An Alternate Method of Natural Drug Production: Elciting Secondary Metabolite Production Using Plant Cell Culture
Plant cells are factories of chemical compounds produced for carrying out biochemical pathways of survival and propagation. Primary metabolism involves biochemical processes for normal anabolic and catabolic pathways which results in assimilation, respiration, transport and differentiation whereas secondary metabolism involves generation of by-products which are the main defense elements of plants against pathogens, herbivore attacks and physical stress such as UV radiation. The secondary metabolites are plant pigments (such as alkaloids, isoprenoids etc.) responsible for the colors, flavors and smell in plants which also act as a source of drugs, fine chemicals, insecticides, dyes, flavours and fragrances. Plant derived secondary metabolites have played an essential role as medicine for thousands of years. Currently, secondary metabolites with bioactivity are being isolated and used either directly or after chemical modification. Their pharmacological value is increasing due to the constant discoveries of their potential roles in healthcare and as lead compounds for new drug development. This review highlights and discusses ways of improving the production of plant secondary metabolites and also focuses on plant cell culture which is considered as a promising alternative for producing bioactive compounds that are difficult to be obtained by chemical synthesis or plant extraction. It would be appropriate to consider the properties of plant cells in culture, in particular the relationship between growth and the expression of the pathways leading to the synthesis of secondary metabolites.
Plants have been the source for many important drugs because they are able
to produce various chemical entities and bioactive molecules through the process
known as metabolism. Plant cell carries out both primary and secondary metabolism.
Primary meta bolism involves synthesis of polysaccharides, proteins, lipids,
RNA and DNA through utilization of sugars, amino acids, common fatty acids and
nucleotides whereas secondary metabolism is activated only during particular
stages of growth and development or during periods of stress, limitation of
nutrients or attack by micro-organisms (Yazaki et al.,
2008; Yeoman and Yeoman, 1996).
Secondary metabolites are generally derived from primary metabolites through
modifications, such as methylation, hydroxylation and glycosylation. Therefore,
secondary metabolites are naturally more complex than primary metabolites and
are classified on the basis of chemical structure (e.g., aromatic rings, sugar),
composition (containing nitrogen or not), their solubility in various solvents
or the pathway by which they are synthesized. They have been categorised into
Terpenes (composed entirely of carbon and hydrogen), phenolics (composed of
simple sugars, benzene rings, hydrogen and oxygen) and nitrogen and/or sulphur
containing compounds (Chinou, 2008) (Table
1). It has been observed that each plant family, genus and species produces
a characteristic mix of these metabolites.
All plants produce secondary metabolites which are often specific to an individual
species or genus during specific environmental conditions making their extraction
and purification difficult. As a result, commercially available secondary metabolites
(pharmaceuticals, flavours, fragrances and pesticides) are generally considered
high value products as compared to primary metabolites and they are considered
to be fine chemicals with their cost ranging from $500 to $10000 per kilogram
(Ravishankar and Rao, 2000; Balandrin
et al., 1985) (Table 2).
Secondary metabolites have played a significant role in medicine since ancient
period. Plants have been directly used as food and herbs in the organised traditional
medical systems such as Ayurveda, Unani and traditional Chinese medicine for
the treatment of various diseases for thousands of years (Mukherjee
et al., 2007; Fabricant and Farnsworth, 2001).
Recently, the ethyl acetate extract of Alpinia officinarum has been reported
interestingly of possessing dual property of anti-microbial and anti-inflammatory
(Subramanian et al., 2008) and the methanol extract
of Ocimum Basilicum has been reported for anti-inflamatory activity (Selvakkumar
et al., 2007a). Drug discovery pursued using traditional knowledge
helps reduce time and developmental cost to identify bioactive molecules (Patwardhan,
2000; Patwardhan et al., 2004; Jachak
and Saklani, 2007). An anti-diabetic molecule has been identified from the
methanolic extract of Costus pictus and currently investigation is on
to isolate an anti-adipogenic molecule from the same extract (Shilpa
et al., 2009).
The plant based drug discovery gained interest with the development of anticancer
and anti-infectious agents and now contributes to new bioactive molecules which
are being isolated for the treatment of various other diseases including metabolic
disorders like Diabetes and Obesity (Saklani and Kutty, 2008)
such as Metformin, a commercially available anti-diabetic drug derived from
Galega officinalis (Oubre et al., 1997).
|| Classification of secondary metabolites
|| Cost estimation for plant-derived secondary metabolites of
commercial importance in pharmaceutical industry
|Ravishankar and Rao (2000)
Cinnamic acid, Methyl tetracosanoate, Tannins, Caffeoyl derivatives, 3β-taraxerol,
Aloe emodin and Stigmasterol have been isolated showing good anti-diabetic potential
and 12-ursene 2-diketone has been isolated which exhibited anti-inflammatory
property in in vitro models (Lakshmi et al.,
2009; Muthusamy et al., 2008, 2010; Shilpa
et al., 2009; Sangeetha et al., 2010;
Anand et al., 2010; Sujatha
et al., 2009; Gayathri et al., 2007).
