Metabolomic profiling of Mp leaves at different development stages
In present study, samples collected from bud, tender and mature leaves of Mp at different developmental stages were analyzed by metabolomics. According to the primary classification of substances (Table S1), a total of 957 metabolites were identified from the leaves of Mp, including the most abundant compounds, 185 phenolic acids, 155 lipids and 109 terpenoids. In addition, 85 organic acids, 81 flavonoids, 78 amino acids and their derivatives, 66 nucleotides and their derivatives, 33 lignans and coumarins, and 31 alkaloids were also detected. The overlay analysis of the QC-TIC diagram (Fig. S1A-B) and the sample multi-peak detection diagram (Fig. S1C-D) showed that the data recorded in this study had a good repeatability and reliability. Additionally, the three biological replicates of each sample exhibited relatively higher correlation coefficients ranged from 0.89 to 0.99 (Fig. S2), which demonstrated that the samples possessed good homogeneity.
PCA and OPLS-DA for Mp
In the PCA plot, leaves at three different maturity stages were apparent separated, and the three biological replicates of each group were closely clustered together, with 51.96% and 14.84% variation explained by PC1 and PC2, respectively, indicating that the entire analysis was repeatable and reliable, and there were significant differences among the three groups (Fig. 2A). A total of 957 substances were identified from all the leaves, as showed in the heatmap, which manifested the visible hierarchical clustering between groups. The content of substances in the bud leaves showed obvious changes compared with that in the tender and mature leaves, among which, around half of samples whose content was relatively higher, while the other examples was lower (Fig. 2B). According to the abundance of the first-level classification (Fig. 2C), the contents of lipids and phenolic acids in the leaves of Mp were the highest, whose levels in the bud leaves were relatively higher than that in the tender leaves and mature leaves. The total amount of organic acids, terpenoids and alkaloids was also relatively abundant in the bud leaves, with example substances including choline, and 2α,3β,9α,23,24-pentahydroxyolean-12-en-28-oic acid 28-O-glucoside,. However, the total amount of nucleotides, amino acids, lignans, coumarins and flavonoids accumulated significantly with the maturation of leaves. The corresponding compounds of alkaloids, lipids, nucleotides, and terpenoids, including choline, stearic acid, adenosine, and 2α,3β,9α,23,24-pentahydroxyolean-12-en-28-oic acid 28-O-glucoside, showed the highest abundance in tender leaves; while, in addition to the three abovementioned substances, adenosine was also most abundance in mature leaves. According to the abundance of secondary classification of substances, phenolic acids, free fatty lipids and organic acids with the highest content were observed in the bud leaves (Fig. 2D). It was also found that the total amount of phenolic acids, saccharides and alcohols, and other substances was relatively higher in tender leaves, while, the content of free fatty lipids, nucleotides and their derivatives, amino acids and their derivatives, coumarins, and flavonols showed the higher abundance in the mature leaves. These results indicated that growth and development strongly influenced the metabolite profiling of Mp leaves at different development stages.
Additionally, OPLS-DA (orthogonal partial least squares-discriminant analysis) was used to screen the variables responsible for differences among these three groups [19]. In the current study, the OPLS-DA model was used to compare metabolites of the samples in the comparisons to evaluate the differences in MpBud vs. MpTen (R2X = 0.691, R2Y = 1, Q2 = 0.969), MpTen vs. MpMat (R2X = R2X = 0.576, R2Y = 1, Q2 = 0.922), and MpBud vs. MpMat (0.736, R2Y = 1, Q2 = 0.979) (Fig. 3). The Q2 values of all the comparisons were greater than 0.9, indicating that these models were stable and credible. OPLS-DA score plots exhibited that these three stages were separated from each other, demonstrating there were significant differences in metabolic phenotypes of the Mp leaves at different developmental stages. These results indicated that mature stage was the most essential process in the evolution of metabolites, followed by bud stage, with tender stage produced the least impact.
