2.2 Ideas and Principles
2.2.1 Identi fication [ 1 ]
Identification is one of the important study contents of pharmacognosy. The main work of it is to discriminate the genuine and false crude drugs and to evaluate the excellent and inferior in quality of crude drugs. The crude drugs mainly originate from plants, and partly originate from creatures and minerals. It is very important to identify and evaluate the crude drugs because the crude drugs from various species will lead to the difference both in contents and categories of active ingredients. The identification is often based on the following characters of crude drugs: the morphology (character identi fi cation), the tissue structure (microscopic identi fi cation), the secondary metabolites (physical and chemical identi fi cation), the genetic information (molecular identification), and other aspects of characters. With the development of science and technology, the research area of the identification technology is constantly evolving. The methods of pharmacognosy study mainly include (1) identification of botanical origin, (2) trait identification, (3) microscopic identification, (4) physical and chemical identification, and (5) molecular identification. The current identification scope of pharmacognosy includes:
Related Species Identification:
Many crude drugs and Chinese herbal medicines are multisource species. This part of crude drugs needs to be identi fi ed. At present, the closely related species identi fi cation and quality evaluation of crude drugs are mainly made by using the traditional identi fi cation methods. Because of the similarities in closely related species on morphology, tissue structure, and chemical composition, their identi fi cation is very dif fi cult. As technology advances, the traditional identification methods combined with molecular biology techniques such as the popular DNA bar coding technology will be an effective means to identify the closely related species.
Identification for Analogs of Valuable Chinese Materia Medica:
The rare and valuable Chinese materia medica (CMM) is one of treasures of traditional Chinese medicine; meanwhile, it is also the object of counterfeit medicines easily produced. The resources of many rare CMM such as Cordyceps sinensis, antler, agallochum, saffron, Dendrobium candidum , musk, etc. are limited, and they are more expensive. Identi fi cation of these herbs is often difficult. Take herbs of agallochums for example. Most of them are imported from the Southeast Asian countries. Both inferior and counterfeit varieties are frequently found in the market.
Because the shapes between the spurious and genuine are very similar, in addition that the spurious’ 95% ethanol extract is higher than that of the genuine one, the common identi fi cation method is sometimes dif fi cult to entirely ensure the identi fi cation results. Recently, with the DNA bar coding technology, E-bionic technology, and GC-MS technology widely used, the authenticity of identi fi cation for valuable CMM has been greatly improved.
Identi fi cation for the Genuineness of CMM:
The genuineness refers to the study of Chinese materia medica which is produced from specific areas and recognized as the authentic and famous in clinic effects. Genuine herb is the medicine of the original species with long-term breeding in specific environmental conditions and specific production process and eventually becomes a certain crude drug in good quality. The formation of genuine medicine can be summarized as genetic model oriented, eco-oriented, and so on. From a species perspective, authentic herbs can originate from both single varieties and multi-varieties. But even if the single varieties of herbs are considered, their original genetic characteristics of species will continue to divide and form a local special variation of the base of gene. As a result, it is the formation of a genuine herb because of this genetic diversity and variability. Since authentic species of medicinal herbs and non-genuine ones are often very similar in morphology, properties, and chemical composition of crude drugs, their identi fi cation is often very difficult. As an important means of modern molecular biology techniques, the DNA molecular markers and DNA bar coding will play a key role in explaining the biological mechanism in the formation of the genuineness of CMM.
2.2.2 Pharmaphylogeny [ 2 ]
When it comes to the field of study in pharmacognosy, there is a very important part
that aims at exploring the relations among the distribution of biologically active substances, efficacy and plant evolution. Xiao Pei-gen fi rstly put forward the theory of pharmaphylogeny which was concluded from the theory of certain links among plant genetic relationship, chemical composition, and ef fi cacy. It provides a theoretical guidance for the development of medicinal plant resources. Pharmaphylogeny is the study of the correlations among genetic relationships, chemical components, and ef fi cacies of medicinal plants. It is a new interdisciplinary subject in the field of medical plants study, which involves plant taxonomy, plant phylogenetics, phytochemistry, pharmacology, numerical taxonomy, genomics, information science, and other computer technology-related disciplines (Fig. 2.1).
