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:
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
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.
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,
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
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.
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