39 Analysis of metabolic variations throughout growth and development of adventitious roots in Silybum marianum L. (Milk thistle), a medicinal plant

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Plant Cell, Tissue and Organ Culture(PCTOC)Journal of Plant Biotechnology ISSN 0167-6857Volume 123Number 3 Plant Cell Tiss Organ Cult (2015)123:501-510DOI 10.1007/s11240-015-0854-8

Analysis of metabolic variationsthroughout growth and development ofadventitious roots in Silybum marianum L.(Milk thistle), a medicinal plant

Mubarak Ali Khan, Bilal Haider Abbasi,Naseer Ali Shah, Buhara Ycesan &Huma Ali

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ORIGINAL ARTICLE

Analysis of metabolic variations throughout growthand development of adventitious roots in Silybum marianum L.(Milk thistle), a medicinal plant

Mubarak Ali Khan1 Bilal Haider Abbasi2 Naseer Ali Shah3 Buhara Yucesan4

Huma Ali5

Received: 20 May 2015 / Accepted: 13 August 2015 / Published online: 21 August 2015

Springer Science+Business Media Dordrecht 2015

Abstract Silybum marianum L. is a medicinal plant used in

the treatment for jaundice and liver diseases. In this study, an

adventitious root culture was developed for the production of

health promoting phytochemicals. Adventitious roots were

induced from nodal explants on solid Murashige and Skoog

medium supplemented with 1.0 mg l-1 of a-Naphthaleneacetic acid. Growth kinetics of the roots was investigated

every week, for 8 weeks of culture period. Highest fresh

biomass formation (153 mg l-1) was observed in 6-week old

cultures. Adventitious roots were harvested from different

growth stages as control (CTR), lag phase (LAG), logarith-

mic phase (LOG) or stationary phase (STN). Metabolite

profiling of the samples was investigated using electro spray

ionization time of flight mass spectrometry. Significant

phenylpropanoids such as cinnamic acid and di-hydro

kaempferol were predominantly found in LOG phase,

whereas the highest amount of malonic acid was detected in

STN as compared to other growth phases. More sucrose

content was detected in CTR, while the tryptophan content

was higher in LOG phase. Among the vital fatty acids,

prostaglandin A1 and phenyl acetic acid were at highest

levels in STN phase. However, more brassicasterols were

observed in LAG phase than other growth phases. Punicic

acid and lignan pinoresinol were detected abundantly in the

LOG phase. Biochemical characterization revealed signifi-

cant correlations between silymarin content and DPPH as

well as TPC and TFC in the growth curve. Interestingly,

among all growth stages there was no correlation of PAL

activity with TFC and silymarin content.

Keywords Silybum marianum L. Silymarin Adventitious roots Mass spectrometry Plant secondarymetabolites

Abbreviations

PGR Plant growth regulator

TPC Total phenolic content

TFC Total flavonoid content

PAL Phenylalanine ammonia lyase

FRSA Free radical scavenging activity

TOF Time of flight

ESI Electro spray ionization

PCA Principal component analysis

Introduction

Silybum marianum (L.) Gaertn. (milk thistle) belongs to

Asteracea family is an important medicinal herb used in

treatment of liver diseases with a history of use spanning

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11240-015-0854-8) contains supplementarymaterial, which is available to authorized users.

& Mubarak Ali [email protected]

& Bilal Haider [email protected]

1 Biotechnology Program, Department of Environmental

Sciences, COMSATS Institute of Information Technology

(CIIT), Abbottabad, Pakistan

2 Department of Biotechnology, Quaid-i-Azam University,

Islamabad 45320, Pakistan

3 Department of Biosciences, COMSATS Institute of

Information Technology, Islamabad, Pakistan

4 Department of Seed Science and Technology, Faculty of

Natural and Agricultural Sciences, Abant Izzet Baysal

University, 14280 Bolu, Turkey

5 Department of Biotechnology, Bacha Khan University,

Charsadda, KP, Pakistan

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Plant Cell Tiss Organ Cult (2015) 123:501510

