Metabolomics analysis during chilling induced dormancy transition
To study the metabolic changes during chilling-induced dormancy release in tree peony, flower buds were picked at five time points after 0-4ºC treatment for 0, 7, 14, 21 and 28 d, respectively, and metabolic profiling 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 profiles (Fig. 1b). As shown, quality control (QC) samples were separated from tested samples, and the two principal components (PC) accounted for 28.8% (PC1) and 14.5% (PC2) of the total variance, respectively. The samples were clearly separated into the 5 different subgroups by PC1 and PC2 (Fig. 1b).
A total of 535 metabolites were detected in the metabolomic analysis, and 511 of them were annotated with MassBank, KNAPSAcK, HMDB [37], MoTo DB and METLIN [38](Table S1). The metabolites were divided into eleven groups, including flavonoids, 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). Notebly, 123 flavonoids and derivates and 15 phytohormones were annotated (Table S1), which benifited to analyze their function. The results of metabolic pathway analysis showed that they were mapped to 14 KEGG pathways (Fig.1c), of them, totally 183 metabolites were assigned to the pathway of global and overview, followed by amino acid metabolism with 85, biosynthesis of other secondary metabolites numbered 82, metabolism of cofactors and vitamins numbered 33, nucleotide metabolism, carbohydrate metabolism amounted 33, 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 treatments (Fig. S1). Totally, 118 DMs were obtained when the chilling treated groups were compared with that of 0 d samples. The results also indicated that the amounts of up-regulated metabolites increased along with the increase of chilling days (Fig. 2a). In detail, the number of up-regulated metabolites were 4, 18, and 29 when 14 d vs 0 d, 21 d vs 0 d, and 28 d vs 0 d, respectively. There were not regular trend for the number of down-regulated metabolites when compared with 0 d. The results indicated that some metabolism pathways were activated by the prolonged chilling exposure. Interestingly, the number of up-regulated metabolites decreased when comparing 14 d vs 0 d with 7 d vs 0 d. To better understand the metabolites changes caused by different chilling durations, the venn diagram of 118 DMs was constructed, showing that four metabolites were common among the four comparisons, i.e. 7 d vs 0 d, 14 d vs 0 d, 21 d vs 0 d, and 28 d vs 0 d (Fig. 2b; Fig. S2). Besides, 30 metabolites were unique in 28 d vs 0 d, implying that wider changes happened in this group. The KEGG pathway annotation of 118 DMs was performed and the DMs involved in amino acid metabolism (24 in number), nucleotide metabolism (16), and biosynthesis of other secondary metabolism (15), 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 also 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 (Fig. S3).
The metabolic processes related to dormancy release in tree peony
To study the crucial metabolic processes related to endodormancy release in tree peony, DMs of 14 d vs 7d and 21 d vs 7 d were screened. Totally, 50 DMs were obtained and presented in a clustering heatmap, which showed the metabolites change from the dormancy to dormancy release (Fig. 3a). Among them,DMsDMsDMs fluctuated with a peak at 14 dThe KEGG analysis of the 50 DMs showed that 25 DMs participated in the metabolic pathway, which accounted for 83.33% of all the 30 annotated metabolites (Fig. 3b, Table S2). The KEGG enrichment anaylsis of 25 DMs showed that they involved in the pathways of glucose metabolism (glucose 6-phosphate), amino acid metabolism (aspartate) and hormone metabolism (ABA) (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 flow during dormancy release of tree peony (Fig. 4). The levels of glucose at 0, 7, 14 and 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 significant upward at 14 d. In terms of amino acid metabolism, some amino acids increased with the release of endodormancy, such as leucine, proline, etc., while some others decreased, such as valine, aspartic acid, glutamic acid, etc. (Fig. 4). In the shikimic acid pathway, the level of shikimic acid and phenylalanine increased after chilling exposure (Fig. 4).