Differentially accumulated metabolites screening, functional annotation and enrichment analysis among the Mp leaves at different developmental stages
To elucidate the impact of each stage on the metabolites present at the different developmental stages of Mp leaves, the crucial differences among these processes were investigated. A total of 317 Differentially accumulated metabolites (DAMs) were screened, of which 202 DAMs were found in MpBud vs. MpTen (Fig. 4A, 74 up-regulation and 128 down-regulation), 54 DAMs were observed in MpTen vs, MpMat (Fig. 4B, 27 up-regulation, and 27 down-regulation), and 254 DAMs were recorded in MpBud vs. MpMat (Fig. 4C, 107 up-regulation and 147 down-regulation).
The Venn diagram exhibited that there were both common and unique metabolites among the comparisons (Fig. 4D, Table S1). Particularly, MpBud vs. MpTen and MpTen vs. MpMat possess 29 common substances (i.e., 3 phenolic acids, 10 flavonoids, 4 terpenoids, 1 lipid, 2 amino acids and derivatives, 3 alkaloids, 5 lignans and coumarins, and 1 other metabolite), which were simultaneously affected by the growth and development of Mp leaves. 149 common metabolites were also observed in MpBud vs. MpTen and MpBud vs. MpMat (i.e., 26 phenolic acids, 12 flavonoids, 22 terpenoids, 7 lipids, 11 organic acids, 15 amino acids and derivatives, 8 alkaloids, 16 nucleotides and derivatives, 6 lignans and coumarins, and 26 others), which illustrated that these common metabolites accumulated more under the bud to tender stages, especially phenolic acids, flavonoids, terpenoids; while 27 common metabolites were also found in MpTen vs. MpMat and MpBud vs. MpMat, which were less affected by the growth and development of Mp leaves under the tender to mature stages.
The 90 metabolites that were unique to MpBud vs. MpMat were only affected by growth and development of the leaves. 36 metabolites (i.e., 24,30-Dihydroxy-12(13)-enolupinol, 3β,19α-Dihydroxyolean-12-en-28-oic acid, and 2α,3β,19α-Trihydroxyurs-12-en-23,28-dioic acid-28-O-glucoside) were only biosynthesized and accumulated under bud to tender stages, and these components maybe participate in the leaf color change of Mp; while ten metabolites (i.e., 4-Hydroxybenzoic acid, 5,6-Dihydroxyindole-5-O-β-glucoside, and Kaempferol-3-O-sophoroside) were only biosynthesized and accumulated under tender to mature stages (Fig. 4D). We proposed that the DAMs found in MpBud vs. MpTen and MpTen vs. MpMat may contribute to biosynthesis of functional and nutrition ingredients, and the leaf color change in Mp.
Multiple comparative analysis showed that among the comparisons (MpBud vs. MpTen, MpTen vs. MpMat, and MpBud vs. MpMat) shared only 12 different metabolites such as Benzamide, 2-Phenylethanol, Pinocembrin-7-O-glucoside (Pinocembroside), Mussaenoside, and 1β,2α,3α,19α-Tetrahydroxyurs-12-en-28-oic acid (Fig. 4D). These DAMs were the most active metabolites during the growth of Mp leaves, and may be closely related to the changes of leaf color, biochemical components and functions. These results suggested that the metabolites that caused the differences among bud leaves, tender leaves, and mature leaves were greatly different. Therefore, the various functional and nutrition ingredients were not present or its content were low under the different stages (tissues or cells) during the growth and development of Mp leaves, while these components were continuously biosynthesized and accumulated with the growth and development of leaves. These results further confirmed that the unique metabolites existed in mature leaves were not only particularly critical for the transformation of the metabolites, but also more than other leaves during Mp leaves growth and development, since the photosynthesis promoted the hydrolysis, isomerization, oxidation, and other reactions of the metabolites.