DOI 10.1007/s11240-015-0854-8

Author's personal copy

more than centuries (Khan et al. 2013). It is of a great

value for its unique compound called silymarin having

several pharmacological activities including anti-inflam-

matory, anti-hepatitis, anti-viral and antioxidant (Khan

et al. 2014). Silymarin is an isomeric flavonoid mixture

containing silybin, silibinin, silidianin and silychristin

isolated from milk thistle (Abbasi et al. 2010). These

flavanolignans are well known for their antioxidant

capacity due to their redox properties, and it has been

assumed that a diet rich in flavonoids is inversely pro-

portional to cell aging, lipid peroxidation and cancer

(Ferreira et al. 2010). Unfortunately, the quality and

quantity of these metabolites are strictly species depen-

dent; thus show a high diversity with respect to the

physiological age, plant tissue type and environmental

conditions (Khan et al. 2015). The biotechnological pro-

duction of the natural compounds is an attractive

approach, alternative to the conventional extraction pro-

tocols using whole plant material without disturbing nat-

ural habitats (Ali et al. 2013). Especially, plant cell and

organ culture systems provide an insight through the

production of value-added plant-specific metabolites

(Ahmad et al. 2014). Establishment of adventitious root

culture can serve as a promising source of nutritionally

and pharmaceutically important metabolites. The main

advantages of adventitious root culture are faster growth

rate, easier maintenance and handling with high homo-

geneity in genetic makeup (Nagarajan et al. 2011). It has

also advantageous over hairy root culture since adventi-

tious roots do not produce any toxic chemicals like opines

(Cui et al. 2010). Flavonoids have a plethora of diverse

functions in plants ranging from physiological processes

such as pigmentation, seed coat and pollen tube devel-

opment to the plant responses against abiotic and biotic

stresses (Stracke et al. 2007; Misra et al. 2010). Moreover,

due to their health-promoting effects including antiox-

idative, anticancer, anti-inflammatory, cardioprotective,

and neuroprotective activities in mammalian tissues, fla-

vonoids are also considered to be of pharmaceutical

interest (Ferreira et al. 2010).

Electro spray ionization (ESI) mass spectrometry is a

rapid, highly sensitive and soft ionization technique which

mainly produces the protonated molecular species for a

wide range of compounds; thereby making data interpre-

tation easier (Smedsgaard and Nielsen 2005). During

adventitious root cultivation, morphogeny of the cells in

each growth phase as mentioned in this study is likely

influenced by expression of some hardly visible key

metabolites; thus, phytochemical profiling seems crucial

for our understanding on metabolic pattern of the root

growth.

In the present study, we established adventitious root

culture system, and investigated the key metabolites

responsible for the growth of adventitious roots through

ESI/TOFMS analysis. Furthermore, total phenolic and

flavonoid contents, antioxidant potential, PAL activity and

silymarin content were also evaluated during the growth

cycle of adventitious roots of milk thistle.

Materials and methods

In vitro seed germination

Mature seeds of S. marianum were collected from the main

campus of Quaid-e-Azam University Islamabad in 2012.

The seeds were rinsed quickly with 70 % (v/v) ethanol for

5 min prior to surface sterilization with 0.1 % (w/v) freshly

prepared mercuric chloride solution (HgCl2) for 3 min.

After rinsing three times with sterile distilled water, all the

seeds were placed into germination medium as described in

Khan et al. (2013) under in vitro conditions.

Adventitious root induction

Leaf explants (*25 mm2), cotyledon explants (*25 mm2),root segments (*5 mm) and nodal explants (*5 mm) wereexcised from 4 weeks old in vitro germinated seedlings, and

were cultured on solid MS (Murashige and Skoog 1962)

medium supplemented with 0.5 mg l-1 NAA in Petri plates.

Due to high morphogenetic potential, nodal explants were

selected for subsequent experiments. To study the effects of

plant growth regulators on adventitious root formation,

nodal explants were incubated on MS media containing

4.0 % sucrose (w/v) and 0.8 % (w/v) plant agar in 150 ml

conical flask supplemented with various concentrations (0.5,

1.0, 1.5 or 2.0 mg l-1) of indole-3-butyric acid (IBA),

indole-3-acetic acid (IAA) or NAA. The pH of media was

adjusted to 5.8 prior to autoclaving (121 C, 20 min at1 atm.). All cultures were incubated in a growth chamber at

16 h photoperiod with a light irradiance of *40 lmolm-2 s-1, and temperature was maintained at 25 1 Cwith 70 % relative humidity. In all set of experiments, PGR

free solid MS medium was used as a control treatment. After

4 weeks of cultivation, the frequency of adventitious roots

(%), mean number of roots and average biomass were

recorded respectively.

