Metabonomic analysis reveals EMP pathway activation and avonoid accumulation during dormancy transition in tree peony

Abstract

Tree peony (Paeonia suffruticosa Andr.) is one of the most ancient ornamental and medicinal plants in the world. As a woody plant, it must undergo a period of low temperature to ensure the sprouting and owering in the next year. Due to the short and concentrated orescence every year, its anti-season production becomes an essential content of the tree peony industry. Until now, the primary method of anti-season production is to provide su cient low temperature exposure alone or combining with gibberellin application. Therefore, it was of great value to understand the mechanism of chilling induced dormancy release in tree peony. Our previous study characterized the relationship between chilling accumulation and dormancy status: the physiological status of tree peony 'Luhehong' after 14 d chilling treatment was regarded as the transition stage from endodormancy to endodormancy release, and that after 21 d chilling treatment was de ned as dormancy release, and after 28 d chilling as a state of ecodormancy [34]. GA pathway plays a crucial role in endodormancy release induced by chilling [35]. And the activity of PPP pathway also increased during the process, suggesting that it played a role in dormancy release of tree peony [8]. As known, traits are more closely related to metabolites, which may provide a new perspective for the understanding of dormancy transition in tree peony.
Here, metabolic changes of tree peony buds during the chilling induced dormancy transition were analyzed. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that differential metabolites were involved in various metabolic pathways such as carbon metabolism, secondary metabolite synthesis, and hormone metabolism. It was revealed that starch degradation and EMP activity were enhanced during dormancy release. Interestingly, avonoid anabolism was also activated by chilling accumulation, and its increasement might in return promote ower bud development. Furthermore, the variations of plant hormones (abscisic acid, jasmonic acid, and indole-3acetic acid) during the dormancy transition were also evaluated in this research. Signi cantly, the roles of avonoids were rstly discussed during the dormancy transition in perennial plants. All results would provide valuable information for the molecular mechanism of dormancy transition in tree peony.

Metabonomic analysis during chilling induced dormancy transition
To study the metabolic changes during chilling-induced dormancy release in tree peony, ower buds were picked at ve time points after 0-4 °C treatment (0, 7, 14, 21 and 28 days), and metabolic pro ling was analyzed by ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) (Fig. 1a). The Principal Component Analysis (PCA) analysis was employed to evaluate the repeatability of the metabolite pro les (Fig. 1b). As shown, quality control (QC) samples were separated from tested samples, and the two principal components accounted for 28.8% and 14.5% of the total variance, respectively (Fig. 1b).
A total of 535 small molecules were detected in the metabonomic analysis, and 511 of them were annotated with MassBank, KNAPSAcK, HMDB [36], MoTo DB and METLIN [37] (Table S1). The metabolites were divided into eleven groups, including avonoids, amino acid and its derivatives, lipids, organic acids and its derivates, nucleotide and its derivates, alkaloids, hydroxycinnamoyl derivatives, carbohydrates and alcohols, phytohormones, coumarins, and vitamins ( Table 1). The metabolic pathway of these metabolites was analyzed using KEGG databases. The results showed that the metabolites were mapped to 14 KEGG pathways during dormancy release (Fig. 1c). Of them, most metabolites were assigned to the pathway of global and overview, followed by amino acid metabolism, biosynthesis of other secondary metabolites, metabolism of cofactors and vitamins, nucleotide metabolism, carbohydrate metabolism, and so on (Fig. 1c).