Pharmaphylogeny is characterized by multidisciplinary and infiltration and the scope of their research must be the combination of multidisciplines. Therefore, the study also involves multidisciplinary content:
Fig. 2.1 The framework of pharmaphylogeny
2.2.2.1 Information Science and Intelligence Science Research
With thousands of years of experience in using traditional Chinese medicine, in combination with a large number of international research data on active ingredients, pharmacological effects, and clinical ef fi cacy of medicinal plants, the key task that pharmaphylogeny tries to fulfill is how to use these valuable documents and resources of medicinal plants. Medicinal plants research information system is about the use of modern computer technology, information processing technology, and especially the “Knowledge Discovery in Database, KDD” technology, combination with the research results of chemistry, mathematics, biology, and Chinese medicine to establish and improve the database of medicinal plants, database of natural chemistry, and knowledge base of Chinese medicine. The goal is to dig the information of efficacy and related components in medicinal plants, combined with knowledge of plant systems to explore the law of distribution of active components in medicinal plants.
2.2.2.2 Distribution Law of Chemical Components in Plants
It is the basis to develop new drugs that find the distribution law of chemical components in plant system. Biological diversity leads to the variety of secondary metabolites, which makes it possible to provide human a variety of drugs. However, there is no theory to introduce the new drugs creation, which is bound to do less effectively. Therefore, the main task of pharmaphylogeny is to explore the distribution law of chemical components in plants and guide the screen and development of new drugs.
2.2.2.3 Chemosystematics
As the plant secondary metabolites of medicinal plants, active components in plants subject to gene regulation. It also has the genetic characteristics of the plants. The components’ accumulation in plants is closely related to species of plants and their phylogenesis. Therefore, the chemical components of plant can be thought as a strong evidence of plant systematics. So another task of pharmaphylogeny is to study the chemical evidence of plant systematics and enrich and improve the chemical taxonomy of plants.
2.2.3 Ingredient Phenotype [ 3 ]
As to the study of pharmacognosy, it is the focus of the quality formation in herbs. And the quality of herbs is based on the content of effective component (mainly the secondary metabolites) of herbs, whose production is closely related to secondary metabolism in medicinal plants. The secondary metabolism is controlled by enzymes in some related secondary metabolism pathway. Therefore, the core of the molecular pharmacognosy study is to disclose the relationship between genes encoding enzymes in the secondary metabolism pathway and secondary metabolites.
When it comes to the use of molecular biology techniques, such as gene cloning, transgenic technology to study the quality of tradition Chinese medicine, the greatest dif fi culty encountered, compared with the crop, is unknown or little known about the genetic information of medicinal plants. In another word, the unclear genetic information especially about genes in secondary pathway in medicinal plants has become a serious constraint of genetic engineering about secondary metabolism in medicinal plants. Because of the cloning of unknown functional genes in the unknown downstream of secondary pathway related to active components biosynthesis, it will become a dif fi cult job but an important task that has to be completed. Although the structure of active components is clear, their biosynthesized secondary metabolic pathways in medicinal plants are not clear, which brings the most dif fi culties in the related functional gene cloning and genetic engineering study. Currently, the rice and Arabidopsis genome sequencing have been completed. As the same with the human genome plan coming into post-genomic era focused on functional genomics, the post-genomic era of plant is also focused on the function of genes. With the reference to Arabidopsis genetic information, many important plant functional genes will be found. However, because of the big difference between the active components of medicinal plants and the structure of secondary metabolites in Arabidopsis, added with the unknown secondary metabolic pathway, it is dif fi cult to design and synthesize primers to clone functional genes related to the secondary metabolisms based on the reference genome information of Arabidopsis. Since most of secondary metabolic pathways for active ingredients biosynthesis in medicinal plant are not clear, it will be extremely dif fi cult to clone genes according to similar information of sequence of genes from other plants.