Growth kinetics of adventitious root culture

To establish adventitious root culture, 4-week-old fresh

adventitious roots were transferred to MS liquid media

502 Plant Cell Tiss Organ Cult (2015) 123:501510

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supplemented with 1.0 mg l-1 NAA. For each treatment,

500 mg fresh root was cultured into 40 ml media in 250 ml

flasks. All cultures were incubated under 24 h dark with a

continuous shaking in gyrator shaker at 110 rpm at room

temperature. Data on biomass accumulation was recorded

weekly by weighing adventitious roots in sterile conditions.

Duration of the study was 8 weeks, and the flasks in trip-

licate were used in all experiments.

Metabolite profiling

The samples were denoted on the basis of growth stage

during adventitious root culture as CTR (Control: fresh

adventitious roots used as inoculum for root suspension

culture), LAG phase (adventitious roots harvested on day

14 of root suspension culture), LOG phase (adventitious

roots harvested on day 35 of root suspension culture), STN

phase (Stationary; adventitious roots harvested on day 52

of root suspension culture). For each growth stage, three

replicates were collected. All samples were transferred to

air tight vials, flash frozen in liquid nitrogen, and stored at

-80 C for phytochemicals extraction.

Extraction of metabolites

Extraction was carried out according to an earlier pro-

tocol established by Overy et al. (2005). Briefly, 1000 llof solvent mixture A (methanol:chloroform:water,

2.5:1:1 at -20 C) was added to the centrifuge tube(2 ml) containing fine powder (10 mg) of each sample.

Samples were mixed with vortex for 25 s, and kept on ice

for 5 min prior to centrifugation at 14 9 103 rpm for

5 min at 4 C. The supernatant was collected and trans-ferred into a new pre-chilled storage tube (2 ml) and

labeled as supernatant A. The remaining pellet was re-

extracted with 500 ll of the pre-chilled solvent B(methanol:chloroform, 1:1 at -20 C) followed by slightmixing and centrifugation at 14 9 103 rpm for 5 min at

4 C to obtain the supernatant B. In the next step,supernatant A and B were combined together, and the

supernatant (methanolic extract) was decanted into a new

pre-cooled microcentrifuge tube. Then organic layer was

separated from aqueous layer by adding 250 ll chilleddistilled water into the mixture followed by centrifuga-

tion for 2 min. Both aqueous and organic phases were

then stored at -80 C until analysis.

Electrospray ionization time of flight mass spectrometry

(ESI-TOF MS)

ESI-TOF MS was performed on a LCT spectrometer (API

Q-Star, Waters Corporation, Milford, USA.) based on the

methods described by Davey et al. (2008) and Walker

(2011). The mass spectrometer was operated at a resolution

of 4000 (FWHM) in positive mode with a capillary voltage

of 4800 V, extraction cone at 3 V and sample cone at 20 V

with a range finder lens voltage of 75 V chosen for

detection of masses from 50 to 800 Da. Source temperature

was 110 C, and desolvation temperature was 120 C.Flow rates were 100 l h-1 for nebulisation and 400 l h-1

for desolvation. Spectra were collected in centroid mode at

a rate of one spectrum s-1 with 180 summed over a 3-min

period without background subtraction or smoothing.

Samples were loaded using a syringe pump (Razel, Con-

necticut, USA) at a flow rate of 20 ll min-1.