Differential metabolites analysis
The Orthogonal Projection to Latent Structures-Discriminant Analysis (OPLS-DA, VIP ≥ 1) and the Student's t test (P < 0.05) were applied to detect the differential metabolites (DMs) among different groups (Fig. S1). The results indicated that the amounts of up-regulated metabolites increased along with the accumulation of low temperature when compared with 0 d (Fig. 2a) Fig. 2b; Fig. S2). Besides, 30 metabolites were unique in 28 d vs 0 d, implying that this group had more variation than other groups. The KEGG pathway annotation of 118 DMs was performed and the DMs were involved in amino acid metabolism, nucleotide metabolism, and biosynthesis of other secondary metabolism, and so on (Fig. 2c). Additionally, KEGG enrichments between each two treatments were listed in Fig. S3. Carbohydrate metabolisms, such as pyruvate metabolism, starch and sucrose metabolism, PPP pathway and so on, were frequently presented in the different enrichments analysis. Plant hormone signaling transduction was enriched in 7 comparable groups. Besides above, several amino acid metabolisms, pyrimidine and purine metabolism, nitrogen metabolism and others were also frequently enriched in the comparations.
The metabolic processes related to dormancy release in tree peony To study the crucial metabolic processes related to dormancy release in tree peony, totally, 50 DMs of 14 d vs 7d and 21 d vs 7 d were identi ed and presented in a clustering heatmap, which re ected the metabolites change between the endodormancy and the endodormancy release transition (Fig. 3a). The heatmap showed that 21 DMs were signi cantly up-regulated, 18 DMs were down-regulated after prolonged chilling enduration, and the other 11 DMs uctuated with a peak at 14 d. The KEGG analysis of the 50 DMs showed that 25 components participate in the metabolic pathway, which accounted for 83.33% of all the 30 annotated metabolites (Fig. 3b, Table S2). The 25 DMs which identi ed by KEGG enrichment anaylsis involved in the pathways of glucose metabolism (glucose 6-phosphate), amino acid metabolism (aspartate) and hormone metabolism (abscisic acid) ( Table S3), suggesting that these metabolic pathways might play a critical role during dormancy release in tree peony.

Carbon metabolism during dormancy transition in tree peony
A metabolic network containing EMP pathway, TCA cycle, shikimate pathway, and amino acid metabolism was presented to visualize the carbon ow during dormancy release of tree peony (Fig. 4). The levels of glucose at 0, 7, 14, 21 d were lower than that at 28 d. However, glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P) had the higher levels at 7 and 14 d (Fig. 4), indicating that the EMP was activated during chilling induced endodormancy release in tree peony. In the TCA cycle, the levels of citrate declined until 14 d and then climbed. The succinic acid amount showed an signi cant upward at 14 d. In terms of amino acid metabolism, some amino acids increased with the release of endodormancy, such as leucine, proline, etc. In contrast, the others decreased, such as valine, aspartic acid, glutamic acid, etc. (Fig. 4). In the shikimic acid pathway, the level of shikimic acid and phenylalanine were increased after chilling exposure (Fig. 4).

The variation of carbohydrates during dormancy transition
Sugars play critical roles in energy metabolism and substance metabolism, whose variation might re ect the status of bud dormancy after chilling exposure. The levels of 15 sugars in metabolic pro les were analyzed throughout the same process (Fig. 5a). Some monosaccharide (G6P, glucosamine, and trehalose 6-phosphate) and polysaccharides (maltotetraose, melezitose, and melezitose O-rhamnoside) were up-regulated after 7 d chilling exposure, then their contents reduced (Fig. 5a). The monosaccharides (fucose, glucose, and arabinose) had the maximum level at the stage of ecodormancy (28 d chilling treated, Fig. 5a), indicating a well preparatory status for the following regrowth. A Gas Chromatography-Tandem Mass Spectrometry (GC-MS/MS) measurement was then employed to further analyze the change of sugars during the chilling process in tree peony, and the results were similar to the metabonomics data. Fructose, glucose, and inositol were the most abundant three kinds of monosaccharides. The content of maltose signi cantly decreased after 7 d chilling treatment. The content of sucrose was the hightest of all the 13 tested sugars, which signi cantly increased and reached a maximum of 92.9 mg/g at chilled 14 d, and then declined rapidly (Fig. 5b), suggesting that it might play a vital role in the whole process. To further investigate the role of sucrose in dormancy release of buds, the expression patterns of sucrose synthase (PsSUS1 and PsSUS2) and sucrose invertase genes were analyzed (Fig. 5c). The expression of PsSUS1 continued to decline after 7 d chilling treatment, but PsSUS2 was up-regulated at 7 and 14 d chilling. The expressions of cytoplasmic invertase (PsCIN), vacuolar invertase (PsVIN), and cell-wall invertase (PsCWIN) were signi cantly increased at different chilling periods (Fig. 5c). Taken together, it was presumed that sucrose catabolism was dominant during chilling duration process, to provide su cient sugars for respiratory metabolism and energy metabolism.
Starch, the main storage carbohydrate in higher plants, was measured during dormancy transition in tree peony. The results indicated that starch content decreased after chilling exposure and reached its minimum at 14 d, which might correspond to the activity of amylases (AMY) during the same process ( Fig. 5d).
The changes of avonoids during dormancy transition , were dominant in ower buds of tree peony, and the others were relactively rare ( Fig. 6a and b). In detail, cyanidin-base anthocyanins (Cy3Glu5Glu) accounted for the majority, followed by Cy3Glu ( Fig. 6a and b). Two EBGs (PsCHS and PsF3H) and two LBGs (PsANS and Ps3GT) showed the similar patterns. Their transcripts were relactively abundant at the beginning (0 d), and dramatically declined at 7 d chilling, but prolonged chilling promoted their expression compared to 7 d treatment. The exception was PsDFR, which remained very low expression level till dormancy release period, but increased by ten folds at 28 d (Fig. 6c). The expression patterns of the raltaed genes were accordance with their content variations during chilling duration period.

