As an important medicinal and dual-use flower tea, Chrysanthemi Flos is widely popular. It usually needs to be dried before being used. The drying processes include shade drying, in which fresh Chrysanthemi Flos heads are placed in a cool, ventilated location until completely dry, and there is oven drying, in which fresh Chrysanthemi Flos heads are placed in a 50 °C oven, followed by complete drying. Previous studies have confirmed that the choice of different drying methods for fresh Chrysanthemi Flos has significant effects on metabolites and enzymes [6]. However, the effects of drying Chrysanthemi Flos using shade drying or oven drying methods have not yet been elucidated.
By comparing metabolomics and proteomics analyses, we found significant effects on primary and secondary metabolism when Chrysanthemi flos was processed via shade drying versus oven drying, which indicates that Chrysanthemi Flos undergoes complex metabolic regulation during the drying process. We described the differential regulation of key pathways (Fig. 5), including α-Linolenic acid metabolism, cyanogenic amino acid metabolism, glycine, serine, and threonine metabolism, phenylpropanoid biosynthesis, purine metabolism, pyrimidine metabolism, and starch and sucrose metabolism. The subsequent sections will discuss the functional classification of the accumulated metabolites and abundant proteins, as well as their associated metabolic reactions. These findings contribute to clarifying the intricate mechanisms of metabolic regulation in Chrysanthemi Flos throughout the drying process.
Fig. 5Changes in the biosynthesis of purine metabolism, pyrimidine metabolism, glycine, serine, and threonine metabolism, cyanoamino acid metabolism, α-Linolenic acid metabolism, starch and sucrose metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis in the FCF, SCF, and DCF groups. Red symbolizes up-regulation, while green symbolizes down-regulation; squares represent the differential protein expression trend of SCF vs. FCF; circles represent the differential protein expression trend of DCF vs. FCF (SurE: 5′/3′-nucleotidase; AK: adenylate kinase; AGT3: alanine: glyoxylate aminotransferase 3; GCSH: glycine cleavage system H protein; CDP: cytidine 5'-diphosphate; dCMP: deoxycytidylic acid; LOX2S: lipoxygenase; SUS: sucrose synthase; INV: β-fructofuranosidase; PAL: phenylalanine ammonia-lyase; 4CL: 4-coumarate-CoA ligase; F5H: ferulate-5-hydroxylase; CAD: cinnamyl-alcohol dehydrogenase; HCT: shikimate O-hydroxycinnamoyltransferase; CCoAOMT: caffeoyl-CoA O-methyltransferase; POD: peroxidase; CHI: chalcone isomerase)
Effects of drying methods on nucleotide metabolism in Chrysanthemi FlosNucleotides are one of the most important nitrogen compounds in any organism. The biosynthesis and metabolism of nucleotides are essential for plant growth and development. Purine and pyrimidine nucleotides are involved in many biochemical processes in plants. They are metabolites involved in bioenergetic processes and the synthesis of macromolecules including polysaccharides, phospholipids, and glycolipids [7]. In our study, we found that Chrysanthemi Flos showed a down-regulation of nucleotide metabolites, including 3′ 5′-Cyclic GMP, Guanine, Xanthine, L-Glutamine, Adenosine, Adenine, Inosine, Uridine, CDP, and dCMP. In the SCF and FCF comparison, AK expression was found to be down-regulated. In contrast, SurE was significantly up-regulated in DCF vs. FCF.
AK, an enzyme widespread across diverse life forms, facilitates the reversible transphosphorylation (2ADP ↔ ATP + AMP), essential for energy metabolism regulation and adenylate pool equilibrium [8]. Additionally, AK is instrumental in plant responses to environmental stressors [9, 10]. From our results, we found that AK was markedly down-regulated in SCF vs. FCF, which may be due to the reduction of its own energy consumption to maintain normal physiological activities in response to the stressful effects of low-temperature and low-drying environments during shade drying. However, prolonged darkness enhances purine catabolism, which gradually supplies the normal activities of mitochondria, chloroplasts, and other tissue cells by enhancing nucleotide metabolism [11]. During the process of drying in the shade, Chrysanthemi Flos is actually in a slow physiological process of "dying", gradually consuming nucleotide compounds to supply its own energy consumption to resist this abiotic stress response, which is in accordance with our metabolomics data.