The plant as a source for important drug molecules is witnessed through the
discoveries of various bioactive molecules such as Taxol, Vincristine, Vinblastine,
Metformin, Morphine etc. The synthetic aspirin used by the modern world today
is a derivative of a plant-based drug (Raskin and Ripoll,
2004). Many new plant-based drugs such as Prostratin, CAPSOROLS, CCS and
over 60 anti-cancer compounds are under active preclinical trials alongwith
with the introduction of herbals in the form of nutraceuticals and dietary supplements
(Corcoran and Spraul, 2003; Steinbeck,
2004; Ganesan, 2002; Saklani
and Kutty, 2008).
Important plant-derived drugs are still obtained commercially by extraction
from their whole plant sources. A loss in activity has been observed when these
compounds have been attempted to be chemically synthesized; this could be because,
the secondary metabolites generally have highly complex structures with many
chiral centers which may contribute to their biological activity and such complex
compounds are difficult to synthesize economically (Pezzuto,
1995; Kolewe et al., 2008).
Currently, the natural habitats of many plants are disappearing due to environmental
and geopolitical instabilities, therefore making it very difficult to acquire
important secondary metabolites and in the process leaving many potentially
useful compounds left undiscovered. Plant cell culture is considered as a promising
alternative for producing bioactive compounds that are difficult to be obtained
by chemical synthesis or plant extraction (Kolewe et
al., 2008). Plant cell culture studies have been carried out based on
the fact that each plant cell in the culture exhibits totipotency, wherein the
cell has the full set of genes necessary for all the functions of a plant, including
secondary metabolism (Verpoorte et al., 1999).
Cell culture systems are useful in large-scale culturing of plant cells, which
form a continuous and reliable source of secondary metabolites and can be purified
easily due to the absence of significant amounts of pigments. This method removes
all seasonal constraints and eliminates the geographic barriers for production
of secondary metabolites (Karuppusamy, 2009). Plant
tissue culture has been applied for the production of secondary metabolites
on a commercial scale since late 1950s, when atropine from the roots of
Atropa belladonna was synthesized and accumulated in roots and in callus
(West-jr and Mika, 1957).
One Major limitation in the production of secondary metabolites by plant cell
culture technology is the low yield of secondary metabolites. This could be
improved by: 1) increasing the productivity of cultured plant cells and the
necessary metabolite through standardization of culture environment (Ramachadra-Rao
and Ravishankar, 2002); 2) manipulation of plant cell cultures to improve
productivity of target compounds employing elicitors, abiotic stresses and other
approaches, regardless of their mechanisms (Zhong, 2001;
Zhao et al., 2005); 3) studying signal transduction
pathways underlying various effective strategies leading to the biosynthesis
of target secondary metabolites (Memelink et al.,
2001; Zhao et al., 2005).
MANIPULATION OF CULTURE ENVIRONMENT FOR IMPROVED BIOMASS AND SECONDARY METABOLITE PRODUCTION
In order to obtain products at high concentrations for commercial manufacturing,
standardization of chemical factors like media components, plant growth regulators
etc and physical factors like pH, temperature, light, etc. play an important
role (Dornenburg and Seydel, 2008; Karuppusamy,
2009). Enhancement of the yield of biomass and secondary metabolite production
through plant cell culture also requires selection of proper genotypes and high-yielding
clones (Barz and Ellis, 1981; Mulabagal
and Tsay, 2004).
Selection of Proper Genotypes and High-Yielding Cell Clone
It has been observed that collection of cells from source explant as inoculum
to the production medium is crucial. Cell cultures derived from wild plants
have been observed to accumulate large amount of secondary metabolites which
could be a good source for the isolation of the bioactive secondary metabolite.
After the cell culture has been established, plant cells undergo a continuous
process of epigenetic or genetic changes during culture which represents a heterogeneous
population. It is therefore essential to select cell clones which produce high
yields as well as accumulate high amount of the desired metabolite (Ogino
et al., 1978). This strategy resulted in an increased accumulation
of anthocyanins and berberin compared to that of the mother culture in Euphorbia
milli and Coptis japonica, respectively (Yamamoto
et al., 1982; Yamada and Sato, 1981).
Medium standardization has been an important parameter in plant cell culture
technology since the composition of the culture media is known to influence
the biomass yield as well as secondary metabolite production. Medium composition,
culture conditions and exogenous phytohormone combinations together influence
the metabolite accumulation in the cell (Seabrook, 1980).
Although Murashige Skoog (1962) Medium is one of the
most widely used medium for rapid growth of callus, it has not been observed
as an ideal medium for inducing secondary metabolite accumulation.
Macro and micro nutrient composition in plant tissue culture media has foremost
effect on both primary and secondary metabolism of cells (Dougall,
1980) and a medium which leads to rapid cell division and limits early cessation
of exponential growth is thought to be helpful for secondary metabolite formation.
An overall level of total nitrogen has been found to affect production of secondary
metabolites. For example, reduced levels of ammonia and increased nitrate levels
were found to promote the production of shikonin and betacyanins, whereas higher
ammonia to nitrate ratio was found to increase the production of berberine and
ubiquinone (Bohm and Rink, 1988; Nakagawa
et al., 1984; Fujita et al., 1981;
Ikeda et al., 1977). It has also been observed
that reduced levels of total nitrogen improved the production of anthraquinones
in Morinda citrifolia and anthocyanins in Vitis species (Yamakawa
et al., 1983; Zenk et al., 1975).
