox3. (I) Expression of SLGA20ox4. (J) Expression of SLGA3ox1. (K) Expression of SLGA2ox1. (L) Expression of SLGA2ox2. (M) Expression of SLGA2ox4. (N) Expression of SLGA2ox5. The values represent the means ± SD of three replicates, and the different letters above the bars represent significant differences; P < 0.05.

PpMYB52 interacts with PpMIEL1

To further explore the regulatory mechanism of PpMYB52, a number of proteins that may interact with PpMYB52 were screened from the peach dormancy-associated SSHcDNA library (Supplementary Table 2). Five genes (Supplementary Table 2) were screened and subsequently tested for their ability to interact with PpMYB52. The full-length cDNA of these genes was inserted into a pGADT7 vector as prey, and BD-PpMYB52 (1–300 bp) was used for transformation into yeast receptor cells in pairs for point-to-point verification. The RING-type E3 ligase MYB30-INTERACTING E3 LIGASE 1 (PpMIEL1; Prupe.1G141000) gene was found to interact with the Myb_DNA-bind_6 domain (1–300 bp) of PpMYB52 (Figure 1B), which might function in ubiquitination-mediated degradation of target proteins. We performed a BiFC assay to confirm the interaction between PpMYB52 and the PpMIEL1 protein in vivo. PpMYB52 was fused to the N-terminus of enhanced YFP (NYFP), and PpMIEL1 was fused to the C-terminus of enhanced YFP (CYFP). These constructs were subsequently transformed into onion epidermal cells and expressed transiently. Nuclear fluorescence was detected when PpMYB52 was coexpressed with PpMIEL1, but in the control experiments, no YFP fluorescence was detected. Taken together, these results indicate that PpMYB52 interacts with the PpMIEL1 protein in vivo (Figure 1C).

PpMIEL1 may positively regulate peach flower bud break

The expression of PpMIEL1 was maintained at a low level in the endodormancy stage, increased in the process of bud break, and decreased to the highest level before bud break (Figure 1D), which contrasts with the expression of PpMYB52 (Figure 2B). Tissue-specific analysis showed that the PpMIEL gene was highly expressed in the flower petals, stamens, and pistils and was expressed the lowest in the peach buds during endodormancy (Figure 1E), which is quite different from the results of PpMYB52. Overall, these results showed that PpMIEL1 may positively regulate peach flower bud break.

Discussion

PpMYB52 is a negative regulator of bud break in peach

In previous studies, MYB TFs have been found to be closely related to seed germination. Specifically, AtMYB44, AtMYB96, and ZmMYB59 play a negative regulatory role in seed germination (Nguyen et al., 2012; Lee and Seo, 2015; Zhai et al., 2020). In this study, we found that the expression of PpMYB52 was maintained at a high level in the endodormancy stage, continued decrease throughout bud break, and decreased to the lowest level before bud break (Figure 2B). Further research showed that the overexpression of PpMYB52 inhibited the germination of transgenic tomato seeds (Figures 4E,F). These results suggest that PpMYB52 is a negative regulator of bud break in peach.

The role of GA in promoting dormancy release and bud break has been widely studied (Gillespie and Volaire, 2017). GA3 treatment has been shown to be a good method for breaking bud dormancy in several tree species (Zhuang et al., 2015). In poplar, the GA2ox4 gene, which is downregulated during temporal events that lead to poplar bud break, is involved in GA catabolism (Gómez-Soto et al., 2021). In Salix pentandra, long-day induced bud break is associated with transiently increased levels of GA1 (Olsen et al., 1997). In Japanese apricot, exogenous GA4 promotes flower bud break (Zhuang et al., 2015). In peach, GA3, GA4, and GA5 promote dormancy release (Li S. et al., 2021). In chrysanthemum, Zhu et al. (2020) found that CmMYB2 interacts with CmBBX24 to regulate flowering by influencing GA synthesis. In this study, overexpression of PpMYB52 inhibited the expression of SLCPS, SLKS, SLKAO1, SLKAO2, and SLGA20ox1 in tomato (Figures 5A–D,F) and promote the expression of SLGA2ox1 and SLGA2ox2 (Figures 5K,L). CPS, KS, KAO and GA20ox are key enzymes in the biosynthesis of GAs, and GA2ox is key enzyme in the deactivation of GAs (Hayashi et al., 2006; Yamaguchi, 2006; Huang et al., 2012). In addition, the height of the PpMYB52-overexpressing transgenic tomato lines was lower than that of the wild type (Figures 4C,D), which might mean that the PpMYB52-overexpressing transgenic tomato lines had lower GA contents. These results suggest that overexpression of PpMYB52 might inhibit the germination of transgenic tomato seeds by inhibiting GA synthesis and promote GA deactivation.

Gibberellin and abscisic acid regulate the expression of PpMYB52

Numerous studies have shown that plant hormones, especially ABA and GAs, play important roles in regulating dormancy and bud break (Horvath et al., 2008; Aksenova et al., 2013; Gillespie and Volaire, 2017). The role of GA in regulating dormancy release and bud break is described above. ABA is generally considered to be a key hormone involved in regulating bud dormancy (Cooke et al., 2012; Vergara et al., 2017). Moreover, ABA can delay the germination of seeds until more suitable growing conditions occur, increasing survival (Richardson et al., 2019). Continued in situ ABA biosynthesis is required for the maintenance of bud dormancy (Le Bris et al., 1999).