The variation of phytohormones during dormancy transition
Phytohormones play vital roles in plant growth and development, owering, stress response, and so on. With metabolomics analysis, 14 phytohormones or analogues, including salicylic acid (SA), jasmonic acid (JA), gibberellin (GA), and abscisic acid (ABA), were detected in dormant buds of tree peony, and their contents during chilling duration were shown in a heatmap (Fig. 7a). Nine metabolites presented the highest contents at chilled 0 d (nonchilled period), and the other ve hormones peaked at chilled 28 d (Fig. 7a). Cluster analysis divided them into three subgroups. The rst subgroup showed a up-regulated tendency, including SA, GA15, JA, methyl jasmonate (MeJA) and jasmonic acid-isoleucine (JA-Ile), all with a peak at 28 d. Dihydrozeatin, salicylic acid O-glucoside, trans-zeatin N-glucoside and ABA were categorized as the second subgroup with an obviously down-regulated tendency. The others uctuated during chilling exposure process with a peak at non-chilling point.
The contents of JA, SA, indole-3-acetic acid (IAA) and ABA were evaluated by LC-MS/MS analysis, which was similar to the metabonomic data. JA level was relatively low from 0 to 21 d chilling, and dramatically peaked to 299.167 ng/g with a ten fold increase at chilled 28 d (Fig. 7b). The MYC2 (myelocytomatosis protein 2) transcription factor plays a central role in JA signal transduction [38]. Consistent with the variation of JA content, the transcript of PsMYC2 increased sharply at chilled 28 d (Fig. 7c). The contents of SA was down-regulated from 0 d to 21 d, and then recovered the initial content of 0 d.
The content of IAA uctuated during the chilling duration process, reached the highest level of 44.57 ng/g at 14 d, and its content of 28 d was also higher than 0 and 21 d (Fig. 7b). The expression pattern of two auxin receptors genes (TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX PROTEIN2,TIR1/AFB2) were analyzed during the chilling duration. PsAFB2 was down-regulated in the whole process, and PsTIR1 was up-regulated at 7 d, and then down-regulated from 7 to 28 d (Fig. 7c). The content of ABA persistently decreased from 1140.37 ng/g at chilled 0 d to 803.83 ng/g at chilled 28 d ( Fig. 7b), indicating that ABA was an inhibitor of dormancy release in tree peony buds, and chilling treatment could gradually reduce the content of ABA.