Data processing and metabolite identification

For each sample run, the summation of 180 centroid mode

spectra were exported from MassLynx data systems as text

file peak lists. These were imported into Microsoft Excel

(Microsoft Corp, USA), and an in-house macro program

was used to compare the accurate masses of three technical

replicate analyses of each sample. For the identification of

real masses within these profiles from background noise,

three replicates of mass spectra from each individual

sample were obtained. Once the peak was selected as a true

peak, the mean of the three masses over the three replicate

scans was used as the accurate mass, and this value along

with the corresponding average intensity made up the

metabolite profile. Finally the text files contained mass

spectra of respective samples were analyzed by Simca-

P ? (version 12.0). Principal component analysis (PCA)

was carried out using Pareto scaled 0.2 Da binned data sets

in Simca-P v12.0 software (Umetrics, Sweden). Identifi-

cation of putatively known metabolites was performed

through the comparison of monoisotopic masses likely to

be present in extracts, including [M?H]?, [M-H]- and

[M?Na]? against the list of metabolites in the plantcyc

database (http://pmn.plantcyc.org/).

Analysis of biochemical parameters

Fresh (FW) and dry weight (DW) of biological samples,

determination of electrical conductivity (EC) of residual

media were evaluated according to Baque et al. (2010).

Total phenolic content (TPC) and total flavonoid content

(TFC), were determined according to Velioglu et al. (1998)

and Chang et al. (2002), respectively. The radical scav-

enging activity of adventitious root cultures was deter-

mined by using 1,1-diphenyl-2-picrylhydrazyl (DPPH)

according to Abbasi et al. (2010). Phenylalanine ammonia-

lyase (PAL, EC 4.3.1.5) activity and silymarin content

were determined by methods described in Khan et al.

(2013).

Plant Cell Tiss Organ Cult (2015) 123:501510 503

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Experimental design and data analysis

All experiments were conducted in a factorial experimental

design with three replicates and were repeated twice. Mean

values of the treatments were subjected to the analysis of

variance (ANOVA), and significant difference was shown

with Duncans Multiple Range Test (DMRT) using SPSS

(Windows version 7.5.1, SPSS Inc., Chicago).

Results and discussion

Adventitious root induction

In preliminary studies, leaf, root, cotyledon or nodal

explants were cultured into solid MS medium supple-

mented with 0.5 mg l-1 NAA for adventitious roots for-

mation. Of the explants tested, nodal explants were

effective for root induction (56 % of rooting); however,

leaf explants failed to induce viable roots (data not shown).

This could be due to the different morphogenic responses

derived from different explants at same growth treatments

(Abbasi et al. 2010). Of the treatments concerning adven-

titious root formation and biomass accumulation,

1.0 mg l-1 NAA was the most effective with a frequency

of 73.4 % rooting and producing 34.3 roots per explant as

compared to 38.2 or 28.5 % of rooting and 12.7 or 13.2

roots per nodal explant produced from MS medium con-

taining 1.0 mg l-1 IAA or IBA, respectively (see Table 1).

Similarly, biomass accumulation of the adventitious roots

was also directly dependent on auxin concentrations.

Increase in auxin concentration from 0.5 to 1.0 mg l-1 (for

any auxin used) resulted in a rise in biomass accumulation

(for fresh and dry biomass, ca. 60 % increase in IBA, ca.

90 % in IAA); however, a tremendous increase in biomass

was observed when NAA only was taken into account

(14.8 to 61.2 mg fresh- and 0.9 to 4.6 mg dry biomass for

NAA; Table 1). On the other hand, increase in NAA

concentrations (from 1.0 to 2.0 mg l-1) clearly showed a

significant decline in terms of root number per explant

(compare 1.0 with 1.5 or 2.0 mg l-1 NAA producing 34.3,

21.4 or 10.9 roots per explant, respectively). Morphological

observations revealed that the resulting adventitious roots

were thick, numerous and mostly shorter while few were

longer without lateral root formation on MS medium

containing 1.0 mg l-1 NAA. Although few adventitious

roots with profound lateral branching were induced on MS

media supplemented with 1.5 mg l-1 IAA. For IAA and

IBA at 1.5 mg l-1, frequency of rooting, mean number of

root as well as biomass of the roots produced from nodal

explants were significantly higher than other concentra-

tions tested (0.5 or 1.0 mg l-1). Similarly, Lee et al. (2011)

observed optimum adventitious root production in Aloe

vera at low concentrations of NAA (0.51.0 mg l-1). It is

conspicuously established that addition of NAA to solid/

liquid medium foster adventitious root induction in a

variety of medicinal herbs. Enhanced biomass in hairy

roots of S. marianum was observed clearly on MS medium

supplemented with 0.5 mg l-1 NAA (Hasanloo et al.