We found that the SurE of Chrysanthemi Flos was up-regulated during the oven-drying process. SurE efficiently catalyzes the dephosphorylation of 5'-ribonucleotides and 5'-deoxyribonucleotides, yielding nucleosides and inorganic phosphate, and demonstrates broad specificity by acting on various phosphorylated substrates. SurE can be involved in physiological processes such as purine and pyrimidine salvage pathways, inter-cellular signaling, and the control of ribonucleotide and deoxyribonucleotide pools [12]. The up-regulation of SurE in DCF vs. FCF may have resulted from the fact that DCF was exposed to high temperatures, which could resist this high-temperature drought stress response by promoting the metabolism of purines and pyrimidines and generating more energy.
Effects of drying methods on α-linolenic acid metabolism in Chrysanthemi FlosIn α-Linolenic acid metabolism, α-Linolenic acid can be liberated from the several complex fatty acids located mainly in the membranes of organelles such as chloroplasts. α-Linolenic acid modulates the transcription of genes implicated in diverse abiotic stress responses, including those to drought, salinity, and physical damage. α-Linolenic acid is involved in a number of pathways that are not only directly related to biological responses but also to a variety of abiotic stress conditions, suggesting that it plays a very important role as a signaling mediator [13]. α-Linolenic acid can be converted to 13(S)-hydroxylinolenic acid and 9(S)-hydroxylinolenic acid through a reaction catalyzed by LOX2S [14]. A recent study found that methyl jasmonate applied to post-harvest peaches mitigated their vulnerability to cold injury when stored at low temperatures [15]. Methyl jasmonate has been shown to stimulate α-linolenic acid metabolism, leading to enhanced membrane lipid unsaturation via a progressive reduction in unsaturated fatty acids (USFAs) coupled with a rise in saturated fatty acids (SFAs). α-Linolenic acid metabolism enhancement may contribute to the reduction in cold injury in peaches during cold storage. Intriguingly, we found that LOX2S was down-regulated in DCF vs. FCF. This may be due to the inhibition of LOX2S enzyme activity in Chrysanthemi Flos under high-temperature environments. Meanwhile, stearidonic acid was found to be down-regulated in both SCF and DCF, indicating that Chrysanthemi Flos reduces α-Linolenic acid consumption during drying. In plants under abiotic stress, lipids are broken down into free fatty acids [16]. As a free fatty acid, α-linolenic acid exhibits potent antioxidant properties and serves as a substrate for jasmonic acid production, which in turn functions as a signaling molecule, triggering subsequent stress mitigation pathways [17]. Chrysanthemi Flos resisted abiotic stress by maintaining a high concentration of α-Linolenic acid under the two drying methods, which was confirmed by the results of metabolite NO. 1457 (α-Linolenic acid) expression trend in FCF, SCF and DCF groups in Table S1.
Effects of drying methods on amino acid metabolism in Chrysanthemi FlosAmino acid metabolism is intricately linked with the dynamics of energy and carbohydrate fluxes, carbon and nitrogen allocation, and the biosynthesis of proteins and secondary metabolites [18]. According to our proteomics and metabolomics studies, glycine, serine and threonine metabolism and cyanoamino acid metabolism, namely, the amino acid metabolic pathways of Chrysanthemi Flos, were significantly affected when the flowers were processed using shade drying and oven drying.
Regarding glycine, serine, and threonine metabolism, threonine showed different degrees of down-regulation in both SCF vs. FCF and DCF vs. FCF. AGT3 was up-regulated in SCF vs. FCF, and GCSH was down-regulated in DCF vs. FCF. Threonine is an obligatory (amino acid) metabolite that is readily interconnected with methionine and isoleucine and reciprocally converted with glycine and serine [19, 20]. Threonine signals the plant's own receptors and converts them to specific functional outputs (for example, changes in plant growth, development, and metabolism, the expression of storage protein genes, abiotic stress tolerance, cell growth and division) when the plants are subjected to unfavorable conditions (e.g., abiotic stress tolerance) and other physicochemical factors [21, 22]. As shown in Fig. 5, threonine was lower in both DCF and SCF than in FCF; meanwhile, AGT3 was up-regulated, which may be due to the fact that the drying environment caused Chrysanthemi Flos to produce more threonine for resisting this stress on the one hand, and on the other hand, as one of the metabolic branches of the aspartic acid family pathway, threonine can be easily converted to glycine for subsequent amino acid metabolism, which provides nutrients for protein synthesis, secondary metabolism and other life activities [19, 23]. We found that GCSH, an aminomethyl-carrying intermediate in the glycine cleavage system (GCS) that carries hydrogen through the thiooctanoyl portion of the prosthesis, was down-regulated in SCF vs FCF [24]. The GCS comprises the T-protein, P-protein, L-protein, and H-protein, though the stability of their complex formation remains a subject of debate [25, 26]. Critical for photorespiration in C3 plants and for a recovery pathway in photosynthesis, the GCS's activity is notably low in senescing pea leaves but can surge by an order of magnitude upon light exposure [26]. When Chrysanthemi Flos is in a dry environment sheltered from light, the down-regulation of GCSH may be due to the inhibition of the glycine cleavage system reaction in SCF vs. FCF.