Apart from the nitrogen source, phosphates were also found to play an important
role in increasing the secondary metabolite production. It had been observed
that added phosphates in the culture medium resulted in prolonged cell growth
in Catharanthus Roseus cultures (Mac-Carthy et
al., 1980), similar results were obtained in a separate study for the
production of betacyanins in callus cultures of Beta vulgaris (Bohm
and Rink, 1988). Increased phosphate also stimulated the production of digitoxin
in Digitalis purpurea (Hagimori et al., 1982).
Cell cultures are also influenced with simple sugars as carbon source along
with inorganic supply of other nutrients. Sucrose and glucose are most preferred
amongst other carbon sources for plant tissue culture media. The carbon source
has been known to influence cell growth and yield of secondary metabolites in
many cases (Dornenburg, 2008). It has been observed
that a range of 2 to 8% of carbon source is appropriate for optimal secondary
metabolite production in plant cell cultures (Ramachadra-Rao
and Ravishankar, 2002). The maximum yield of rosmarinic acid produced by
cell suspension cultures of Salvia officinalis was 3.5 g L-1
when 5% of sucrose was used but it was 0.7 g L-1 in the medium containing
3% sucrose (Whitaker et al., 1984).
Plant Growth Regulators
In a plant cell and/or tissue culture study, growth regulator concentration
plays a crucial role in secondary metabolite accumulation (Di-Cosmo
and Towers, 1984). The concentration of auxin and cytokinin individually
or in combination significantly alters both the growth and the secondary metabolite
accumulation in cultured cells (Mantell and Smith, 1984).
The growth regulator 2, 4-dichlorophenoxyacetic acid (2, 4-D) has been found
to play an important role in callus initiation, but it has been found to inhibit
the production of secondary metabolites in a large number of cases. However,
there have also been reports where cultures grown in the presence of 2, 4-D
have shown secondary metabolite induction for eg. carotenoid and anthocyanin
production in callus cultures of Daucus carota (Rajendran
et al., 1992) and Oxalis linearis (Meyer
and Staden, 1995), respectively. In cases where secondary metabolite production
is inhibited, elimination of 2, 4-D or replacement of 2, 4-D with other auxins
such as Naphthalene Acetic Acid (NAA) or Indole Acetic Acid (IAA) has been found
to enhance the production of nicotine in suspensions of Nicotiana tabacum
and shikonin in suspensions of Lithospermum erythrorhizon (Sahai
and Shuler, 1984; Fukui et al., 1983). Cytokinins
have been observed to have different effects depending on the type of plant
species and metabolite. Kinetin has been found to stimulate the production of
anthocyanin in Haplopappus gracilus but inhibited the formation of anthocyanins
in Populus cell cultures (Seitz and Hinderer, 1988;
Mok et al., 1976). Apart from auxins and cytokinins,
other plant growth regulators such as Gibberellic acid and abscisic acid have
been reported to suppress production of anthocyanins in a number of cultures
(Bohm and Rink, 1988; Seitz and Hinderer,
pH, Light and Temperature
In addition to the necessary salt, sugar and exogenous phytohormone concentrations
in media, pH also plays an important role on cell growth and metabolite production.
Optimum growth of plant cells in the culture is usually obtained between pH
range of 5.5 and 6.0. It has been known for some time that the pH of the culture
medium can influence the uptake of nutrients and precursors, the permeability
of membranes and release of products from the vacuole to the culture medium.
Concentration of hydrogen ions in the medium is known to change during the development
of the culture. The medium pH decreases during ammonia assimilation and increases
during nitrate uptake (Mc-Donald and Jackman, 1989).
Photoautotrophic cell suspension cultures of Chenopodium rubrum L. showed
an increase in biomass between pH 5.0 to 6.0 (Husemann et
There are reports of light stimulateSelvakkumard secondary metabolites production
(Whitaker et al., 1986) and for each plant species
different temperatures favour the production of secondary metabolite. It has
been observed that white light has a significant effect on cell growth as well
as on chlorophyll and kalata B1 synthesis in Oligodon affinis cell cultures.
In callus cultures the optimum irradiation intensity for growth was found to
be lower than that required for production of secondary metabolite, which was
found to increase with increasing intensities of light (Dornenburg
and Seydel, 2008). Light was found to stimulate accumulation of anthocyanin
in cell cultures of Daucus carota and Vitis hybrids (Seitz
and Hinderer, 1988), on the contrary, elimination of light prompted the
accumulation of monoterpenes in callus cultures of Citrus limon (Mulder-Krieger
et al., 1988).
Temperature ranging between 17-25°C are normally used for induction of
callus and growth of cultured cells but Toivonen et al.