The classic study of plant secondary metabolic pathways is isotopic tracer method. Because this method has some dif fi culties in operation, along with isotopes of radioactive pollution and other unforeseen factors, the isotopic tracer method is limited to be widely used to some extent. To this point, we introduce a new idea and method, the “ingredient difference phenotypic cloning,” for secondary metabolic pathway of active ingredient and its related functional genes cloning.
This method does not require clear information of pathway for active ingredients biosynthesis and sequence information of homologous genes. It has lots of advantages, such as high-speed and high-flux cloning, without clear information about gene sequence of secondary metabolism-related enzymes and the pathways. This will undoubtedly provide new ideas and effective means to solve the bottleneck problem of active related ingredients. Its concepts, mechanism, and methods are introduced as follows:
2.2.3.1 Concept
“Ingredient difference phenotypic cloning,” a sort of phenotype cloning, is an effective strategy and method for the cloning of genes encoding key enzymes that regulate the secondary metabolisms. It is based on the phenotype of differences in ingredients (secondary metabolites) to clone functional genes that belong to some unknown secondary metabolic pathways by the technology of gene differential expression. The phenotypes of differences in ingredients include the difference in the content of them and the presence or absence of certain ingredients. This method has advantages of high fl ux, fast, and high ef fi ciency in gene cloning, and the most predominance of it is to clone the unknown functional genes in unclear secondary metabolic pathway.
2.2.3.2 Postulate
Under external stimulation, such as a variety of environmental stresses and elicitors’ stimulation, levels of gene expression in plant cells often abnormally increase, and it may lead to dramatic increase in secondary metabolites’ accumulation.
Since ingredients (secondary metabolites) in medical plants are easily tested, We can regard it as the phenotype of differential expression of genes and clone some objective genes in a certain secondary metabolic pathway by some technologies, such as differential display, suppression subtractive hybridization, and cDNA microarray.
2.2.3.3 Method
When it comes to the design of ingredient difference phenotypic cloning, the most important factor that has to be considered is how to make the biggest difference of active ingredients in plant cells between dealt groups of plants and the control ones. The operation in details is as follows: Firstly, add elicitors like heavy metals and other biological elicitors into the culture medium to make the biggest difference of active ingredients in the dealt groups of plant cells compared with that of control ones. After dealing with elicitors, the activity of secondary metabolism in plant cells will be stimulated and enhanced quickly, and the production of secondary
Fig. 2.2 Steps of ingredient difference phenotypic cloning
of metabolism, the active ingredients will be accumulated rapidly at the same time. Secondly, determine the content and categories of ingredients in different groups of cultured plant cells with HPLC or LC-MS and select pair groups with the biggest different content of ingredients for the study of “ingredient difference phenotypic cloning.” Finally, screen the objective fragment of genes with differential expression, clone the full length of cDNA, analyze the sequence, and verify the function. The steps of the method can be shown in Fig. 2.2 .
In this study, the first to do is to acquire the biggest variability of ingredients. Several methods can be used in ingredients analysis. For determination of the most active ingredients, HPLC method is usually used, but for some of volatile ingredients, GC method can be used, and for the unknown structure of ingredients, LC-MS is an ideal determination method. As to gene cloning method in the study of “ingredient difference phenotypic cloning,” the mRNA differential display reverse transcription PCR (DDRT-PCR), the suppression subtractive hybridization (SSH), cDNA microarray, and so on will be good methods available.
Advantages: Firstly, it has the advantages of high fl ux, fast and high ef fi ciency in gene cloning. Secondly, it will effectively clone the functional genes without necessity to know the exact homologous sequence information for multi-gene cloning and the ingredient-related biosynthesis pathway.