Cyanoamino acid metabolism is positively correlated with biotic stress tolerance [27]. L-isoleucine and L-valine are nutrients in the branched-chain amino acid (BCAA) group that are essential for humans and animals [28]. BCAAs play a key role as an osmotic regulator in plant stress tolerance [29]. In a study of Arabidopsis thaliana’s tolerance to drought stress, the branched-chain amino acid catabolic pathway was found to be required for its dehydration tolerance process [30]. In our study, isoleucine and valine showed various degrees of down-regulation in SCF vs. FCF and DCF vs. FCF, and it might be the case that these amino acids can be most efficiently used as alternative respiratory substrates during gradual senescence to death or carbohydrate starvation in Chrysanthemi Flos [31]. Meanwhile, the proteomics data indicated that β-glucosidase was down-regulated in DCF vs. FCF, potentially due to the heightened catabolic activity of branched-chain amino acids during high-temperature drying. The metabolomic data affirmed that more lotaustralin and linamarin were synthesized to combat this environment.
Effects of drying methods on sugar metabolism in Chrysanthemi FlosIn plants, sucrose constitutes the principal photosynthetic output, acting as a crucial substrate for energy and a regulatory signal that modulates plant development. Additionally, sucrose plays a crucial role in responding to various abiotic stresses [32]. Exposure of Chrysanthemi Flos to desiccation stress under low or high thermal conditions alters the plant's carbon allocation and metabolism, leading to a depletion of energy reserves and a consequent decline in yield [33]. The accumulation of sugars in response to drought may influence the regulation of sugar and carbohydrate metabolism as well as their translocation within the plant [34, 35]. Our research indicates that the dehydration process markedly affects the dynamics of the starch and sucrose metabolism in Chrysanthemi Flos. Moreover, sucrose metabolism is crucial not only for plant development but also for abiotic stress responses [36, 37]. SUS was significantly up-regulated in SCF vs. FCF, while INV was markedly down-regulated in DCF vs. FCF. Furthermore, the differences between trehalose and inulin were also observed in SCF and DCF.
Starch and sucrose metabolism are pivotal in the growth and developmental processes of plants. The assimilation of sucrose into cellular metabolism is facilitated by two key enzymes: SUS and sucrose convertase [38]. Acting as a glycosyltransferase, SUS plays an integral role in the synthesis of starch and proteins, as well as in the generation of energy, predominantly within storage tissues [34, 35]. It catalyzes the reversible conversion of sucrose into fructose and nucleotide diphosphate glucose, either uridine diphosphate glucose (UDP-G) or adenosine diphosphate glucose (ADP-G). The metabolites produced through SUS-mediated sucrose breakdown are crucial for a host of metabolic functions, including energy generation, primary metabolite biosynthesis, and the formation of complex carbohydrates [39]. Research examining the effects of water deficit on the accumulation, translocation, and breakdown of carbohydrates in the foliage and roots of two soybean cultivars with comparable fertility levels revealed a notable elevation in SUS activity under conditions of water scarcity [32]. Moreover, it has been observed that SUS exhibits optimal activity at a temperature of 37 °C and maintains stability up to 50 °C [40]. The heightened activity of SUS in SCF relative to FCF may be ascribed to the enhanced enzymatic function in response to cooler, arid conditions.