Discussion
The dormancy transition of woody plants is a complicated process, which was synergetically regulated by photoperiod and low temperature [39]. Chilling treatment as an effective method to promote dormancy release had been veri ed in many species including tree peony [3][4][5]. Transcriptomics, proteomics and microRNAs analysis had been performed to investigated the concealed mechanism of chilling induced dormancy release in tree peony, which revealed the roles of GA and carbohydrate metabolism [8,35,40].
Here, substance changes were detected by metabolomics analysis during the chilling duration process.
Starch degradation and EMP pathway were enhanced during dormancy release Carbohydrate is the basic energy substance of primary metabolism and secondary metabolism. Sugars metabolism and signal transduction involving in bud dormancy process had been revealed by RNA-seq [41]. Sugar metabolism genes (beta-amylase 5,alpha-amylase-like 1, sucrose synthase 3, and trehalosephosphatase/synthase 7), sugar transporter genes (GT-2 like 1, sucrose-proton symporter 2, protein O-mannosyltransferase 5, and senescence-associated gene 29) and sugar signal transduction genes (glucose insensitive 2 and beta-fruct 4) had been proven to involve in dormancy release in poplar, grape and P. mume. [41][42][43]. In this study, the content of carbohydrates was continuously measured during the chilling induced dormancy transition of tree peony. Starch and maltose showed a downward trend during dormancy release, which were consistent with the increasing of amylase activity and sugar catabolism (Fig. 5b, d). Our recent results also revealed that the transcripts of PsAMY and PsBMY were up-regulated [8,41]. At the same time, the enzyme activity of AMY gradually increased. Together, these results indicated that chilling treatment promoted the degradation of starch during dormancy release of tree peony. After endodormancy release (21-28 d), the buds maintained active respiratory metabolism and amylase activity, but the starch and maltose contents did not decrease signi cantly (Fig. 5b, d). It was speculated that a considerable amount of carbohydrates were transported from other parts of the plant to the ower buds, such as the root system (tree peony has a developed succulent root system), to meet the consumption of ower buds. The reopening of the material transport channel also enables the longdistance transport of carbohydrates [11].
The content of sucrose was the most abundant among all the measured sugars, which implied that sucrose might also be used to transport of assimilates in tree peony rather than sorbitol as in apple because sucrose was about 3 200 times higher than sorbitol. Sucrose was up-regulated till to 14 d and decreased afterward according to GC-MS/MS results (Fig. 5b). The starch was rapidly degraded at the beginning of the chilling duration due to high amylase activity, and sucrose is a kind of its intermediate product.
Taken together, we speculated that sucrose mainly came from starch degradation. It was also found that sucrose accumulated, and sucrose synthase genes were up-regulated at the early stage of dormancy process in poplar and P. mume [41]. Therefore, sucrose might be used as an energy center to ensure the supply of glucose in dormancy transition.
In addition, extensive degradation of starch should lead to glucose accumulation, but glucose and fructose were at a low level or down-regulated until to ecodormancy stage (Fig. 5a, b). Meanwhile, the content of F6P and G6P increased signi cantly after chilling exposure, but decreased thereafter (Figs. 4 and 5a). Also, Our previous study showed that the transcripts and enzyme activities of Hexokinase (HK) and glucose 6-phosphate isomerase (G6PI) were also signi cantly up-regulated [8,41]. These results indicated that the EMP pathway was activated during dormancy release of tree peony. Metabolome results showed that dormancy release was an energy-consuming process. A large number of carbohydrates were broken down to produce enough substances and energy to promote dormancy release while also providing a carbon chain for secondary metabolism.

Flavonoids accumulation at ecodormancy stage
As secondary metabolites, avonoids play important roles in many processes of plant growth and development, such as color formation, stress resistance, etc. [29]. However, the changes and functions of avonoids during dormancy transition were still poorly understood. Variations of avonoids were observed during chilling duration process in our work. Flavonoids (e.g. quercetin, kaempferol, and apigenin) synthesized in the early step of the avonoid biosynthesis pathway were down-regulated in the whole endodormancy stage, i. e. the treatments before 21 d, due to the decreasing expression level of PsCHI (Fig. 6). Subsequently, their levels were sharply upregulated at 28 d, an ecodormancy stage (Fig. 6a). The primitive high level of avonoids might be related to cold acclimation for winter survival. It was considered that the initial products of the avonoid biosynthesis pathway, such as quercetin, kaempferol, apigenin, etc., inhibited the transport of polar auxin and regulate plant development [44]. Also, the correlation between pollen fertility and avonoids had been found in maize and peanuts [29], and the silencing of chalcone synthase gene resulted in parthenocarpy in tomato [45]. Flavonols (in particular quercetin) is essential for pollen germination [32]. In our results, avonols showed higher levels at ecodormancy stage (Fig. 6a), when the ower buds are well-differentiated, and stamens are clearly visible [46]. Therefore, avonols might be involved in ower bud development at ecodormancy stage in tree peony.
In a recent study, Gu et al. suggested that anthocyanin accumulation occurred 10 d before anthesis in tree peony 'Qing Hai Hu Yin Bo' [47]. Here, we found anthocyanins were up-regulated after endodormancy release, accumulating in large amounts at ecodormancy stage in tree peony 'Luhehong' (Fig. 6). The results implied that the oral pigments might begin their synthesise before the bud entering into endodormancy with a early peak at ecodormancy period. Cyanidin-based glycosides such as Cy3Glu and Cy3Glu5Glu were the most abundant anthocyanins in the petal blotches of 35 cultivars [48], and they were also the most abundant anthocyanins in the buds of 'Luhehong' (Fig. 6b). We hypothesized that dormancy release induced by chilling duration synchronously activated anthocyanin synthesis and accumulation.
Taken together, Flavonoid accumulation during chilling induced dormancy release might accelerate ower bud development by promoting ower organ development (petal and stamens). To our knowledge, it was the rst report to describe the changes and role of avonoids during dormancy transition in perennials.