Table 2.1 Genes of key enzymes, precursors, and production of secondary metabolism in Salvia miltiorrhiza
2.2.3.4 Applications
Clone of Genes Encoding Key Enzymes of Secondary Metabolic Pathways With the introduction of “ingredient difference phenotypic cloning” strategy, we have cloned six cDNA fragments of genes encoding key enzymes involved in secondary metabolism of Salvia miltiorrhiza hairy roots dealt with elicitors of yeast extract by cDNA microarray analysis (Table 2.1). Among these six functional genes, five of them (SmAACT, SmCMK, SmIPPI, SmFPPS, SmKSL) encoded the enzymes of tanshinone biosynthesis and one (Sm4CL) was involved in the biosynthesis of salvianolic acid by blastx analysis, which involved secondary metabolic pathway analysis with online software of KEGG. After the analysis, full-length cDNA of these 5 genes were cloned by 3’race-PCR and 5’race-PCR method. The five genes involved in tanshinone biosynthesis were registered in GenBank online database.
Their GenBank code was shown to be in the order: F635969, EF534309, EF635967, EF635967, EF635968, EF635966. Help to Systemically Reveal Between the Biological Network of External Stimulating Factor and Secondary Metabolism and Elucidate the Mechanism of Gene Expression Regulation of Secondary Metabolisms
Here we elucidate the effects of elicitors on secondary metabolism as an example; to begin with, the elicitors bind with membrane receptors and introduced changes in membranes of which lead to changes of membrane’s permeability and internal ion distribution in membranes. At the same time, G-proteins may be coupled to receptors and mediate elicitor-induced ion channel activation. Ion fl uxes, especially Ca2+influx, cause cytosolic free Ca2+ spiking which causes activation of protein kinases, peroxidases, NADPH oxidases, and phospholipases, which further generate other signaling messengers, such as reactive oxygen species, DAG, IP3, cAMP, lysoPC, JA, ethylene, NO, cADP ribose, and SA. All these messengers compose paralleling or cross-linking pathways to integrate these signals into regulation of transcription factors (TFs) [ 4 ]. Various transcription factors integrate these signals to activate gene expression by transcription machinery. Most genes for secondary metabolite synthesis are late response genes. The response genes’ expression levels then affect their encoding enzymes in regulating secondary metabolite synthesis. That is the theory to explain the biological network relations between external stimulating factor and secondary metabolism.
With the development of functional genomics, proteomics, and metabolomics, many new and powerful tools could be applied to plant secondary metabolism study and improve overall understanding and practical manipulation of plant secondary metabolite production. The ingredient difference phenotypic cloning is a useful and powerful method mainly involved in transcript-profi ling differential analysis and secondary metabolites related genes cloning in medical plants. This method helps to identify more genes involved in biosynthesis of secondary metabolites but also facilitates isolation of some possible signal transduction components such as transcription factor and other regulation genes.
Taking advantages of “ingredient difference phenotypic cloning” methods, we have at the same time acquired signal transduction proteins, sulfate transporter, DNA binding/transcription factor, and other secondary metabolism-related genes.
The results and established methods lay a useful groundwork for future study of regulation mechanism of genes for ingredients biosynthesis (Table 2.2). The ingredient difference phenotypic cloning method makes people’s attention once again focus on the close relations between phenotype of secondary metabolites and genetic information in medicinal plants. To minimize the impact of external uncontrollable factors and make the maximum differential of secondary metabolites, the ideal strategy of the study system is to culture the tissue and cells of medicinal plants under the controlled condition and stimulate plant cells to produce the utmost differential phenotype by adding the elicitors into the culture medium. According to the phenotype difference, a great body of differentialexpressed objective genes would be efficiently cloned with high-throughput techniques of microarray.
Since secondary metabolism of plant is often affected by external environmental factors, profiling a group of secondary metabolites from plants under various environmental conditions helps to understand metabolic fl ux and the related regulatory mechanisms. Because of the close relationship between geographyrelated environmental condition and biosynthesis of secondary metabolites for defense, plant cells have various strategies to control metabolic flow directions.
This regulation is mainly controlled at enzyme and gene expression levels. Thus, the “ingredient difference phenotypic cloning” method can be widely used in the study on the formation mechanism of geo-authentic medical material at molecular level.