In plant storage tissues, sucrose can be cleaved by both SUS and INV. SUS catalyzes the cleavage of sucrose into glucose and fructose in a reaction that is generally irreversible under physiological conditions [35]. In contrast, INV mediates a reversible reaction that converts sucrose and UDP into UDP-glucose and fructose. Sucrose requires more ATP to enter metabolism via INV than via SUS, providing more opportunities to coordinate this process with the carbon requirements of cellular metabolism [41]. Notably, abiotic stress suppresses the expression and activity of INV. Severe stress can cause plants to cease photosynthesis entirely, reducing the translocation of sucrose to the reservoir organs and leading to the repression of certain INV genes [35, 42]. The impact of drought stress on INV activity in soybean pods was confirmed by a study [43]. This finding is consistent with DCF where INV was down-regulated. Alginate, an intermediate product of starch and sucrose metabolism, plays a crucial role in reducing damage [44]. The marked up-regulation in alginate observed in DCF vs. FCF may be due to the fact that Chrysanthemi Flos reduces environmental damage to itself by increasing sugar metabolism under high-temperature conditions.
Sucrose must be broken down by the SUS enzyme to UDP-G and fructose, or by the INV enzyme to glucose and fructose. The need for these two sugar-degrading enzymes in plants is still not fully understood [39], as they may have different or complementary functions and energy requirements. SUS requires less energy for degradation than INV. It is common for them to operate in an apparently inefficient cycle, which could be significant for facilitating sucrose import and utilization or as a potential source of inefficiency [45]. Furthermore, both forms of sucrose catabolism present a promising energy source for energy production in Chrysanthemi Flos in response to various factors such as abiotic stress and metabolic regulation [46].
Effects of drying methods on phenylpropanoid biosynthesis in Chrysanthemi FlosChrysanthemi Flos contains abundant flavonoids, phenylpropanes, and other phenolic compounds, of which flavonoids and phenylpropanes are the main active components, with biological functions such as anti-inflammatory, antimicrobial, antioxidant, blood-pressure-lowering, cholesterol-metabolism-accelerating, and immune-modulating activities [2]. Phenylpropanoid biosynthesis is a crucial secondary metabolic pathway in Chrysanthemi Flos, contributing significantly to its growth, development, and interactions with the environment [47]. The reactions initiating phenylpropanoid biosynthesis are PAL, C4H, and 4CL, catalyzing phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and 4-coumarate-CoA ligase. Generally, phenylalanine, which is the initial substrate of phenylpropanoid biosynthesis, undergoes conversion to cinnamic acid via PAL. Cinnamic acid is transformed into p-coumaric acid by C4H and subsequently activated by 4CL, yielding p-coumaroyl-CoA. This compound generates precursors for various downstream branches of the metabolic pathway [48]. Our discussion emphasizes the lignin and flavonoid pathways, which are the two primary branches of phenylpropanoid biosynthesis.
In our investigation, a comparative analysis between SCF and DCF against FCF revealed that within the phenylpropanoid biosynthetic pathway, the levels of sinapic acid and sinapyl alcohol were suppressed, while caffeoyl shikimic acid concentrations increased. Concurrently, the expression of enzymes PAL, 4CL, β-glucosidase, CAD, and POD inhibited, whereas F5H, HCT, and CCoAOMT enhanced significantly. Corroborating these findings, another study demonstrated that the spike organ of wheat under drought stress exhibited enhanced tolerance associated with the phenylpropanoid pathway, evidenced by elevated activities of PAL, C4H, and 4CL, alongside an increase in the antioxidant enzyme POD's activity [49]. In addition, the lignin synthesis pathway plays a key role under low temperature [50].