MYB TFs have been found to respond to plant hormone signals and are widely involved in hormone-regulated plant growth and development (Jaradat et al., 2013; Yin et al., 2020). In rice, the OsGAmyb gene was shown to be induced in response to GA signaling and participates in seed germination (Gubler et al., 1997). In addition, by interacting with SLENDER RICE 1 (SLR1), a DELLA repressor of GA signaling, OsMYB103L is involved in GA-mediated regulation of the cellulose synthesis pathway (Ye et al., 2015). In Lolium temulentum, LtGAMYB is upregulated in response to GA3 in the seed and participates in the flowering process (Gocal et al., 1999). In this study, we found that PpMYB52 can also respond to GA signaling and that exogenous GA3 treatment can inhibit the expression of PpMYB52 (Figure 3A). In addition to GA signaling, many studies have found that MYB TFs can also respond to ABA signaling. In Arabidopsis, AtMYB30 and AtMYB52 are involved in the ABA response (Park et al., 2011; Zheng et al., 2012). In grapevine, the expression of VvMYB60 increases in response to ABA (Galbiati et al., 2011). In this study, we found that PpMYB52 can also respond to ABA signaling and that exogenous ABA treatment can promote the expression of PpMYB52 (Figure 3B). Endogenous GA levels increase during the onset of bud break, and the ABA concentration decreases with dormancy progression (Gillespie and Volaire, 2017). Therefore, we hypothesized that the continuous downregulation of PpMYB52 from endodormancy to bud break might be regulated by ABA and GA.

PpMIEL1 may positively regulate peach flower bud break by interacting with PpMYB52

MIEL1 is a RING-type E3 ubiquitin ligase that plays an important role in ubiquitin-mediated degradation of target proteins (Chen et al., 2021). In this study, we used Y2H assays to identify the interacting protein of PpMYB52, namely, the RING-type E3 ubiquitin ligase PpMIEL1, which was confirmed by BiFC analysis (Figures 1B,C). An et al. (2017b) found that ectopic expression of MdMIEL1 in Arabidopsis produced early-germinating phenotypes relative to those of wild-type plants. In this study, we found that PpMIEL1 expression was maintained at a low level in the endodormancy stage, increased during bud break, and decreased to the highest level before bud break (Figure 1D), which was quite different from the pattern of PpMYB52 (Figure 2B). These results showed that PpMIEL1 may positively regulate peach flower bud break. Therefore, we speculate that the expression of PpMIEL1 increased during the bud break process in peach flower buds, which interact with PpMYB52 and may promoting its ubiquitination-mediated degradation and bud break.

Conclusion

In summary, the results of our study demonstrate that PpMYB52, which is upregulated by ABA and downregulated by GA, negatively regulates peach bud break. In addition, we found that PpMYB52 interacts with a RING-type E3 ubiquitin ligase, PpMIEL1, which is upregulated in the process of bud break and may positively regulate peach bud break by ubiquitination-mediated degradation of PpMYB52 (Figure 6). This research characterized a potential mechanism of the involvement of MYB TFs in peach bud break, highlighting a new way for dormancy-related molecules to avoid bud damage in perennial deciduous fruit trees.

FIGURE 6.

FIGURE 6

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Model for PpMYB52 regulation of bud break in peach flower buds. PpMYB52 is upregulated by ABA and downregulated by GA, which negatively regulates peach bud break by inhibiting GA synthesis. And PpMYB52 interacts with PpMIEL1, which is upregulated in the process of bud break and may positively regulate peach bud break by ubiquitination-mediated degradation of PpMYB52.

Data availability statement

The original contributions presented in this study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s.

Author contributions

LL, XF, and YZ designed the study. YZ, QT, NW, XM, HH, BW, WX, XC, and DL performed the experiments and analyzed the data. YZ wrote the manuscript. All authors contributed to the article and approved the submitted version.

Acknowledgments

We thank LL, XF, WX, Changxing Nie, and DL, for providing reagents, materials, and analysis tools.

Abbreviations

ABA

abscisic acid

BiFC

bimolecular fluorescence complementation

cDNA

complementary DNA

CDS

coding sequence

DNA

desoxyribose nucleic acid

GFP

green fluorescent protein

YFP

yellow fluorescent protein

MS

murashige and skoog medium

OD

optical density

ORF

open reading frame

qRT-PCR

real-time quantitative

RNA

ribonucleic acid

X-α-gal

5-bromo-4-chloro-3-indolyl-α-D-galactoside

Y2H

yeast two-hybrid.

Funding

This work was partially supported by the National Key Research and Development Plan (No. 2018YFD1000104), the National Natural Science Foundation of China (No. 31872041), Shandong Province Agricultural Good Seed Project grant (Nos. 2020LZGC007 and 2020LZGC00702), and Shandong Provincial Fruit Industry Technology System—Cultivation and Soil Fertilization Post (No. SDAIT-06-04).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2022.971482/full#supplementary-material

Click here for additional data file. (345.6KB, docx)
Click here for additional data file. (36KB, XLS)

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Supplementary Materials

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Data Availability Statement

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