2008). The efficiency of NAA for the production of

adventitious roots is due to its rapid absorption by plant

cells during in vitro growth conditions as compared to

other auxins. Peeters et al. (1991) reported that the uptake

of NAA is six times faster than IAA in tobacco explants.

Table 1 Effect of different auxins at different concentrations on induction of adventitious roots from nodal explants of S. marianum

S.no. MS?PGRS (mg l-1) Root induction frequency (%) Roots per explant (mean) Fresh bio mass (g/l) Dry bio mass (g/l)

1 MS (0) 0 0 0 0

2 IBA (0.5) 21.3 1.1b,c 8.4 0.9b,c 30.1 2.1b,c 3.7 1.8b

3 IBA (1.0) 28.5 1.6b 12.7 1.2b 48.3 3.8a,b 4.8 0.6a,b

4 IBA (1.5) 36.4 2.2a,b 17.8 1.8a,b 50.6 4.1a,b 5.3 0.3a

5 IBA (2.0) 23.2 1.4b,c 13.2 1.4a,b 36.2 2.3b 3.1 0.7b,c

6 IAA (0.5) 26.2 1.5b 6.3 0.4b,c 10.1 0.9 cd 0.8 0.3 cd

7 IAA (1.0) 38.2 2.4a,b 13.2 1.4a,b 19.3 1.1c 1.4 0.8b

8 IAA (1.5) 45.1 3.1a,b 15.4 1.4b 31.5 2.2b,c 2.9 1.9b,c

9 IAA (2.0) 22.4 1.2b,c 6.5 0.7b,c 17.3 1.2c 1.2 0.9b,c

10 NAA (0.5) 39.6 2.9a,b 20.2 1.2a,b 14.8 1.1c 0.9 0.2c

11 NAA (1.0) 73.4 4.3a 34.3 2.2a 61.2 5.4a 5.9 1.4a

12 NAA (1.5) 47.2 2.3a,b 21.4 1.4a,b 58.3 5.1a 4.6 0.4a,b

13 NAA (2.0) 23.6 1.6b,c 10.9 1.1b 38.1 2.8b 2.4 0.4c

Values are mean per explant standard error of three replicates

Values with different letter/s are significantly different at P\ 0.01


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Growth kinetics of adventitious root culture

Adventitious roots established in suspension culture dis-

played a relatively quick growth curve which was charac-

terized by lag phase at first week, followed by log phase for

4 weeks (from 2nd to 5th week), and a subsequent sta-

tionary phase for last three weeks of cultivation (from 6th

to 8th week). Maximum fresh biomass (FBM) and dry

biomass (DBM) of 167 5.91 and 10.73 0.384 mg l-1,

respectively, were observed on 42th day (Fig. 1). FBM was

almost seven times higher than the initial inoculum culture.

Similarly, Kim et al. (2004) observed FBM of adventitious

roots in Panax gingeng on MS liquid medium by using

2.0 mg l-1 NAA. A linear decrease in culture volume was

inversely correlated with an increase in fresh weight of

roots depending on the number of sub cultivations (Wu

et al. 2008). Gradual decrease in electrical conductivity

(EC) of residual medium with consumption of nutrients by

adventitious roots was clearly observed between 3rd and

8th week of cultivation (Fig. 1). At initial phase, there was

only a minor increase in EC that might be due to excretion

of metabolites to the medium. The decrease in EC of

residual medium strongly confers the increase in biomass

of adventitious root; therefore, it can be correlated with

intake of nutrients (PO4-, NH3

?, NO3-, etc.) from the

culture medium (Liu et al. 2006).