The complex regulatory mechanism of phenylpropanoid biosynthesis in Chrysanthemi Flos varies under different processing modes, as depicted in Fig. 5. PAL, the initial enzyme in the phenylpropanoid pathway, mediates the diversion of metabolites from the shikimic acid pathway to various phenylpropanoid branches by facilitating the synthesis of trans-coumaroyl-CoA from phenylalanine [51]. In contrast, 4CL facilitates the generation of p-coumaroyl-CoA in an ATP-dependent manner. Additionally, it promotes the attachment of multiple phenylpropanoid compounds to CoA, and various 4CL homologs display varying enzymatic preferences for distinct phenylpropanoid substrates [52]. Both light quality and intensity affect the expression of the genes responsible for phenylpropanoid biosynthesis in plants, with UV light exhibiting more significant inducible effects on PAL genes than white light [53]. PAL and 4CL also show clear co-expression characteristics [54]. In our study, PAL and 4CL were down-regulated in SCF vs. FCF and DCF vs. FCF, possibly attributed to light avoidance inhibiting the expression of PAL and 4CL in Chrysanthemi Flos. Additionally, CAD is integral to lignin biosynthesis, catalyzing the transformation of cinnamic aldehyde to its alcohol form, a process that contributes to lignin's accumulation and structural heterogeneity. Meanwhile, POD facilitates the last stage of lignin biosynthesis by promoting the polymerization of lignin’s phenylpropanoid precursors [55]. Both CAD and POD were down-regulated in SCF vs. FCF and DCF vs. FCF, which may potentially lead to the gradual senescence and eventual death of Chrysanthemi Flos. However, it is noteworthy that sinapyl alcohol showed an up-regulation in SCF vs. FCF, which contrasts with the trend of down-regulation observed in CAD expression in SCF. Although CAD is identified as the main enzyme catalyzing the biosynthesis of monolignol, SAD (sinapyl alcohol dehydrogenase) is actually involved in lignin biosynthesis [56, 57]. When CAD is down-regulated, its function may be compensated by an enzyme with similar activity. Preisner et al. utilized semi-quantitative PCR to analyze the gene expression in vitro cultures of CAD-reduced flax [57]. They found that CAD silencing resulted in a decrease in the activity of CAD genes in CAD27 and CAD33. Surprisingly, rational overexpression of the SAD gene compensated for the effects of CAD silencing on flax. The trend towards up-regulation of sinapyl alcohol, a CAD-regulated downstream metabolite, in SCF may be attributed to the compensatory mechanism of SAD. Meanwhile, the F5H enzyme, which is the third P450 enzyme involved in lignin monomer biosynthesis, facilitates nicotinamide adenine dinucleotide phosphorylation and the O2-dependent hydroxylation of a range of phenylpropanoid metabolites [58, 59]. The up-regulation of F5H was observed in both SCF and DCF, suggesting that this change may enhance the ability of Chrysanthemi Flos to resist the abiotic stresses that increase the synthesis and consumption of sinapic acid. Notably, the metabolic pathway from Coumaroyl-CoA to Sinapoyl-CoA showed an up-regulation of both metabolites and proteins. HCT, a bifunctional enzyme, facilitates the partial transfer of caffeoyl back to CoA, directing metabolic flow away from the general phenylpropanoid pathway towards lignin monomer biosynthesis [60, 61]. Additionally, CCoAOMT catalyzes methyl transfer reactions in lignin monomer biosynthesis [62]. Interestingly, HCT can cause a shift in metabolic flow towards flavonoid biosynthesis by activating lignin biosynthesis. This highlights HCT's key function as a gateway enzyme for lignin biosynthesis [63, 64]. We hypothesize that Chrysanthemi Flos will primarily exhibit resistance to low-temperature drying or high-temperature arid environments through this pathway during drying.
Flavonoid synthesis represents a significant segment of the phenylpropanoid pathway, yielding a vast array of polyphenolic compounds. Characterized by a C6–C3–C6 skeleton that includes two aromatic rings (A and B) and a central heterocyclic pyran ring (C), these metabolites are pivotal in plant defense, offering protection against various biotic and abiotic challenges including UV-B radiation, cold stress, and water scarcity [65]. There are two forms of CHI present in plants, and type I CHIs are found in Chrysanthemi Flos. They primarily convert chalcone to flavanones using 6′-hydroxychalcone as a substrate [66, 67]. Our investigation revealed that Chrysanthemi Flos’ flavonoid biosynthesis was active during processing, resulting in the up-regulation of CHI. The hydroxylation of flavonoids was found to improve their metabolic stability, membrane permeability, solubility, and antioxidant properties [68]. This could be because Chrysanthemi Flos promote the hydroxylation of flavonoids during drying to counteract their own negative effects [69]. In summary, phenylpropanoid biosynthesis plays a crucial role in the growth and development of plants as well as their interactions with the environment. Phenolic compound synthesis in plants escalates as a ubiquitous defensive response to stress, providing a shield against a spectrum of abiotic threats [70]. These compounds also play a vital role in a host of physiological functions, enhancing the plant's resilience and adaptive capacity to less-than-ideal environments [71,72,73]. The phenolic compounds in Chrysanthemi Flos contribute significantly to the plant’s resistance to the drying environment. However, as a natural consequence of harvesting, the inflorescences of Chrysanthemi Flos are detached from the chrysanthemum plant, leading to their eventual demise as discrete organs.
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