(1992) found that lowering the cultivation temperature increased the total
fatty acid content per cell of dry weight. Studies have also shown that at 32°C,
tobacco cell cultures produced higher yield of ubiquinone when compared to either
24 or 28°C (Ikeda et al., 1977). Cell cultures
of Catharanthus roseus produced 12-fold higher crude alkaloids at 16°C
as compared to the normal 27°C (Courtois and Guren, 1980).
MANIPULATION OF PLANT CELL CULTURES FOR INCREASED SECONDARY METABOLITE PRODUCTION
Altogether it is the appropriate combination of all the above mentioned parameters, which would aid in a cell culture capable of producing high biomass yield along with increased secondary metabolite production (Table 3). After medium optimization, the subsequent strategies vary based on the type of product needed, like organ cultures, product absorption and secretion, biotransformation, cell immobilization, mutagenesis, metabolic engineering etc.
It is generally found that in plants high concentrations of secondary metabolites
tend to accumulate in specific cell types at specific developmental stages,
therefore tissue cultured cells from these plants typically accumulate large
amounts of secondary compounds under specific conditions (Robins
et al., 1986). Although, a wide range of secondary metabolites are
produced through de-differentiated callus, there exist certain metabolites which
are organ specific and require differentiated organ cultures (Table
3) when the metabolite is produced only in specialized plant tissues or
glands in the wild plant for example, Hypericum perforatum (St. Johns
wort), accumulates the anti-depressant hypericins and hyperforins in foliar
glands and have no reports of their ability to accumulate phytochemicals in
undifferentiated cells (Smith et al., 2002).
It has also been observed that Nicotiana tabacum (tobacco) roots are
source for biosynthesis of lysine to anabasine, followed by the conversion of
anabasine to nicotine in leaves. Herein the callus and shoot cultures of tobacco
have been found to produce only trace amounts of nicotine because they lack
the organ-specific compound anabasine (Karuppusamy, 2009).
Product Absorption and Secretion
Secondary metabolites produced during cell culture studies have been reported
to be accumulated intracellularly and there are also reports of metabolites
which are released into the culture medium (Misawa, 1994).
In certain cases the product of interest might be accumulated intracellularly
and its production might sometimes inhibit its own synthesis through product
inhibition and repression. In some cases higher level of product can be produced
when secreted into the medium. In such situations permeabilization of cell membranes
has been attempted using permeabilizing agents such as DMSO, Triton X-100, electroporation
or ultrasonication, for enhancing the product release, but has achieved limited
||In vitro secondary metabolites production from plant
cell, tissue and organs cultures using different media and plant growth
Viability of the cells was observed to be lost in most cases when permeabilizing
agents and electroporation were used (Brodelius, 1988).
Chenopodium rubrum cultures which secrete amaranthin (red betacyanin
pigment) when incubated with low concentrations of chitosan (0.01%) and DMSO
(5.7%) for 96 h showed no significant effect on cell viability, but a longer
incubation period (196 h) was found to have a deleterious effect (Knorr
and Berlin, 1987). Ultrasonication was found to be a better method in comparison
wherein Beta vulgaris cells upon ultrasonification for 20 to 60 sec was
found to aid the release of betanin with no apparent effect on cell viability
(Kilby and Hunter, 1990).
Most secondary metabolites produced in cell cultures are generally accumulated
intracellularly, but some metabolites have also been reported to be secreted
into the media such as taxol produced by Taxus brevifolia (Collins-Pavao
et al., 1996). In such cases XAD-7 (resin) is added in the suspension
medium and the metabolites (pigments) are found to be absorbed into it. Addition
of XAD-7 (resin) into its suspension culture of Chinchonaledgerina also
stimulated the production of anthraquinones with a 15 fold increase compared
to the medium without resin (Robins et al., 1986).
Similarly, active charcoal in the medium was also found to stimulate the product
yield (Misawa, 1994)
Biotransformation is a process through which the functional groups of organic
compounds are modified either stereo specifically or region specifically by
living cultures, entrapped enzymes or permeabilized cells to a chemically different
products. The advantage of this method includes the production of novel compounds,
enhancement in the productivity of desired compound and overcoming the problems
associated with chemical synthesis. The production of fine chemicals and pharmaceuticals
can be achieved through biological catalysts in the form of enzymes and whole
cells (Ravishankar and Rao, 2000; Meyer
et al., 1997).
A suitable substrate compound can be biotransformed to obtain a desired product
using plant cells. There are two major reasons to choose plant cells for biotransformation
purposes. To start with, these cells are generally able to catalyze the reactions
stereospecifically resulting in chirally pure products. Secondly, they have
the ability to perform regiospecific modifications that can not be easily carried
out through chemical synthesis or by microorganisms (Hamada
and Furuya, 1996). Plant cells perform reactions such as reduction, oxidation,
hydroxylation, acetylation, esterification, glucosylation, isomerization, methylation,
demethylation, epoxidation, etc. This process should meet four important prerequisites
that the culture must have the necessary enzymes. the substrate or precursor
must not be toxic to the culture and that the cellular compartment of the cell
and importantly the rate of product formation must be faster than its further
metabolism (Table 4).