Metabolic analysis of adventitious root culture

Mass spectra and principal component analysis (PCA)

The raw mass spectrum profiles of both aqueous and

organic fractions differed in peaks detected at varying sig-

nals/intensities (Fig. S1&S2; Supplementary). The spectra

of aqueous fractions were dominated by some common

peaks at m/z 242 and m/z 146; however, many uncommon

peaks were also detected with a highest peak at m/z 96 in

STN. Nevertheless, the mass spectra in organic fractions

were dominated by uncommon peaks only. The highest

peaks at m/z 178.045, m/z 233.89, m/z 178.044 and m/z

178.11 were detected in CTR, LAG, LOG and STN,

respectively. The common peaks represent the masses in the

biological samples which are thoroughly expressed during

growth of adventitious roots, whereas the uncommon peaks

represent the rare masses which are responsible for sepa-

rating one growth stage from the rest in the growth cycle

(Walker 2011).

These mass spectrum profiles were then converted into

text files by using in-house software and were then sub-

jected to PCA analysis to determine the putatively known

metabolites responsible for growth and development of

adventitious roots. PCA loading scatter plots distinguished

the samples on the basis of differences in growth stages

during root formation, and enabled the detection of several

bin masses responsible for separating samples from dif-

ferent growth stages (Fig. 2a, b). The principal components

PC1 and PC2 accounted for 76 % variation in aqueous

fractions and 52 % variation in organic fractions. The

loadings scatter plot for a PCA analysis can provide a list

of the metabolite bins, as to whether they show an

increasing or decreasing pattern in content. The bins which

are closest to the origin in the loading plot are the bins that

changed the least. Conversely, the bins furthest away from

the origin are those that changed most, suggesting that

these bins contain compounds which might be useful for

the differential responses at different growth stages during

adventitious root formation (Khan et al. 2015).

Bin masses

The bin masses having high PCA loadings derived from

four different growth stages were selected, and their total

ion counts/intensities were summed up (Fig. 3a, b;

S3A&B, Supplementary). Bin mass 159 was found in the

loading scatter plot influencing the separation of the STN

lines and bin mass 337 was found contributing in the

separation of the LAG lines in all aqueous fractions.

Similarly, the organic fractions of the biological samples

showed bin mass 148 separating the LOG lines from the

other plant lines (Fig. 2a, b). Bin masses 214 and 377

contributed high score scatter loadings of aqueous and

organic fractions respectively; nevertheless, these bins

were not identified by the in-house bin program. Therefore,

those aforesaid bins that were not identified by the in-house

bin program were ignored at all, and the selected bins

identified by the in-house bin program were compared

Fig. 1 Growth kinetics of Adventitious root culture of Silybummarianum L. in relation to electrical conductivity of residual medium

on MS medium supplemented with 1.0 mg l-1 NAA. Values are

mean standard error of three replicates


123


Fig. 2 PCA Loading Scatter plots of biological samples evaluated by SIMCA-P ? (12.0). a Aqueous fractions and b Organic fractions


123


among the biological samples. Bin masses 159 and 337.2

were detected with sequential increase in total ion counts/

average intensity from CTR to LOG (Fig. 3a, b; S3A&B,

Supplementary). However, increased signals of the bin

masses 149 and 381.2 were found in LOG followed by

STN and LAG while the signals decline was detected in

CTR. Bin mass 365.2 was found to be at sequential decline

in peak signals from CTR to STN. Average intensities

calculated also depicted the same behavior. Increased sig-

nals of the bin mass 437.4 were found to be higher in LAG

phase among all the bins tested in the organic fractions

(Fig. 3a, b; S3A&B, Supplementary).

Evaluation of key metabolites in different growth stages

during root culture

Based on the values of total ions count (%), and average

intensity of selected bins in all biological samples, puta-

tively known metabolites were obtained from in-house bin

program (Fig. 4; Table S1, Supplementary). Significant

phenylpropanoids from shikimate pathway in plants such

as cinnamic acid and di-hydro kaempferol (DHK), were

found to be at high amount in LOG phase. These phenolic

acids are produced as intermediary products of phenyl-

propanoid metabolic pathway, and can be anticipated for

stimulating role in biomass formation during in vitro root

culture in present study. They are also reported for their

role in oxidative phosphorylation and photophosphoryla-

tion, stimulation of RNA synthesis, bud development as

well as prevention of senescence due to their strong

antioxidant nature (Rathod et al. 2014). Both cinnamic acid

and di-hydro kaempferol activate antioxidant enzymes

SOD and POD against reactive oxygen species (ROS),

under stress conditions (Szalai and Janda 2009).When

taken into account total ion counts (Fig. 4), malonic acid

showed a growing trend from CTR to STN (from 0.8 to

4.8 %). Among the organic acids, malonic acid is proven as

a preferential plant defensive chemical against a variety of

abiotic stresses (Kim 2002).