Combining the approaches of transcription profiling proteomics and secondary metabolite profiling, the method of “ingredient difference phenotypic cloning” offers the most powerful tool ever in studying all aspects of plant secondary metabolism as a whole.
2.2.4 Systems Biology
Secondary metabolite is a kind of micromolecular organic compound produced during the growth and development of plants along with the adaptation of outer environment. It has been estimated that the amount of secondary metabolites in plants is more than 100,000, including terpenoids, phenols, alkaloids, polyacetylenes, etc. [5] . So far, the studies on medicinal plant secondary metabolites have concentrated on such aspects as separation of chemical composition, structure determination, bioactivity, pharmacological actions, etc. However, the content of secondary metabolites in medicinal plants is relatively low, and the resources of natural medicinal plants are limited, which affect the quality control of medicinal plants and the exploitation of active ingredients. So it is obviously important to study the biological formation of secondary metabolites; to unearth genes of relevant enzymes, signal factors, or enviromental factors; and to systematically illustrate biosynthetic pathway, signal transduction pathway, and biological formation mechanism as well as the interaction of them.
Systems biology is a new fi eld that aims at system-level understanding of biological systems, and it is the fi rst time that we may be able to understand biological systems grounded in the molecular level as a consistent framework of knowledge after genomics, proteomics were put forward [ 6 ] . Different from molecular biology focusing on the individual ingredient, systems biology concentrates on constitution of all the compositions (such as gene, RNA, protein, etc.) in a biological system and the correlations of these compositions under specific condition [ 7 ] . It is a powerful tool to explore biology fully, and the thought and approach applying to secondary metabolites in medicinal plants is an effective way to fully proclaim the process from genes to secondary metabolites [ 8 ] .
2.2.4.1 Biological Process of the Formation of Secondary Metabolites in Medicinal Plants
Biological process of the formation of secondary metabolites in medicinal plants, which are regulated by various biotic and abiotic factors either from gene or environments, is very complex. Now, there are various hypotheses existing in induced mechanism of the production and accumulation of secondary metabolites, including growth/differentiation balance (GDB), carbon/nutrient balance (CNB), optimum defense(OD), resource availability(RA), etc. [ 9– 12 ] . From the angle of systems biology, the formation of secondary metabolites is a systematic biological process,
Fig. 2.3 The possible biological processes of secondary metabolites formation in medicinal plants
which consists of three portions, the stimulation of environmental factors (inner and outer environments), signal transduction, and the biological process catalyzed by gene expression and protease translation. The speci fi c process is as follows: environmental factors stimulate receptors out of plant cells, making receptors activated. These receptors activate intracellular signaling cascade and then transcription factors to start the expression of specific genes. Afterward, genes are transcribed and translated into relevant proteases, in order to catalyze the production of secondary metabolites (Fig. 2.3 ).
2.2.4.2 Technology Platform and Basic Methods of Systems Biology
Compared with the method of reduction theory that molecular biology adopts, systems biology applies the method of systems science to quantitative studies on organisms as an entire system instead of isolated parts [ 13 ] . Classic molecular biology research is a vertical research to study a single gene or protein by various approaches. First, fi nding speci fi c genes on the DNA level and then studying the function of genes by methods of gene mutation or gene knockout; on this basis, studying the space structures, modi fi cation of proteins, or protein-protein interaction. Genomics, proteomics, and the others are horizontal researches to study thousands of genes or proteins simultaneously by a single method. But the method of systems biology integrates both of them to be a three-dimensional study [ 14 ] . It can take full advantage of omics technologies to study the molecular difference among biosystems, infer the mechanism of environmental chemistry in biosystems, establish mathematical models to assess the modification or diversity of mRNA, protein, illustrate the holistic biological effects, and describe biological functions, phenotypes, and behaviors.