|| Production of pharmaceuticals in plant cell cultures using
Immobilization is a technique, which confines a catalytically active cell
on a fixed support and prevents cells entry into the suspension culture
(Yeoman, 1987). This technique involves entrapment of
cells in some kind of gel or combination of gels, which are allowed to polymerize
around them, e.g., of gels include calcium alginate, agar, agarose, gelatin,
carrageenan and polyacrylamide; alternative supports include polyurethane foam
and hollow fibre membranes. Amongst them, gels of alginate are most widely used
because of their simplicity and lack of toxicity. There is a necessity that
immobilized cells should maintain prolonged viability and biosynthetic capability
with high rates of secondary metabolite production. Catharanthus roseus had
been subjected to two types of immobilization system, one was immobilization
in polysaccharide beads (Brodelius and Nilsson, 1980)
and the other polyacrylamide sheets (Lambe and Rosevear,
1982), both of which exhibited alkaloid release with no loss in viability.
Capsicum frutescens cells immobilized on polyurethane were found to release
capsaicin completely into the medium, though other species immobilized by the
same method showed retention of their product intracellularly (Lindsey
and Yeoman, 1984). Chenopodium rubrum cells when immobilized in alginate
beads were found to secrete the red betacyanin pigment, amaranthin into the
medium (Knorr and Berlin, 1987). De novo biosynthesis
of many important secondary metabolites is carried out through single and/or
multistep biotransformation of precursors on immobilized plant cells. Product
synthesis should not be strictly growth associated as growth of cells can lead
to disintegration of the immobilization matrix, which may lead to interruption
of the process and it is essential that a significant amount of product is released
into the medium. There has been many more studies conducted using immobilization
(Table 5a, b).
Elicitation of Secondary Metabolites
Various studies towards enhancement of secondary metabolite production in
cell cultures include treatment with various precursors, elicitors, signal compounds
and abiotic stresses.
It has been noticed that addition of precursors or related compounds (which
intermediate at the beginning of a secondary metabolite biosynthetic route)
to the culture media sometimes stimulate secondary metabolite production (Mulabagal
and Tsay, 2004). This is thought to occur as any precursor would stand a
good chance of increasing the yield of the final product. For example, Salvia
officinalis cell suspension cultures and Taxus cultures stimulated
rosmarinic acid and taxol production respectively upon addition of phenylalanine
(Ellis and Towers, 1970; Fett-Neto
et al., 1993). This approach could be advantageous if the precursors
||Use of immobilized plant cell systems for production of secondary
|| Use of immobilized plant cell systems for production of secondary
metabolites through De novo synthesis
Plant secondary metabolites are majorly produced as a response to protect
plants from environmental stresses. The environmental stresses (microbial, physical
or chemical factors) leading to an increase in secondary metabolism are known
The use of elicitors in cell cultures has been developed as one of the main
strategies to improve the yield of secondary metabolites wherein elicitation
is induced by the addition of trace amounts of elicitors (Radman
et al., 2003; Roberts and Shuler, 1997).
Elicitors are signals triggering the formation of secondary metabolites and
are classified based on their nature into abiotic or biotic elicitors (Mulabagal
and Tsay, 2004; Namdeo, 2007).
Elicitors of non-biological origin are called abiotic elicitors, which predominantly consists of physical and chemical stresses such as UV radiation extremes of temperature, ethylene, fungicides, antibiotics, salts of heavy metals or high salt concentrations etc. (Table 6).
Other class of elicitors are biotic elicitors; these are substances with biological
origin such as polysaccharides derived from plant cell walls (pectin or cellulose)
and micro-organisms (chitin or glucans) and glycoproteins or intracellular proteins
which act by activating or inactivating a number of enzymes or ion channels
(Rokem et al., 1984). This class of elicitors
are categorized into two types based on their origin, exogenous and endogenous
elicitors (Table 7). The elicitors which have originated outside
the cell like polysaccharides, polyamines and fatty acids are known as exogenous
elicitors whereas endogenous elicitors have originated inside the cell like
galacturonide or hepta-β-glucosides etc. (Namdeo, 2007).
Evidently, in certain circumstances elicitation can be used to obtain better
consequence in increasing product yield and has also been found to have commercial
During the process of elicitor study, it should be noted that cells showing
failure to elicit necessary secondary metabolites does not necessarily mean
that the metabolic pathway cannot be triggered. Inappropriate combination of
medium and elicitor, as well as unsuitable concentration of the elicitor could
be a cause for unsuccessful elicitation, which indicates that a successful elicitation
is a very challenging process requiring intense standardizations (Namdeo,
2007; Zhao et al., 2005). In several studies
it has been observed that elicitor treatments performed at the late log phase
result in the high biomass yields along with secondary metabolite production
as almost all the elicitors, when used at early log phase showed immediate increase
in the secondary metabolites while suppressing the biomass leading to overall
low productivity. For commercial production of secondary metabolites, apart
from the secondary metabolite enhancement, it is equally important to study
the signals involved in the process of elicitation, which might help in choosing
the appropriate elicitor (Zhao et al., 2005).
|| Abiotic elicitors used for production of secondary metabolites
|| Biotic elicitors used for the production of secondary metabolites
Stress Related Signal Transduction Pathways
Plants subjected to stresses (including various elicitors or signal molecules)
lead to an accumulation of a variety of secondary metabolites. It is important
to study these stress related signal transduction thereby helping in the development
of strategies for commercial production of the target compounds by either activation
or suppression of certain metabolic pathways making it a powerful tool to investigate
pathway regulation based on gene expression.