The roles of phytohormone during dormancy transition
In perennial woody plants, ABA and GA have been widely proven to regulate bud dormancy. Recent researches revealed the antagnism between ABA and GA in bud dormancy. A MADS-box (DAM) family gene, SHORT VEGETATIVE PHASE-like (SVL) had been shown to play a key role in ABA-mediated bud dormancy in poplar. In SVL RNAi strain, the expression of FT1 was signi cantly up-regulated, and GA biosynthesis gene GA20ox was up-regulated to promote bud rupture [18]. Further research found that SVL could directly bind to the promoters of GA2ox8 and CALS1 to induce their expression, reduce the level of active GAs and control the closure of plasmodesmata, thereby maintain dormancy status [49]. In our work, ABA was down-regulated along with the chilling duration (Fig. 7). Gibberellin 15, the precursor of bioactive gibberellins, was up-regulated gradually (Fig. 7a). Meanwhile, the expression of PsGA20ox, a key enzyme in GA biosynthesis, was up-regulated, and the content of GAs increased with chilling accumulation [50,51]. Therefore, there might be similar mechanisms between ABA-regulated dormancy in poplar and chilling-induced dormancy in tree peony. Chilling accumulation reduces the level of ABA, which in turn suppresses the expression of SVL and promotes the biosynthesis of GAs, and nally break bud dormancy [35].
Auxins and CKs play antagonistic roles in meristems of many plants [52]. Previous researches had shown that CKs play a positive role in hydrogen cyanamide-induced bud dormancy release in grape [53], but that of IAA is still ambiguous until now. However, in our study, the level of CKs (dihydrozeatin, trans-zeatin Nglucoside and kinetin 9-rboside ) decreased after chilling exposure, and IAA was up-regulated by chilling, con rmed by metabonomics and LC-MS analysis (Fig. 7). TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX PROTEIN (TIR1/AFB) family are known as auxin receptors [54]. The transcript of PsTIR1 was signi cantly induced and consistent with IAA variation (Fig. 7c), which implied a positive role of IAA during dormancy release in tree peony. These results were also different from the upregulation of CKs in P. kingianum and P. mume during dormancy release [55,56]. Therefore, the regulations of CKs and IAA in dormancy release were not a common mechanism in different perennial plants, and they might not be the key factors in dormancy regulation.
Usually, SA and JA are regarded as stress response hormones, rather than function on dormancy regulation [57,58]. Recently, Ionescu et al. proposed that the upregulated JA-Ile induced the expression of MYB21 and MYB108 to participate in the ower development process during HC-induced dormancy release in sweet cherry [56]. In our results, jasmonates were dramatically upregulated when entering ecodormancy period, along with a transcriptional climb of PsMYC2, a key transcription factor for JA signal transduction (Fig. 7). The results indicated that JA signal transduction was activated after su cient chilling duration. In recent studies, the mechanism of promoting the anthocyanins accumulation by JA was revealed in Arabidopsis. When the JA content increased, the inhibitory effect of JAZs protein on MYB-bHLH-WD40 complex was released, which promoted the expression of DFR and ANS, and the accumulation of anthocyanins [59].
Interestingly, PsDFR and PsANS also signi cantly increased, and anthocyanins presented higher levels at 28 d chilling treatment in our study (Fig. 7). It was hypothesized that jasmonate was additionally involved in anthocyanin accumulation after endodormancy release in tree peony. Additionally, SA involve in the response to low-temperature stress, for SA and glucosyl SA accumulating after low-temperature exposure [57]. The use of exogenous SA improves the cold tolerance of corn, cucumber, and rice [58]. The accumulations of SA and Salicylic acid O-glucoside were also observed during chilling induced dormancy release in tree peony (Fig. 7), it might be the response of buds to low-temperature stress.