From the changes of metabolite abundance (Fig. 4E), the contents of metabolites in Sub Class 1, 4, 5, 7 and 9 were higher in the bud leaves, with a total of 176 metabolites, mainly including 2-Isopropylmaleic Acid, 2-Phyllethylamine, LysoPC 18:1 (2n isomer), and L-Cyclopentylglycine; Sub Class 3 and 6 contained 118 metabolites, which almost increased with leaves maturity, mainly including Adenosine, L-Isoleucine, Quercetin-3-O-galactoside (Hyperin); In Sub Class 2, the content of 20 metabolites in tender leaf was significantly higher, such as Caffeine, Kaempferol-3-O-rutinoside-7-O-glucoside, Quercetin-7-O-rutinoside-4'-O-glucoside, and Mussaenoside. Compared with tender leaves, the amount of Dehydrocastus lactone, which belongs to sesquiterpenoids, decreased to 48.2%; Mussaenoside in the sesquiterpenoids increased by 2.3 times, while Penstemonoside and Deacetylasperosidic acid decreased to 49.1% and 35.8%, respectively; Triterpenoids showed a downward trend, with a decline range of 13.1%~46.0%, and the most significant decrease was observed in 1β, 2α, 3α, 9α-Tetrahydroxurs-12-en-28-oic acid and the least in 23-Hydroxybetulinic acid; In flavonoids, the glycoside of Kaempferol increased while that of Quercetin decreased, in which Kaempferol-3-O-rutinoside-7-O-glucoside increased by 15.5 times, and Quercetin-3-O - (6 '' - O-acetyl) glucoside decreased to 17.8%. Compared with mature leaves, the Isohyperoside and Quercetin-3-O-glucoside (Isoquercitrin)* of flavonol and Scopoletin-7-O-glucoside (Scopolin) of coumarin increased by 2.1–2.3 times in tender leaves; The content of 2-Phenylethylamine, which belongs to alkaloids, decreased more, and the mature leaves were only 20.7% of the tender leaves, while the mussaenoside of sesterpenes and Kaempferol-3-O-rutinoside-7-O-glucoside of flavonols were only 13.1% and 5.2% of the tender leaves; In addition, the triterpene 2,3-Dihydroxy-12-ursen-28-oic acid increased by 2.1 times.
The differential metabolites for MpBud vs. MpTen, MpTen vs. MpMat, and MpBud vs. MpMat were involved in 66, 37 and 75 pathways and the major pathways were presented in bubble plots (Fig. 4, Table S2). Most noteworthy, the top ten metabolic pathways including "ABC transporters", "Purine metabolism", and "Galactose metabolism" were significantly up-regulated (p-value < 0.05) in MpBud vs. MpTen (Fig. 4F). The top ten metabolic pathways including “Flavone and flavonol biosynthesis”, “Nitrogen metabolism”, “Phenylalanine metabolism”, “Folate biosynthesis”, “Sphingolipid metabolism” were enriched in the comparison of MpTen vs. MpMat (Fig. 4G). Whereas, “ABC transporters”, “Nicotinate and nicotinamide metabolism”, “Fructose and mannose metabolism”, “Metabolic pathways”, “Arginine biosynthesis”, “Biosynthesis of cofactors”, “Biosynthesis of amino acids”, “Aminoacyl-tRNA biosynthesis”, “Monobactam biosynthesis”, “Indole alkaloid biosynthesis” showed a p-value < 0.05 in the enrichment analysis of MpBud vs. MpMat (Fig. 4H).
Evaluation of the different metabolites during the growth and development of Mp leaves
A total of 317 different metabolites mainly involved in the 150 primary metabolites of 33 amino acids and derivatives, 21 organic acids, 16 lipids, 31 nucleotides and derivatives, and 49 others, as well as the 167 secondary metabolites involved in 48 phenolic acids, 35 flavonoids, 54 terpenoids, 14 lignans and coumarins, and 16 alkaloids (Fig. 5A), of which 36 DAMs were unique to MpBud vs. MpTen, accounting for 11.4% of the total different metabolites (Fig. 5B), and 90 DAMs were unique to MpBud vs. MpMat, accounting for 28.4% of the total different substances (Fig. 5C).
Secondary metabolites
Secondary metabolites involved in flavonoids, terpenoids, phenolics or alkaloids are small molecular organic compounds derived from primary metabolites such as carbohydrate substances that are not directly associated with growth and development of plant cells and organs, which function as an important health-promoting phytochemicals for functional foods or medicines as well as interact with bioenvironment and for the establishment of defense mechanism [20].