The major technology and platform of systems biology consist of genomics, transcriptomics, proteomics, metabonomics, interactomics, and phenomics [ 15 ]. Genomics is about genome mapping (including genetic map, physical map, and transcription map), nucleotide sequences analysis, gene mapping, and gene function analysis to all genes of a species. And the common analysis methods of transcriptomics are differential display, gene chip, EST, MPSS, cDNA-AFLP, etc. [ 16, 17 ] .
Recently, the German scientist Marc Sultan [ 18 ] utilized deep sequencing to get a brand-new view of the human transcriptome, and it is expected to apply to transcriptomics of other species. The major approaches of proteomics analysis are DEP, MS, etc. Metabonomics is a very important way to study medicinal plants and achieve modernization of traditional Chinese medicine [ 19– 21 ] , with common analysis methods of NMR, GC-MS, LC-MS, FTMS, CE-MS, etc. Genomics, transcriptomics, proteomics, and metabonomics detect and identify various molecules to study their functions on the level of DNA, mRNA, protein, and metabolin, respectively, forming multiple levels of biological information transfer. Interactomics studies interaction of molecules to discover and identify molecular pathways and networks, and to draw interaction maps systematically. Phenomics is regarded as a link between genotype and phenotype.
2.2.4.3 The Application of Systems Biology in Secondary Metabolites Study
Biosynthetic Genes and Pathways of Secondary Metabolites Biosynthetic pathway is the core of study on secondary metabolites in medicinal plants, an extremely complicated process from the genes to biological phenotypes (secondary metabolites). People have had a basic understanding of the main part of secondary metabolic pathway through long-term studies, such as shikimic acid pathway of phenols and IPP pathway of terpenoids [ 5 ]. Because of a variety of metabolites, end products are often generated by structural modification after the formation of basic framework. Currently, except taxol, arteannuin, indole alkaloids in Catharanthus roseus , etc., the majority of secondary metabolic pathways are not yet fully elucidated, waiting to be further illustrated.
Our research group has adopted the thought and approach of systems biology to acquire systemic studies results on biosynthetic pathway of tanshinone as diterpene secondary metabolite in S. miltiorrhiza [ 22– 25 ] (Fig. 2.4 ). Using elicitors to stimulate S. miltiorrhiza hairy root can lead to diverse phenotypes of the tanshinone content. And we analyze the metabolome, proteome, transcriptome of multiple materials with diverse phenotypes. The next step is to obtain full-length genes from the screened gene fragments which are closely related to tanshinone secondary metabolites, by multivariable analysis. SmCPS from clone is the fi rst (+)-CPP synthetase in angiosperm; SmKSL is identi fi ed as a new diterpene synthetase, and it
Fig. 2.4 Systems biology approach to explore tanshinone biosynthesis
can catalyze (+)-CPP into miltiradiene. This is a new and speci fi c branch of tanshinone diterpene biosynthetic pathway, which puts tanshinone diterpene biosynthetic pathway two steps forward.
Excavation of Signal Factors and Study of Signal Transduction Pathways Cells for the intercellular communication and the process of cellular compression reactions receive signals from outside. And these signals are converted into intracellular signals or cascades. The typical signals include hormone, pheromone, heat, cold, light, osmotic pressure, and some other materials, like the appearance or the concentration changes of glucose, potassium ion, calcium ion, and cAMP.
And the significant difference between signal transduction and metabolic process is metabolic process provides the transmission of quality, determined by a series of catalyze reactions but signal transduction undertakes information processing and transferring.