The highest sucrose level was detected in CTR with a

sequential decline through different growth stages, and

lowest value was detected in STN. Generally, sucrose is the

most frequently used carbohydrate acting as a signaling

molecule, osmoticum and a source of carbon and energy in

culture media for following in vitro morphogenetic pro-

cesses such as adventitious rooting, callus organogenesis

and somatic embryogenesis (Khan et al. 2015; Yucesan

et al. 2015). The amount of tryptophan accumulated at

LOG phase was significantly higher than other growth

phases during adventitious root culture (Fig. 4). Since

auxin plays a key role during in vitro rooting in majority of

plant species, its de-novo biosynthesis is controlled by

concomitant production of tryptophan through multiple

routes. Usually it acts as a key precursor for auxin (indole-

3-acetic acid, IAA) biosynthesis through the tryptophan-

dependent pathway (Michalczuk et al. 1992). From our

results, we extrapolated that the accumulation of trypto-

phan in LOG phase results in more biomass accumulation

underlying the essential role of auxin during development

of adventitious roots. Comparison of phenyl-acetic acid

(PAA) among the biological samples revealed its highest

level in the STN followed by LOG (Fig. 4). Although PAA

is a non-indolic, active endogenous auxin present at

physiologically different levels in higher plants, the bio-

logical significance of PAA is not completely clear.

However, with possession for an auxin-like activity, PAA

0

10

20

30

40

50

60

70

80

90

100

c

c

cc

cc

bc

bc bc bcbc

bb

abab

aB

A

Ctr Lag Log Stn

Bin Masses

Tot

al I

on c

ount

s (%

)

437.4

301.228

914

3

Fig. 3 Percentage of total ion counts of the bins (putatively identifiedmetabolites) in CTR, LAG, LOG and STN. Bin masses were selected

from the loading plots sorted by PCA. a Aqueous fractions andb Organic fractions. Data represents the values of the mean stan-dard error from three replicates. Column bars sharing the same letter/s

are similar otherwise differ significantly at P\ 0.05


123


regulates the auxin transport, and has also been shown to

influence morpho-regulation (Korasick et al. 2013). Among

the vital fatty acids, prostaglandinA1was detected at highest

signal (2.5 %) in STNphase. Prostaglandins (PGs) are cyclic

fatty acids, generally called as oxylipins and are considered

as signal compounds in plants and animals. They accumulate

as a consequence of oxidative stress. Notably prostaglandin

A1has an important role in the induction of genes involved in

plant defense and biosynthesis of secondary metabolites

(Loeffler et al. 2005). Besides stress modulation, PGs also

play significant roles in other physiological processes

including cell division, elongation, membrane polarization,

vascular and reproductive development, proton pumping and

photo morphogenesis (Thoma et al. 2003). The highest

amount of brassicasterols was detected in LAG phase than

other growth lines (Fig. 4). As the name suggests, brassi-

casterols are predominately found in the Brassicacea family,

and are reported as plant steroids essential for normal plant

growth and development (Bishop and Yokota 2001). In

contrast to malonic acid and PAA, higher amount of punicic

acid was found in the LOG phase during growth kinetics of

adventitious root culture (Fig. 4). Puccinic acid has some

structural similarities (such as carbon composition, atomic

arrangement and the number of carbon double bonds) to the

conjugated linoleic and a-linolenic acids (Viladomiu et al.2013). Recently, puccinic acid has increasingly attracted

scientific interest because of its several potential health

benefits including antioxidant, antitumor, immunomodula-

tory, anti-atherosclerotic and serum lipid-lowering activities

(Carvalho et al. 2010). Similarly, pinoresinol had also higher

total ion counts in the LOG phase that other growth phases in

all the biological samples (Fig. 4). Pinoresinol is a type of

lignans derived from the enantio-selective dimerization of

two coniferyl alcohol units (Nakatsubo et al. 2008). How-

ever, the exact roles of lignans in plant are not clear, although

it has been suggested that they are involved in plant defense

(Naoumkina et al. 2010).