Upon challenge by the biotic elicitors, plants generally stage an array of
defence or stress responses. The recent studies are focused on studying the
recognition of elicitation stimulus and the subsequent triggered defence response.
Signal transduction of plant defence response primarily involves the host (cell)
recognition to elicitors which initiates the early signalling events such as
protein phosphorylation or dephosphorylation, changes in ion fluxes and oxidative
burst. These processes stimulate the subsequent transcriptional activation of
lant defence genes, which involves activation of enzymes such as Glutathione-S-Transferases
(GST), Phenyl Ammonium Lyase (PAL), Chalcone synthase (CHS) etc., biosynthesis
of endogenous secondary signals such as salicylic acid etc. and activation of
NADPH oxidase leading to complex generation of reactive oxygen species (ROS)
such as O2.-, H2O2 and thereby altering
the redox status of plant cells affecting the defense signalling. ROS have been
found to be generated after any elicitation process. Plants have evolved efficient
antioxidant systems to scavenge ROS (Fig. 1). The induction
/suppression of antioxidant activities provide evidence for occurrence of oxidative
burst and variation of secondary metabolite accumulation (Yang
et al., 1997).
The interaction of elicitors with cellular receptors and subsequent transcriptional
and postranslational activation of transcription factors thereon lead to the
induction of defense genes (Zhu et al., 1996).
In addition to eliciting primary defense responses, elicitor signals may be
amplified through the generation of secondary plant signalling molecules such
as Salicylic Acid (SA) (Durner et al., 1997).
Elicitors and secondary endogenous signals generally activate a diverse array
of plant defense and protector genes, whose products include glutathione S-transferases
(GST), peroxidases, cell wall proteins, proteinase inhibitors, hydrolytic enzymes
(e.g., chitinases and ~-1, 3-glucanases), pathogenesis-related (PR) proteins
and phytoalexin biosynthetic enzymes, such as Phenylalanine Ammonia Lyase (PAL)
and chalcone synthase (CHS) (Hammond-Kosack and Jones, 1996).
Phytoalexins are low-molecular-weight, antimicrobial compounds (e.g., phenylpropanoids,
terpenoids, etc.), whose synthesis is induced following biotic elicitation.
Furthermore, signaling components such as G proteins, NADPH oxidase, H2O2,
SA, mitogen-activated protein kinases (MAPK) and Myb transcription factors have
been found to participate in several plant defense responses (Fig.
2). The appearance of cell death and production of ethylene also play a
regulatory role in the elicitor induced defence responses of plants (Yang et
||Mechanisms of ROS production and scavenging during biotic
or abiotic stresses. Ascorbate peroxidase (APX) and catalase (CAT) are some
of the key ROS scavenging enzymes of plants and heat shock proteins (HSPs);
pathogenesis related proteins (PR); phenylalanine ammonia-lyaze (PAL), chalcone
synthase (CHS); nicotinamide adenine dinucleotide phosphate-oxidase (NAD(P)H
oxidase) are some of the important stress related genes
||Signal transduction involved in plant defense responses. Cell
recognizes elicitors and initiates early signaling events. Subsequent transcriptional
and posttranslational activation of transcription factors leads to induction
of plant defense genes and biosynthesis of endogenous secondary signals.
Additionally, the activated NADPH oxidase complex generates reactive oxygen
species (ROS), which alter the redox status of plant cells and affect defense
Host Recognition of Biotic Elicitors
When cells are incubated with an elicitor, the cell must recognize the elicitor
and subsequent defense responses are initiated so as to limit the potential
damage that could possibly be caused by the elicitor. Receptor (R) genes present
in plants have been found to encode receptors for the recognition of specific
elicitors (Gabriel and Rolfe, 1990), but there are many
fungal and bacterial oligosaccharides, proteins and glycoproteins that have
been found to act as nonspecific elicitors to induce defense responses in the
plants cells carrying no specific R genes (Benhamou, 1996;
Role for Protein Phosphorylation in Intracellular Signalling
Protein kinases and phosphatases have been found to play a pivotal role
in early as well as later events of plant defense signaling pathways which indicates
that phosphorylation and dephosphorylation also play an essential role for defence
responses (Yang et al., 1997). Kinase cascades
of the Mitogen-Activated Protein Kinase (MAPK) class of enzymes play an extremely
important role in plant signalling during various stress conditions. MAPK plays
a key role in induction of defence mechanisms by amplifying pathogen-derived
signals perceived at membrane receptors and transducing these signals into altered
gene expression (Pitzschke et al., 2009) (Fig.