The biosynthesis of secondary metabolites in plants is a series of complex biochemical reactions controlled by correlative intracellular genes. But the environmental factors do not participate in secondary metabolites directly like outside stimulating factors, so there must be relevant intracellular signal molecules and corresponding signal transduction mechanisms to receive and conduct stimuli of external factors. Study has discussed that the correlative signal molecules and signal transduction mechanisms, regulated by secondary metabolites biosynthesis in plant molecules, can contribute to the understanding of the regulation laws of secondary metabolites biosynthesis in molecules, as well as can provide a rationale for improving secondary metabolites output of cultured cells in production practice [ 26 ]. Since there have been in-depth signal transduction researches on plant disease resistance and defense responses, studies on the mechanism of secondary metabolism signal transduction in medicinal plants are still in the initial exploration stage. Xu Maojun research group has made progress on the study on the signal factors and signal transduction mechanism of mediating Forsythia suspense seeds into hypericin and accumulating Ginkgo fl avonoid glycoside in Ginkgo cells [ 27 ]. Additionally, at the foundation of optimizing the cell culture conditions, it fi nds that UV-B is an external environment stress factor, which can induce the synthesis and accumulation of flavonoids in the cells from 5 to 30 h, using the established Hypericum chinense cells as materials, then, it can explore the signal transduction mechanism deeply induced by UV-B. It also considers that this process is in fl uenced by NO and H2O2 signal molecules, and these molecules will have synergetic effects on the process, which is regarded as a new signal interaction phenomenon. Furthermore, the study deems NO mediating UV-B to induce the synthesis and accumulation of flavonoids and is related to the CHS genes expression, but H2O2 is not [ 28 ]. The most feasible application of systems biology is conducting detailed models of cell regulation and focusing on specific signal transductions and molecules at all levels, in order to have a deep understanding of drug discoveries on the basis of mechanism [ 29 ] .
Obviously, these are beneficial explorations via the thought and methods of systems biology to study the secondary metabolite signal transduction pathway. And secondary metabolite signal regulation in plant cells is a quite complex system.
Recently, there have been some defi nite progresses in the relevant researches, but now it is still far from completely knowing the mechanism of secondary metabolite signal transduction pathway. By fi nding signal molecules that can make the phenotype of secondary metabolites different, the application of systems biology methods is ultimate to make high-throughput isolation of mutants related to plant secondary metabolites, to clone the relevant genes of secondary metabolism regulation with studying the functions as well as to discuss about activating transcription factors to start specific genes expressing signal transduction pathways so that it can establish the interaction and network of signal factors, genes, and metabolites to illustrate signal transduction pathways based on the formation and accumulation of secondary metabolites.
Ecology of Medicinal Plant Secondary Metabolism Plants in the growth progress will be influenced or even coerced by various environmental factors, including abiotic factors (such as light, temperature, soil, moisture, atmosphere, etc.) and biotic factors (damage by diseases and insects, herbivores, microorganisms, arti fi cial inferences, etc.). Plants make adaptive responses to these factors at the foundation of morphological structure, physiology, biochemistry, and gene expression. And secondary metabolite is one of significant biochemical regulators, for example, concentration of fl avonoids, terpenes, alkaloids, and organic acids in plant tissues will definitely rise under water deficit conditions [ 30– 33 ] . As a result of coupling with environment for a long period, plant secondary metabolism is playing an important role in enhancing plants self-protection ability and recording more environmental information than primary metabolism. Some scholars [ 34 ] proposed the concept of ecology of plant secondary metabolism. Compared with chemical ecology and plant physiological ecology, ecology of plant secondary metabolism pays much attention to both secondary metabolism itself and the way environmental factors induce the generation of these compounds. Therefore, it is an important task for ecology of plant secondary metabolism to illustrate how to induce activation of relevant acceptors, express genes, and conduct secondary metabolism.
Owing to the fact that secondary metabolism is complicated and plants are usually affected by different environmental factors at the same time, like drought and high temperature often coexist, the work of studying the relationship between ecological factors and secondary metabolism is full of challenge. Recently, there have been more and more related researches on the way to quantitative researches, because qualitative description cannot state the relationship fundamentally.
In terms of signal transduction, gene expression, or metabolites, we can adopt the methods of controlled experiment and systems biology to reveal how different outside stimuli induce and regulate secondary metabolism by receptors and mechanism of intracellular signal transduction. We can also establish a network of enviromental factors-genes-metabolites, and try to find out the pathway of enviromental factors activate related receptors, and the receptors initiate gene expression and regulation for producing the metabolites, which cloud clarify the physiological mechanism of the generation and accumulation of secondary metabolites in medical plants as well.