Biochemical parameters in developmental stages

during adventitious root culture

DPPH-free radical scavenging activity was found to be

highest in STN phase (78 %) while lowest in LAG phase

(31 %).Throughout the growth curve, TFC and TPC were

detected at maximum levels (3.5 mg GAE/g DW and

1.9 mg QE/g DW) in log phase respectively, whereas

silymarin content was observed at highest level

(3.08 0.14 mgg-1 DW) in stationary phase (Fig. 5, 6).

PAL activity test of the biological samples showed its

higher level in the LAG phase and least in STN phase.

Interestingly, the different growth stages exhibited signif-

icant variations in PAL activity and silymarin content

(Fig. 6). Biochemical parameters during growth of adven-

titious root culture in present study revealed significant

correlations (r = 0.96, P = 0.044) between silymarin

content and DPPH as well as TPC and TFC (r = 0.97,

P = 0.032) at P\ 0.05 (Table 2). However, among allgrowth stages, there was no correlation between PAL

Fig. 4 Comparison of the keymetabolites in CTR, LAG, LOG

and STN lines. Putatively

known metabolites were

obtained from in-house bin

program on the basis of their

distribution with total ions count

(%) in the biological samples.

Data represents the values of the

mean standard error from

three replicates. Column bars

sharing the same letter/s are

similar otherwise differ

significantly at P\ 0.05


123


activity with TFC and silymarin content. Therefore, an

understanding of the biosynthesis of flavonoids during the

process of adventitious rooting is important because of

their role in develotpmental physiology and defense

mechanisms. Generally, biosynthesis of flavonoids occurs

through the derivation of their carbon skeletons from two

basic compounds: malonyl-CoA and p-coumaroyl-CoA in

the phenylpropanoid pathway (Heller and Forkmann 1988).

The first committed step for biosynthesis of the phenyl-

propanoid skeleton in plants is the deamination of L-

phenylalanine to yield trans-cinnamic acid and ammonia.

This reaction is catalyzed by phenylalanine ammonia lyase

(PAL, EC 4.3.1.5), and is often regarded as a key step in

the biosynthesis of these flavonoids (Khan et al. 2013). The

inverse correlation of PAL activity with TFC and silymarin

content in our study can be explained in a way that the

specific growth stage during development of adventitious

roots do affect the PAL activity, as the root growth pro-

gresses from one phase to another. Notwithstanding, the

level of PAL activity depends on the genotype, develop-

mental stage and the organ or tissue type of the plant

(Swedan 2013). Moreover, PAL turnover may also be

responsible for the biosynthesis of a wide range of

phenylpropanoid compounds, such as cinnamic acid

malonic acid, DHK and lignin (Khan et al. 2015).

Acknowledgments Financial Support of National Research Pro-gramme for Universities (NRPU), Higher Education Commission

(HEC) of Pakistan is appreciated here. Dr. Khan M.A. acknowledges

Financial Support of Indigenous Scholarship Programme for his Ph.D.

by HEC.

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Analysis of metabolic variations throughout growth and development of adventitious roots in Silybum marianum L. (Milk thistle), a medicinal plantAbstractIntroductionMaterials and methodsIn vitro seed germinationAdventitious root inductionGrowth kinetics of adventitious root cultureMetabolite profilingExtraction of metabolitesElectrospray ionization time of flight mass spectrometry (ESI-TOF MS)Data processing and metabolite identification

Analysis of biochemical parametersExperimental design and data analysis

Results and discussionAdventitious root inductionGrowth kinetics of adventitious root cultureMetabolic analysis of adventitious root cultureMass spectra and principal component analysis (PCA)Bin massesEvaluation of key metabolites in different growth stages during root culture

Biochemical parameters in developmental stages during adventitious root culture

AcknowledgmentsReferences

Documents

39 Analysis of metabolic variations throughout growth and development of adventitious roots in Silybum marianum L. (Milk thistle), a medicinal plant