Flavonoids, the most described phenolic secondary metabolites, are widely distributed in plant, which can be classified into six categories involved in flavonols, flavanones, flavones, flavanols, isoflavones and anthocyanidins [21]. Among the 35 different flavonoids substances examined herein (Fig. S3, Table S3), 21 DAMs significantly altered involved in ten up-regulated differences (e.g., Quercetin-3-O-galactoside (Hyperin) and Kaempferol-4'-O-glucoside) and 11 down-regulated differences (e.g., Quercetin-3-O-Sambubioside-5-O-Glucoside and Kaempferol-3-O-rutinoside-7-O-rhamnoside) across the whole growth stage of Mp leaves (MpBud vs. MpMat). Additionally, a total of 25 metabolites significantly accumulated with 14 differences of Epicatechin and Apigenin-6,8-di-C-glucoside (Vicenin-2) or decreased with 11 differences of Luteolin-7-O-Sophoroside-5-O-arabinoside and Luteolin-7-O-Sophoroside-5-O-arabinoside as the buds growing into the tender leaves (MpBud vs. MpTen). While, 15 significantly changed with the majority of the down-regulated substances such as Kaempferol-3-O-rutinoside-7-O-glucoside and Quercetin-7-O-rutinoside-4'-O-glucoside in the maturation stage of leaves (MpTen vs. MpMat). What’s more, it was found that five compounds were unique to the whole growth stage with the example of Kaempferol-3-O-glucoside (Astragalin) and Quercetin-4'-O-glucoside (Spiraeoside). Several compounds with high accumulation have been proved to have beneficial bioactivities such as kaempferol, quercetin, naringenin and their glycosides [22]. For instance, naringenin and kaempferol-3-O-glucosideis are flavanone and flavonoid, respectively, which possess a variety of bioactivities including antioxidant, anticancer and anti-inflammatory [23, 24]. The highly accumulated flavonoids metabolites as mentioned above were observed in the bud leaves, which confirmed that it was positively related to the antioxidant activity.
Phenolic acids possess various important medicinal compounds such as hydroxybenzoic acid, hydroxycinnamic acid and their derivatives in tea plants [25]. In this research, a total of the 48 different phenolic acid compounds were detected herein (Fig. S3, Table S4), of which 46 exhibited significant alteration across the whole growth stage with 21 up-regulated (e.g., 1-O-Caffeoyl-β-D-glucose*) or 25 down-regulated (e.g., Trans-4-Hydroxycinnamic Acid) DAMs. Among 26 different substances, nine significantly accumulated metabolites were observed for the example of 2-Naphtho and 11 remarkably decreased metabolites were obtained such as Benzoylmalic acid. However, only seven substances involved in five up-regulated (e.g., Ethylparaben) and two down-regulated (e.g., Benzamide) differences were found in the maturation stage (MpTen vs. MpMat). In addition, 18 DAMs were unique to the whole growth stage of leaves (MpBud vs. MpMat), of which eight significantly up-regulated metabolites with the example of Homogentisic acid and ten remarkably down-regulated metabolites with the example of 2-Hydroxybenzaldehyde (Salicylaldehyde). The highly accumulation of these phenolic acid metabolites especially Trans-4-Hydroxycinnamic Acid Methyl Ester in the bud leaves were consistent with the results reported by Wu et al. who found that phenolic acids were higher than those of the old leaves [26].
The various terpenoid metabolites were obtained including monoterpenoids, sesquiterpenoids, triterpene and triterpene saponin (Fig. S3, Table S5). It was found that the 54 different terpenoids substances examined herein, the majority of metabolites presented a declined trend with the highest down-regulation being observed in the growth stage of the leaves (MpBud vs. MpTen), such as Ursolic acid and Morolic acid. While, eight metabolites were observed for the examples including the up-regulation of Dehydrovomifoliol and the down-regulation of Mussaenoside during the maturation stage (MpTen vs. MpMat). In addition, 11 DAMs with the example substances of Dehydrovomifoliol and Asiatic increased significantly, and 27 down-regulated metabolites were observed for Mussaenosidic acid and Mussaenoside in the whole growth stage of Mp leaves. Additionally, almost all five triterpenes (e.g., Asiatic acid), one triterpene saponin of Cadambagenic acid, and one monoterpenoid of Sweroside were remarkably up-regulated, and seven monoterpenoids with the example metabolites including Geniposidic acid and Mussaenosidic acid that were unique to this stage (MpBud vs. MpMat). A large number of terpenoids, especially Mussaenosidic acid and Mussaenoside, observed in the bud leaves were higher than those of the tender leaves or mature leaves, which confirmed that the abundant terpenoid compounds possessed antibacterial, anti-inflammatory and antioxidant effects [27, 28].
