In vitro experimentation on plant tissue cultures gives a wide range of secondary metabolites that are used as therapeutics, flavors, fragrances, colors, and food additives [1
]. For increasing the entire biomass and production of high amounts of secondary metabolites in plant cell cultures, the use of biotic and abiotic elicitors has been recognised as one of the most effective strategies [3
]. Some such pharmaceutical secondary metabolites, the triterpenoids betulin and oleanolic acid, can be extracted from the bark of Betula platyphylla
Suk. Triterpenoids are excellent drug candidates, with antiviral, antibacterial, antitumor, and anti-AIDS activity [5
]. In our previous study, betulin was found in B. platyphylla
cell cultures, and nitric oxide (NO) was shown to be an effective elicitor to stimulate triterpenoid synthesis in Betula platyphylla
cell cultures [8
Nitric oxide (NO) is a small gaseous radical, defined as a multi-purpose molecule, which is present throughout the plant life cycle [10
]. It is involved in diverse physiological processes such as stomatal closure, as well as pathophysiological processes such as biotic and abiotic stress responses [11
]. The biosynthesis of NO and plant secondary metabolites was reported to protect plants from attack by insect, herbivores, and pathogens, or to survive under abiotic stresses, and NO also plays important roles in the accumulation of secondary metabolites in plants [12
], so a better understanding of the role of NO in the biosynthesis of such secondary metabolites is very important for optimizing the commercial production of those pharmaceutically significant secondary metabolites. However, holistic approaches to detect a wide range of metabolite changes due to the effect of NO have not yet been performed for betulin synthesis in B. platyphylla
Metabonomics aims to compare the relative differences between biological samples based on their metabolite profiles. It can provide a snapshot of the entire physiology of an organism [13
]. Metabonomics, therefore, may be a good method to get a ‘holistic view’ of the actual responses of plants to NO elicitor. So, in this study, Proton Nuclear Magnetic Resonance (1
H-NMR)-based metabonomics was employed to characterize the metabolic profile of the intracellular metabolites change in response to NO elicitor. The objective of the work reported here was to carry out the analysis of these key metabolites combined with corresponding pathways, which served as a guide for enhancement production of secondary metabolites. Furthermore, rational exogenous feeding strategies were proposed according to the results of comparative metabolic profiling analysis towards improvement of betulin production.
In a previous report, SNP treatment increased the gene expression of key enzymes in the triterpenoid biosynthesis pathway in suspension cells of birch. For example, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the formation of methylerythritol 4-phosphate is catalyzed by deoxyxylulose phosphate isomerase (DXR), 2,3-oxidosqualene can be synthesized in the presence of squalene epoxidase (SQS) and then formation of oleanolic acid and betulin catalyzed by β-amyrenol and lupeol synthase, respectively [8
]. The main pentacyclic triterpenoids in birch were betulin, betulinic acid and oleanolic acid [15
]. On this study, we verified that SNP treatment enhanced the betulin production. This result was consistent with the above result at transcription level.
In the study betulin production associated metabolites and metabolic remobilization under NO treatment, profiling of metabolites, including sugars, amino acids, organic acids and secondary metabolites (in total, approximately 41 annotated metabolites), was performed using samples obtained from suspension cells of birch. The results showed that the response to NO treatment was much more than the simple induction of production of triterpenoid.
Among the 41 annotated metabolites, only the content of glucose and fructose in carbohydrate metabolism were all enhanced at SNP treatment and cPTIO treatment, and the result of SNP treatment was consistent with that of transcription level. The increased glucose and fructose at SNP treatment and cPTIO treatment was, at a first glance, difficult to interpret. Further statistical analysis showed that VIP value of fructose was greater than 1, and its correlation with betulin content was from −0.866 under cPTIO treatment to 0.530 under SNP treatment. These indicated that fructose could be used as the identified metabolite to differentiate different NO treatments, and had great contribution for betulin production. Combinatorial feeding strategies under SNP and fructose treatment further enhanced betulin production. The above results suggested that fructose as a portion of the diverted carbon was shifted toward betulin production.
The response of 13 amino acids (except for serine, phenylalanine, arginine, glutamine, 4-aminobutyrate and methionine), six organic acids, two purine nucleosides and two alkaloids to different NO treatments were opposite. SNP treatment increased the content of 13 amino acids, six organic acids and two purine nucleosides, and reduced the content of two alkaloids. These results indicated NO treatment altered shikimate, amino acids metabolism, alkaloids metabolism and organic acids metabolism in TCA. The above change in metabolism may change the carbon, nitrogen and energy metabolism, in turn, increase of betulin production.
The shikimate pathway produces the three proteinogenic aromatic amino acids, phenylalanine, tyrosine and tryptophan, which are, in addition to several intermediates of the shikimate pathway, intermediates in the biosynthesis of numerous aromatic natural products in higher plants [16
]. Trigonelline is one of their downstream products. In previous resport, UV-B or salt treatment enhanced the trigonelline production, and increased the phenylalanine ammonia-lyase (PAL) activity [17
], but our study showed that SNP treatment decreased the content of phenylalanine, tyrosine and trigonelline, and had no effect on tryptophan. The other alkaloid choline obtained similar result derived from the serine under SNP treatment. NO was the product of UV-B or salt treatment, and had different effect on trigonelline production. The reason may be a result of using different kinds of samples.
In the secondary metabolites biosynthesis of plant cell cultures, some precursor feeding at suitable concentrations with optimal exposure time can elevate the synthesis of secondary metabolites, but on the other hand, excess precursor concentration with improper exposure time may cause feedback inhibition of metabolite pathways [19
]. Determination of appropriate precursors and their concentration in precursor-feeding is essential to achieve higher production of secondary metabolites [20
]. Our exogenous feeding results based on comparative metabolic profiling showed that fructose or phenylalanine treatment further enhanced the production of betulin under SNP treatment, but myo-inositol had the opposite result. Myo-inositol is a sugar-like carbohydrate produced by most plants, and is important for phosphate storage, cell wall biosynthesis, the production of stress related molecules, cell-to-cell communication, storage and transport of plant hormones [22
]. Its detailed reason for above result in betulin production needs further study.
There also are notable gaps in our understanding the relationship between plant primary metabolism and secondary metabolism under NO treatment. Usually the major secondary metabolism classes produced by plants can be divided into three main groups: firstly, phenolic compounds; secondly, terpenoids/isoprenoids; and thirdly, nitrogen or sulfur containing compounds such as the alkaloids and glucosinolates, respectively. The three major classes of secondary metabolisms are produced from pathways of different primary metabolisms, including glycolysis, the TCA cycle, aliphatic amino acids, pentose phosphate pathway, shikimate pathway and notably the aromatic amino acids (AAAs) [23
]. However, 1
H-NMR analysis could not examine whole metabolites in living cells in our study. Therefore, more qualitative and quantitative information of metabolome are needed to comprehensive understand cellular differentiation pathway. Also metabonomics integration with transcriptome and proteome will give new insights towards the accumulation of secondary metabolism under NO treatment.