3). Fungal elicitors trigger rapid and transient protein phosphorylation
in cell suspension cultures of parsley (Dietrich et al.,
1990) and protein kinase inhibitors like K-252a and staurosporine have been
found to block these elicitor induced changes in protein phosphorylation and
prevent the induction of plant defense responses. In contrast, many protein
phosphatase inhibitors such as calyculin A,cantharadin and okadaic acid, imitate
elicitor action to activate defense responses (Felix et
al., 1994; Levine et al., 1994; Mackintosh
et al., 1994).
||The signaling pathway activated in plants in response to external
application of oxidants. SA, salicylic acid; NO, nitric oxide; H2O2,
hydrogen peroxide; PCD, programmed cell death; HSP, heat shock protein;
MAPK, mitogen-activated protein kinase
Early Signaling Events
In addition to protein phosphorylation, early signaling events in defense
responses involve ion fluxes (ion channels), oxidative burst (ROS), GTP-binding
proteins, phospholipases etc.
Oxidative burst is another prominent event occurring early after elicitation.
It leads to the rapid production and accumulation of ROS, such as O2.-and
H2O2 (Doke et al., 1996;
Low and Merida, 1996). Elicitors induced oxidative burst
appears to be mediated by multiple signaling cascades, which might be associated
with activation of G proteins, Ca2+ influx, H+/K+
exchange, induction of phospholipases and protein phosphorylation (Low
and Merida, 1996). ROS generation is apparently dependent on the activation
of a plasma membrane NADPH oxidase. Specific inhibitors of NADPH oxidase such
as diphenylene iodonium, prevent plant ROS production induced by elicitors.
ROS production occurs in two distinct phases which have been observed during
cell-elicitor interactions. The first burst occurs within minutes whereas the
second, sustained burst occurs after few hours of treatment with elicitors (Laxalt
et al., 2007; Manickavelu et al., 2010).
Oxidative burst generates superoxide anion and H2O2 and
together they play multiple roles in defense responses. Furthermore, they act
as a secondary messengers to induce plant defense-related genes (Orozco-Cardenas
et al., 2001) and hypersensitive host cell death (Levine
et al., 1994; Yoda et al., 2003).
The action of ROS is thought to be mediated through changes in cytosolic Ca2+
levels and generation of lipid peroxides. Several studies have shown that Ca2+
plays an important role in defence signalling. Ca2+ channel blockers
were shown to inhibit ion fluxes as well as defense responses induced by fungal
and bacterial elicitors (Garcia-Brugger et al., 2006;
Mc-Anish and Schroeder, 2009) (Fig. 3).
ROS is found to activate defense gene expression by altering the redox status
of plant cells, thereby modulating the activity of redox-sensitive transcription
factors. ROS has been proven to play an important role in plant defense signaling,
although their mechanism of action is poorly understood. In few cases, GTP-binding
proteins have also found to participate in the induction of ion fluxes and the
oxidative burst by some fungal elicitors (Xing et al.,
1997; Garcia-Brugger et al., 2006).
Endogenous Secondary Signals in Plant Cell Defense
The elicitor signals are often amplified through the production of secondary
signal molecules such as SA, ethylene and jasmonates subsequent to the early
signaling events activated by elicitor treatment. SA is known to play an important
role in the activation of defense responses. Increase in the levels of SA and
its conjugates have been noticed during the activation of defence responses.
The increase in SA levels slightly leads to parallel expression of PR genes
in cells (Malamy et al., 1990). In addition to
SA; ethylene, Jasmonic Acid (JA) and systemin have also been proved to be important
secondary signals for plant defense responses (Kotchoni
and Gachomo, 2006). Ethylene levels have been found to increase during the
HR and have been eminent to induce the expression of PAL and the basic PR genes
(Hammond-Kosack and Jones, 1996).
Integration of Signaling Pathways and Activation of Plant Defense Genes
Metabolic engineering of biosynthetic pathways inside a plant cell to improve
accumulation of a constitutively produced metabolite is an attractive strategy
in which great progress has been made in the past decade (Verpoorte,
1999; Lucker et al., 2007). Secondary metabolic
pathways are extremely complex and still remain partially undefined in most cases.
Genomes of very few plants have been completely sequenced and the among the ones
that have been, are model systems in which secondary pathways are not of great
interest concerning secondary metabolite accumulation and as a result, complete
pathway information is lacking for most medicinal plant species. As many secondary
metabolic pathways are partially understood, elucidating these pathway genes and
their control elements and identification of rate influencing steps within a biosynthetic
pathway is of current research interest. Nevertheless, significant advancements
in overcoming some of the key challenges have been made in recent years (Kolewe
et al., 2008).
The initial recognition and early signalling events may be different for
various elicitors, however, many of these signals are incorporated into one
of the various pathways that lead to the transactivation steps involved in the
interaction between activated transcription factors and elicitor responsive
cis elements in the promoters of defense genes. A single elicitor can activate
multiple transcription factors that interact with different cis elements
in the same or different promoters, leading to activation of many defense genes
(Zhu et al., 1996). Some of these transcription
factors are transcriptionally and/or post-translationally activated by elicitor
or treatment with secondary signals, such as SA.
Techniques are therefore available for the identification of targets (i.e., genes, proteins, metabolites) as well as for their development. Various tools have been employed to identify unknown genes as well as to characterize secondary metabolite pathway regulation, including precursor feeding, gene over expression, application of metabolic inhibitors and mutant selection.