Alkaloids are considered the primary bioactive components in plant chemicals, which possess various bioactivities because of their properties [29]. Among the 16 different alkaloids substances examined herein (Fig. S3, Table S6), 14 differences exhibited remarkable up-regulation (e.g., 3-Indoleacrylic acid) or down-regulation (e.g., Betaine) during the whole growth stage (MpBud vs. MpMat). Nine DAMs involved in the three accumulated metabolites (e.g., Caffeine and N-Acetyl-5-hydroxytryptamine) and six decreased substances (e.g., 2-Phenylethylamine and Indole-3-cyano-2-O-glucoside) were observed in the growth stage of Mp leaves (MpBud vs. MpTen). In addition, only four DAMs with the three down-regulated metabolites such as Caffeine, 2-Phenylethylamine and N-Oleoylethanolamine were found in the maturation stage (MpTen vs. MpMat). While, almost all six substances with the example of 3-Indoleacrylic acid and 2-Glucosyl-glucosyloxy-2-phenylacetic acid amide (except for the metabolite of Betaine) that were unique to the whole growth stage showed an up-regulated trend. The health benefits of tea plant are associated with its abundant content of caffeine. Although the quantities of caffeine in the bud and tender leaves were slightly higher than that in the mature leaves, which result was consistent with a study reported by Koushik et al. who showed that the content of black tea processed by the fresh and tender buds or leaves from spring was higher than that of mature leaves from monsoon and autumn seasons [30].
As for lignans and coumarins, 14 different substances were detected herein (Fig. S3, Table S7), of which eight metabolites significantly changes with five up-regulated differences (e.g., Daphnin) and three down-regulated differences (e.g., 6,7-Dihydroxy-4-methylcoumarin) across the whole growth stage of Mp leaves (MpBud vs. MpMat). While five significantly accumulated metabolites involved in Daphnin and Lariciresinol-4'-O-glucoside, and seven remarkably decreased substances such as 6,7-Dihydroxy-4-methylcoumarin and Scopoletin (7-Hydroxy-6-methoxycoumarin) were observed as the buds growing into the tender leaves (MpBud vs. MpTen). In addition, five DAMs significantly up-regulated (e.g., Lariciresinol-4'-O-glucoside) or down-regulated (e.g., Epipinoresinol*) in the maturation stage (MpTen vs. MpMat). It was found that two up-regulated substances were unique to the whole growth stage of Mp leaves such as Esculin (6,7-DihydroxyCoumarin-6-glucoside) and Syringaresinol-4'-O-(6''-acetyl) glucoside.
Primary metabolites
Primary metabolites involved in amino acids, organic acids, lipids or sugars are essential for maintaining the life activities of cells and function as an important energy resource and some small molecular compounds for secondary metabolism [31].
Free amino acids not only bring fresh and brisk tastes to Mp tea infusion, participating in the formation of aroma substances, but also contribute to nutritional and functional ingredients [32]. Of these 33 different amino acids and their derivatives examined herein (Fig. S4, Table S8), 30 DAMs showed significant changes across the whole growth and development of Mp leaves. 15 DAMs were significantly up-regulated as the buds grow into tender leaves, of which L-Arginine and S-Sulfo-L-Cysteine were most clearly affected by the growth and development of Mp leaves. While, only six metabolites significantly up-regulated or down-regulated in the mature stage of leaves, such as L-Serine, L-Glutamine, and N-Alpha-Acetyl-L-Asparagine. In addition, 12 DAMs were unique during the mature period, including nine up-regulated metabolites (e.g., ophan, γ-Glutamyl-L-valine), and three down-regulated metabolites (e.g., 5-Oxoproline). These majority of up-regulated metabolites during the growth stage might contribute to the formation of taste and aroma substances.