Here we could utilize two strategies to achieve the goal: initially, using controlled experiments which control investigated environmental factors (like temperature factor) strictly, tracking and analyzing the key enzymatic genes expression in secondary metabolism and protein (enzyme) synthesis, detecting the content variation of secondary metabolites, thus positively proclaiming the relationship between environmental factors and secondary metabolism; then, analyzing the content of secondary metabolites with different phenotypes and the environmental factors that may affect the accumulation of secondary metabolites and detecting relevant genes expression. Therefore, through repeated con firmation, we read the relationship between plants and environment via secondary metabolism, cognizing plant secondary metabolism via ecology.
Metabolic Engineering of Medicinal Plant Secondary Metabolites Metabolic engineering is mainly about altering metabolic fl ow, expanding metabolic pathway, or establishing new metabolic pathway to reach the expected target by genetic engineering. And the study has made great development. The group of Professor Kexuan Tang converts genes of PMT (rate-limiting upstream enzyme putrescine N -methyltransferase) and H6H (the downstream enzyme hyoscyamine 6-hydroxylase) into henbane and produces 411 mg/L scopolamine in transgenic henbane hairy root which is over nine times more than that in the wild type (43 mg/L), improving the accumulation of tropane alkaloids greatly [ 35 ] . American Professor Jay D. Keasling et al. produced the antimalarial drug precursor artemisinic acid in engineered yeast by a series of methods of gene regulation [ 36 ] . This introduced single, multiple target genes or an integrated metabolic pathway to produce new target materials or increase the content of target metabolites in organisms.
Moreover, antisense RNA and technologies like RNA interference can reduce the expression level of target genes and thereby restrict competitive metabolism pathway, alter metabolic fl ow, and increase the content of target materials. The study of Allen et al. [ 37 ] shows that blocking the metabolism pathway of morphine production in opium poppy will lead to the accumulation of reticuline and its methylated derivatives.
A new strategy of metabolic engineering is treating signal pathway and transcription factor as regulating targets [ 5 ] . Modifying the transcription factors that control multiple biosynthesis genes will regulate plant secondary metabolism effectively and improve the accumulation of specific compounds. For example, in the biosynthetic pathway of diterpenoid indole alkaloids in Catharanthus roseus, high expression of the transcription factor ORCA3 with AP2/ERF functional domain will result in the overexpression of several relevant genes and accumulation of
diterpenoid indole alkaloids [ 38 ] .
Medicinal plant metabolic engineering aims at improving the content of some important secondary metabolite and its precursor to solve the problem of medicine sources. If the content can be enhanced by the method of gene engineering, there will be enormous economic and social bene fi ts. So far, scientists have paid much attention to developing predictive metabolic engineering, and it utilizes the way of systems biology to integrate the data from metabolomics, proteomics, and transcriptomics and then to carry out repeated systematic simulation on the level of metabolic network, finally to get the result that is closer to true state. The existing database and instrumental analytical methods have made such system analysis possible to some extent.
2.2.4.4 Prospect
Systems biology is a collaborative study on the interaction of components in cellular network and other components in biosystem, the application of highthroughput genome-wide experimental techniques, and the integration of calculation methods and experiment results. Study on the production of secondary metabolites in medicinal plants by the thought and approach adopted in systems biology includes the matriculate way of secondary metabolites and signal transduction of signal factors. The most obvious feature of interactive relationship between generation and accumulation of metabolites and external environment is the holistic study from the reductionism perspective, which can adequately explore genes, transcription factors, signal factors, and environmental factors related to secondary metabolite biosynthesis. Establishing the system model of genes expression and regulation in secondary metabolite biosynthesis provides rationale for fully interpreting molecular mechanism of the production of secondary and the metabolic engineering. And it is of great signi fi cance to explain the cause of active ingredients in traditional Chinese medicine, the formation mechanism of famous-region drug, or the reasonable development and utilization of medicinal plant resources
systematically.