Conclusions
In summary, we systematically revealed the metabolomic changes during the chilling induced dormancy transition of tree peony, and a total of 511 substances and 118 DMs were identi ed. Chilling accumulation promoted the degradation of starch and enhanced the activity of EMP, providing adequate energy and substances for secondary metabolism required by dormancy release and bud burst. Flavonoid was accumulated by su cient chilling duration along with endodormancy release. Furthermore, we also reported phytohormone changes in during the dormancy transition in tree peony. Prolonged chilling exposure declined ABA content, but promoted JA and GA accumulation at the end of dormancy. Taken together, we proposed a work model of dormancy transition induced by chilling according to the metabonomics analysis (Fig. 8). Our results might help people better understand the dormancy transition of perennial plants.

Plant materials
Four-year-old tree peony plants (Paeonia suffruticosa 'Luhehong') were treated with continuous arti cial chilling (0-4 °C) from Nov 12, 2018 as described previously [8]. At 0, 7, 14, 21 and 28 d after refrigerating treatment, buds were picked in each time point, frozen in liquid nitrogen, and stored at -80℃ until further analysis. The samples from each three plants were harvested and mixed in each treatment. Three replicates (3 plants/replicate) per group were set.

Sample preparation and extraction
The freeze-dried samples were crushed at 30 Hz for 1.5 min using a mixing mill with zirconia beads (MM 400, Retsch). Then 100 mg powder was mixed with 1.0 mL 70% methanol solution (containing 0.1 mg/L lidocaine as an internal standard) at 4℃ overnight. After centrifugation at 10,000 × g for 10 min, the supernatant was ltered (scaa-104, aperture 0. LIT and triple quadrupole (QQQ) scans were performed on a triple quadrupole linear ion TRAP mass spectrometer (QTRAP). The AB Sciex QTRAP4500 system was equipped with an ESI-Turbo Ion-Spray interface, ran in positive ion mode, and was operated by the Analyst 1.6.1 software (AB Sciex). Operating parameters were as follows: ESI source temperature was 550 °C; Collision activation dissociation (CAD) was set to the highest; Ion-spray voltage (IS) was 5500 V; The m/z range was set to 50 to 1000. The QQQ scan was obtained as an multiple reaction monitoring (MRM) experiment, with the optimal solution cluster potential (DP) and collision energy (CE) for each MRM transformation.

Qualitative and quantitative determination of metabolites
The qualitative analysis of metabolites was performed based on the public metabolite database (e.g. MassBank and KNApSAcK) and the METLIN database (MWDB) [36,37]. The repetitive signal of K + , Na + ,

Differential metabolites analysis and KEGG analysis
The most distinguishable metabolites between every two groups were ranked by the variable importance of the projection (VIP) score using the OPLS model. The threshold for VIP was set to 1. Besides, Student's t test was used as a univariate analysis to screen different metabolites. Those with P < 0.05 and VIP ≥ 1 were considered as differential metabolites between two groups. The speci c primers used in qRT-PCR were designed by Primer Premier 6.0 according to the full-length cDNA sequences, and were listed in Table S4. The relative expressions of these genes were performed using 2 −ΔΔCt method as described by Livak and Schmittgen [62].   way ANOVA, *P < 0.05, **P < 0.01, and ***P < 0.001). (c) The relative expression levels of sucrose synthase and sucrose invertase genes. The mean ± SD in three biological replicates was shown. *, ** and *** indicated signi cant differences of one-way ANOVA at P < 0.05, P < 0.01 and P < 0.001, respectively.

Measurements of sugar contents
(d) The variations of starch and amylases activity. The mean ± SD in three biological replicates was shown. *, ** and *** indicated signi cant differences of one-way ANOVA at P < 0.05 and P < 0.001, respectively.