Organic acids function as the important intermediate products of carbohydrate catabolism, which contribute to the vinegar taste and fruity flavor of Mp tea, simultaneously restrain the bitterness and sourness [33]. It was found that 21 different organic acids examined herein (Fig. S4, Table S9), 19 substances showed significant changes during the process of buds growing into mature leaves with the majority down-regulation. A total of 11 metabolites exhibited remarkably alteration, among which nine substances down-regulated (e.g., Shikimic acid, Triethyl citrate and 2-Picolinic acid) and two compounds up-regulated (e.g., α-Ketoglutaric acid). While, only two metabolites significantly up-regulated (Citraconic acid) or down-regulated (L-Pipecolic Acid) as the tender leaves grow into the mature. The majority of the down-regulated metabolites detected in our research was consistent with Zhou et al. who reported that the content of organic acids in fresh leaves was obviously higher than that in mature leaves [34]. In addition, eight DAMs involved in three up-regulated (e.g., γ-Aminobutyric acid) and five down-regulated metabolites (e.g., Tartronate semialdehyde*) were unique organic acid substances during the process of maturation, which acted as a crucial role in reconciling the taste of the Mp tea, and contributed to improve the functional and nutritional components.
Lipids in fresh tea leaves are considered to be responsible for the production of flavor and aroma substances [35, 36]. A total of 16 lipids examined herein (Fig. S4, Table S10), among which 11 substances significantly up-regulated (e.g., 13-methylmyristic acid and Palmitoleic Acid) or down-regulated (e.g., LysoPC 16:1 and LysoPC 18:1). While, 12 metabolites were found to be remarkably altered with the most down-regulated differences in the growth stage of Mp leaves (MpBud vs MpTen), such as Methyl linolenate, Hydroxy ricinoleic acid and LysoPC 18:4. However, only two LysoPE 20:3 and LysoPC 18:4 were detected and both showed a downtrend. In addition, only three up-regulated or down-regulated compounds were unique to the mature stage of Mp leaves (MpBud vs MpMat) involved in (5S,8R,9Z,12Z)-5,8-Dihydroxyoctadeca-9,12-dienoate and 1-Stearidonoyl-Glycerol. The significant accumulation of majority LPC compounds, such as LysoPC 15:0, LysoPC 16:1 and LysoPC 18:1, were observed in buds and tender leaves, which was consistent with a study reported by Liu et al. who showed that PC compounds in new shoots were higher than that in mature leaves [37], indicating that highly abundance of lipid metabolites contributed to the formation of tea aroma.
Total phenolics, flavonoids, terpenoids, anthocyanin, and antioxidant activity
Total phenolics, flavonoids, terpenoids, anthocyanin, DPPH radical scavenging activities, radical cation ABTS + scavenging activities, and ferric reducing antioxidant power (FRAP) were displayed in Fig. 6. Total phenolics, flavonoids, anthocyanin, DPPH, ABTS, and FRAP ranged from 4.27 mg/g to 6.24 mg/g, 3.66 mg/g to 5.94 mg/g, 0.82 µg/g to 24.7 µg/g, 9.93–23.59%, 108.32 to 150.87 µmol Trolox/g, and 87.05 to 140.16 µmol Trolox/g, respectively. The contents or activities of bud leaves was significantly higher than those of the other two samples, with tender leaves being the lowest, and there is no significant difference with mature leaves. However, terpenoids in the three samples ranged from 0.59 mg/g to 0.91 mg/g. The contents of mature leaves were significantly higher than those of the other two samples, with bud leaves being the lowest.
These results showed that during the growth and development of Mp leaves, the contents of total phenolics, flavonoids, and anthocyanin gradually decreased, while the contents of terpenoids increased significantly, indicating these components contributed to enhance the functional and nutritional ingredients of Mp leaves. Phenolic compounds involved in phenolics, flavonoids and anthocyanin are considered as the primary antioxidant components of diverse plants, whose contents in bud leaves were higher than those of mature leaves [38]. The relatively abundant contents of total these three components were observed in the buds, which were results that were confirmed in this study. Meanwhile, the high accumulation of anthocyanin was found in the bud leaves with the purple-red color, which was believed to be associated with the formation of leaf color [39]. The comparison of DPPH, ABTS, and FRAP values of the three samples exhibited a remarkable difference in antioxidant activities among Mp leaves, with the antioxidant activities for bud leaves in Mp being higher than that of mature leaves. This finding was positively related to the content changes of total flavonoids, phenolics and anthocyanin, which indicated that the high total phenolic compounds might contribute to the antioxidant activity [40].