The variation of carbohydrates during dormancy transition
Sugars play crutial roles in energy metabolism and substance metabolism, whose variation might reflect the status of bud dormancy after chilling exposure. The levels of 15 sugars in metabolic profiles were analyzed throughout the dormancy 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, and then their contents reduced (Fig. 5a). The monosaccharides (fucose, glucose, and arabinose) had the maximum level at the ecodormancy stage (28 d chilling treated, Fig. 5a), indicating a well preparatory status for the following re-growth. 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 metabolomics data. Fructose, glucose, and inositol were the three most abundant kinds of monosaccharides. The content of maltose significantly decreased after 7 d chilling treatment. Remarkably, the content of sucrose was the hightest of all the 13 tested sugars, which significantly increased and reached a maximum of 92.9 mg/g at 14 d chilling, 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, Fig. S4). 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 significantly increased at different chilling periods (Fig. 5c). Taken together, it was presumed that sucrose catabolism was dominant during chilling duration process, to provide sufficient 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 be related to the activity of amylases (AMY) during the same process (Fig. 5d).
The changes of flavonoids during dormancy transition
In KEGG enriched terms, the biosynthesis of secondary metabolites accounted for a large part (Fig. 3b), of which flavonoids are secondary metabolites that are widely present in plants. Thus a flavonoids metabolic pathway analysis was performed to understand its changes during dormancy transition in buds. Most flavonoids (naringenin, apigenin, luteolin, etc.), flavonols (kaempferol, quercetin, etc.), and anthocyanins (cyanidin 3-O-glucoside, pelargonidin 3-O-glucoside, etc.) showed similar down-regulated tendency at the early stages of dormancy (0-14 d) and up-regulated at 28 d. Thus, their highest contents were usually detected at 28 d, such as cyanidin-base anthocyanins, cyanidin 3-O-glucoside etc. (Fig. 6a). According to the results of LC-ESI-MS/MS, the relative contents of anthocyanins were analyzed at 28 d. The results indicated that five anthocyanins, including cyanidin 3-O-glucoside (Cy3Glu), cyanidin 3-O-rutinoside (CyRut), cyanidin O-syringic acid (CySyr), cyanidin 3,5-di-O-glucoside (Cy3Glu5Glu), and pelargonidin 3-O-glucoside (Pg3Glu), were dominant in flower 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 excluding PsDFR. Their transcripts were relactively abundant at the beginning of dormancy (0 d), and dramatically declined at 7 d chilling, but prolonged chilling promoted their expression compared to 7 d treatment. The PsDFR remained very low expression level till dormancy release period, but significally increased by ten folds at 28 d comparing with 0 d (Fig. 6c, Fig. S4). The expression patterns of the raltaed genes were accordance with their content variations during chilling duration process.
The variation of phytohormones during dormancy transition
Phytohormones play vital roles in plant growth and development, flowering, stress response, and so on. In metabolomics analysis, 14 phytohormones or analogues, including SA, JA, GA, and ABA, were detected in dormant buds of tree peony, and their content variations 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 five hormones peaked at chilled 28 d (Fig. 7a). They were divided into three subgroups by cluster analysis The first subgroup showed a up-regulated tendency with a peak at 28 d, including SA, GA15, JA, methyl jasmonate (MeJA) and jasmonic acid-isoleucine (JA-Ile). Dihydrozeatin, salicylic acid O-glucoside, trans-zeatin N-glucoside and ABA were categorized into the second subgroup with an obviously down-regulated tendency. The others fluctuated during chilling exposure process with a peak at 0 d chilling point.
The contents of JA, SA, IAA and ABA were also evaluated by LC-MS/MS analysis, and the results were similar to the metabolomics data. JA levels were relatively low from 0 to 21 d chilling, and dramatically peaked to 299.167 ng/g with a ten folds increase at chilled 28 d (Fig. 7b). The MYC2 (myelocytomatosis protein 2) transcription factor plays a central role in JA signal transduction [39]. Consistent with the variation of JA contents, the transcript of PsMYC2 increased sharply at chilled 28 d (Fig. 7c, Fig S4). The contents of SA were down-regulated from 0 d to 21 d, and then recovered the initial content of 0 d. The contents of IAA fluctuated 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, Fig S4). The contents of ABA persistently decreased from 1140.37 ng/g at 0 d to 803.83 ng/g at 28 d (Fig. 7b), indicating that ABA was an inhibitor of dormancy release in tree peony buds, and chilling treatment could gradually reduce the contents of ABA.