It is observed that the amount of the enzyme and its activity is directly proportional to the rate limiting step, therefore the amount of product formed is ultimately determined by the flux at the rate limiting step. The flux is generally influenced by substrate availability and end-product inhibition. When the respective biosynthetic pathway is known, then metabolic engineering can be performed to enhance the necessary product by increasing the activity of the enzymes which are limiting in the pathway, regulatory genes expression can be induced; the competitive pathways and catabolism can be blocked.
The former two possibilities require the enhanced expression of genes yielding
active enzymes and the latter two approaches need antisense genes for blocking
the genes involved in competitive pathway or catabolism. The most important
regulatory step in secondary metabolism is transcription of the biosynthetic
genes. Concentration of precursors and end products are determining factors
in the metabolic control of synthesis and breakdown of the compounds, as all
secondary metabolic processes derive their precursors from primary metabolism
(Verpoorte et al., 1999).
Genetic methods can be applied to increase the production of the necessary
metabolite or a group of related compounds through the expression of genes encoding
key enzymes (Dueckershoff et al., 2005; Tian
and Dixon, 2006; Inui et al., 2007; Liu
et al., 2007) or through functional genomics (Goossens
et al., 2003). The end goals of the genetic modifications generally
include enhancement in the levels of necessary secondary metabolites along with
lowered levels of unwanted or generation of novel compounds. Despite successes,
metabolic engineering of the relevant biosynthetic pathways is often hampered
by a lack of knowledge of the enzymes and genes involved (Chaudhuri
et al., 2009).
Hairy Root Culture
The use of Agrobacterium rhizogenes for production of hairy root
has been used as yet another indirect genetic approach for increasing the secondary
metabolite production in a variety of species. Agrobacterium rhizogenes
inserts the Ri plasmid into wounded tissue and induces the growth of very fine
adventitious roots called hairy roots. These roots can be cultured in hormone-free
medium and there are several examples of enhanced accumulation of secondary
products, relative to non-transformed tissue. This transformation occurs through
Ri T-DNA of Agrobacterium rhizogenes, which produces roots that can further
be cultured in vitro, which often show high accumulation of secondary
metabolites (Guillon et al., 2006). Many dicot
species show similar morphological and physiological changes by the Ri T-DNA;
thus, Ri T-DNA allows secondary metabolite production in multiple species without
any knowledge about the mechanism of production (Chaudhuri
et al., 2009) (Table 8).
Apart from the general increase in the secondary metabolite production, Elicitation, precursor feeding, cell permeabilization and immobilization are also considered efficient ways to improvement of secondary metabolite productivity of hairy roots (Table 9). And recently there has been an increased research in the field of hairy root metabolic engineering, where this study is based on integrating genes that encode enzymes of a given pathway between the T-borders of the Ri plasmid, then transferring this construct into plant
|| Pharmaceutical products produced using hairy root cultures
Many plant derived drugs are still obtained commercially by extraction from
their whole plant sources. However, their over-utilization is depleting many
important plant sources, leading to serious ecological problems. Plant cell
culture provides a continuous, reliable source of natural products. There are
only few drugs which are produced commercially using plant cell culture, due
to the limitations such as low yields of desired metabolites and biochemical
and genetic instability of the plant cells. The factors playing an important
role in overcoming the limitations include standardization of chemical and physical
parameters for producing high yield of cell mass as well as the secondary metabolite.
Secondly, plant cells are subjected to precursors and elicitors, so as to increase
the secondary metabolite production. Finally, it becomes important to understand
the mechanism by which the secondary metabolites are triggered or produced during
the above mentioned conditions.
Despite the increasing demands of plant secondary metabolites, much of plant
secondary metabolism is still poorly understood. As secondary metabolites are
expressed in response to a variety of stresses, therefore it becomes important
to focus on regulatory machinery of plant defense responses. Plants utilize
multiple signaling pathways, such as jasmonic acid, ethylene, salicylic acid
signaling pathways, to regulate defense response against elicitor or various
stresses. Therefore, quantitative profiling of signal in response to various
abiotic or biotic elicitors may help detect the initiation of elicitation of
secondary metabolites. Elucidation of the signaling network would help in specific
and efficient engineering of the production of target secondary metabolites.
Molecular biology techniques are being used recently to produce transgenic cultures
and to effect the expression and regulation of biosynthetic pathways. This would
be a significant step towards commercial production of secondary metabolites
using cell cultures. Apart from these methods, there exists another technology
called the hairy root technology. Hairy roots grow easily upon infection with
a bacterium and are also easy to transform. Their genetic and biochemical stability
offer substantial advantages over cell suspensions. Goossensnerally, hairy roots
accumulate increased amounts of secondary metabolite. Therefore, using elicitors
or by performing metabolic engineering to the hairy roots can produce increased
amount of secondary metabolites.
Various studies have shown that plant cell cultures, including both suspension cultures and hairy root cultures, have proven to be an extremely useful platform for production of secondary metabolites and further research needs to be performed to develop fast growing; renewable and cheap source of material for production of economically viable, complex structured pharmaceutically important secondary metabolites.
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