Components of antioxidant system of Picrorhiza kurrooa exhibitdifferent spatio-temporal behavior

Manu Pratap Gangola • Jai Parkash •

Paramvir Singh Ahuja • Som Dutt

Received: 24 January 2013 / Accepted: 14 September 2013 / Published online: 22 September 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Antioxidant system is one of the important

factors in regulating plant growth, development and

adaptation. Thus, in order to have better insights into

molecular mechanisms of growth and adaptation of a plant

it is prerequisite to have known the status of various

components of the antioxidant system of the plant. Here we

studied the status of enzymatic and non-enzymatic com-

ponents of the antioxidant system of picrorhiza (Picrorhiza

kurrooa). Picrorhiza is an important medicinal herb of

western Himalayan region and has been listed in the Red

Data Book as an endangered species. Spatio-temporal

analysis of ascorbic acid and glutathione in leaf, root and

rhizome during different stages of development revealed

differential status of these antioxidant molecules. Of the

three tissues, ascorbic acid was found to be highest in

leaves and lowest in roots. Interestingly, just opposite to

that, glutathione was highest in roots and lowest in leaves.

Using degenerate primers based approach followed by

rapid amplification of complementary DNA (cDNA) ends

method, full length cDNAs of three important genes

namely Picrorhiza kurrooa ascorbate peroxidase (pkapx),

Picrorhiza kurrooa monodehydroascorbate reductase

(pkmdhar) and Picrorhiza kurrooa glutathione reductase

(pkgr) of antioxidant system were cloned from picrorhiza.

Complementary DNAs of pkapx, pkmdhar and pkgr con-

tained 1,049, 2,016 and 1,664 bp, respectively. Expression

analysis showed differential spatio-temporal expression of

these genes. Expressions of all the three genes were found

higher in roots as compared to rhizome and leaves. Tem-

poral expression analysis of pkapx, pkmdhar and pkgr

revealed differential transcript levels. Expression of pkapx

exhibited negative correlation with the light intensity. Just

opposite to the pkapx, expression pattern of pkgr revealed

its positive correlation with light intensity. Expression

pattern of pkmdhar revealed its light independent expres-

sion behavior. The findings may be useful to assess the role

of cloned genes in picrorhiza growth, adaptation and can

further be utilized for transgenic development for desired

trait(s).

Keywords Antioxidant system � Ascorbate

peroxidase � Monodehydroascorbate reductase �Glutathione reductase

Abbreviations

pkapx Picrorhiza kurrooa ascorbate peroxidase

pkmdhar Picrorhiza kurrooa monodehydroascorbate

reductase

pkgr Picrorhiza kurrooa glutathione reductase

RACE Rapid amplification of cDNA ends

ROS Reactive oxygen species

PFD Photon flux density

CDD Conserved domain database

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11033-013-2772-3) contains supplementarymaterial, which is available to authorized users.

Present Address:

M. P. Gangola

Department of Plant Sciences, University of Saskatchewan,

Saskatoon, SK S7N 0P5, Canada

J. Parkash � P. S. Ahuja � S. Dutt (&)

Biotechnology Division, CSIR-Institute of Himalayan

Bioresource Technology (CSIR-IHBT), Palampur 176061,

Himachal Pradesh, India

e-mail: sd_bio@yahoo.com; somdutt@ihbt.res.in

123

Mol Biol Rep (2013) 40:6593–6603

DOI 10.1007/s11033-013-2772-3

Introduction

Picrorhiza (Picrorhiza kurrooa Royle ex Benth.), a small

perennial herb, belongs to family Scrophulariaceae and

tribe Veroniceae. It grows primarily in the north-Western

Himalayan region from Kashmir to Kumaun and Garhwal

regions in India and Nepal at an altitude of 3,000–5,000 m

above mean sea level. Picrorhiza’s leaf, root, bark and the

underground part, rhizome are widely used in the tradi-

tional Indian (ayurvedic) system of medicine due to its

antioxidative, hepatoprotective, antiproliferative, immuno-

modulatory, antibacterial and antiviral activities [1]. The

plant is self-regenerating but unregulated over-harvesting

has caused it to be threatened to near extinction and thus

picrorhiza has been listed in the Red Data Book as an

endangered plant species [2]. Being the plant of high alti-

tude, oxidative stress may be one of the major limiting

factors in its growth and productivity [3]. Oxidative stress

leads to the generation of various highly reactive chemical

forms of oxygen, collectively designated as ‘‘reactive

oxygen species (ROS)’’ [4]. ROS are toxic to biological

organisms because they oxidize lipids, proteins, DNA and

carbohydrates resulting in the breakdown of normal cel-

lular membrane and reproductive functions. Ultimately,

toxic levels of ROS cause a chain reaction of cellular

oxidation, resulting in disease and lethality [5]. Ascorbate

and glutathione, the two major soluble antioxidants in plant

cell, linked via ascorbate–glutathione cycle play an

important role in scavenging the ROS. Ascorbate detoxifies

H2O2, neutralises superoxide radicals (O2�-), singlet oxy-

gen (O�-) or hydroxyl radical (OH�-). In addition, it also

serves as a major redox buffer, signaling molecule,

cofactor (violaxanthin de-epoxidase in xanthophyll cycle),

photo protector, regulator of cell growth and cell division

[6–10]. Ascorbate, a precursor for the synthesis of com-

pounds such as ethylene, gibberellic acid, anthocyanins,

and flavonoids required during different development

stages in plants [7]. Similarly, glutathione is another

essential metabolite which shows multiple functions in

plants. Glutathione is a good candidate for transmission of

ROS signals against pathogen attack [11]. Being a com-

ponent of antioxidant system, it provides resistance against

biotic and abiotic stresses to the plant cells [12]. The pri-

mary function of glutathione is in thiol-disulphide inter-

actions, in which reduced glutathione (GSH) is

continuously oxidized to a disulphide form (GSSG) that is

recycled to GSH.

Ascorbate–glutathione cycle is mediated by various

enzymes. Ascorbate peroxidase (APX; EC 1.11.1.11)

scavenges H2O2 generated both internally and externally

with the help of Ascorbate. Monodehydroascorbate reduc-

tase (MDHAR; EC 1.6.5.4) is involved in the recycling of

the ascorbate by converting the monodehydroascorbate

(MDA) again into ascorbate. Glutathione reductase (GR;

EC 1.6.4.2) is essential for the recycling of glutathione

disulfide (GSSG) into reduced glutathione (GSH) by using

NADPH as an electron donor, and maintained highly

reduced state of GSH/GSSG and ASA/DHA ratios at the

intracellular level [13]. Attempts to reduce oxidative dam-

ages under the stress conditions have included the manip-

ulation of ROS scavenging enzymes by gene transfer

technology. In order to use genetic engineering for modu-

lation of various ROS scavenging constituents, it is essential

to have the comprehensive knowledge of the antioxidant

system in that particular plant at molecular level. There is

no information available on the antioxidant system of pic-

rorhiza at plant, cellular and molecular level. Keeping this

in view, the present work was aimed to identify, clone and

characterize the key genes; pkapx, pkmdhar and pkgr of the

antioxidant system of picrorhiza.

Materials and methods

Plant materials

Picrorhiza (P. kurrooa) plants used in the present study

were collected from its natural habitat at Rohtang Pass

(4,000 m altitude, 32�230N, 77�150E, India) during April–

May when the plants were in dormant stage and brought to

the institute at Palampur (1,300 m altitude; 32�060N,

76�330E, India). These were transplanted in plastic pots and

maintained in the experimental farm of the institute as

described by Kawoosa et al. [14].

Estimation of ascorbate and glutathione

Ascorbic acid and glutathione contents were measured by

following the methods of Foyer et al. [15] and Smith [16],

respectively. Both, total and reduced forms of ascorbic acid

as well as glutathione were estimated in leaf, root and

rhizome tissues. Sampling of the tissues was carried out at

14 days intervals starting from 14 days after transplanta-

tion (DAT) till 140 DAT. Further samplings were not

performed due to rapid advancement of leaf senescence.

For each sampling three biological replicates were used.

Picrorhiza plants were randomly selected and were

uprooted from the pot carefully. Leaves (2nd and 3rd leaf

position, starting from the top), roots and rhizomes of the

plant were harvested, washed with distilled water, blotted

dry, weighed, frozen in liquid nitrogen and stored at

-80 �C till further use. The frozen samples (100 mg) were

ground to fine powder in liquid nitrogen using pestle and

mortar followed by addition of 1.0 ml of 1 M HClO4 with

intermittent grinding for 1 min. On thawing, it was trans-

ferred to eppendorf tube and centrifuged at 12,0009g for

6594 Mol Biol Rep (2013) 40:6593–6603

123

15 min at 4 �C. Supernatant (500 ll) was transferred to

fresh eppendorf tubes and 100 ll of phosphate buffer

(120 mM, pH 7.6) was added. Sufficient K2CO3 was added

to bring the pH of the solution to 4–5. The sample mixtures

were centrifuged to remove insoluble KClO4 and super-

natant was transferred to a fresh tube and kept on ice. The

prepared extract was used for the estimation of ascorbic

acid as well as glutathione.

For estimation of reduced ascorbic acid 25 ll extract

was added in 975 ll of 120 mM phosphate buffer (pH 5.6).

Absorbance was measured at 265 nm wavelength. Subse-

quently 10 ll of ascorbate oxidase (AO; 0.1 unit/ll) was

added and again A265 was recorded for 5 min. The differ-

ence in A265 before and after addition of AO was calculated

and ascorbic acid content was measured with the help of

calibration curve. For estimation of total ascorbic acid

content 25 ll extract and 215 ll of 120 mM phosphate

buffer (pH 7.6) were taken in an eppendorf tube. 10 ll of

20 mM dithiothreitol (DTT) was added in the mixture and

incubated for 30 min at room temperature. The mixture

was assayed as for reduced ascorbic acid. The concentra-

tion of ascorbic acid was expressed in nmol/gram fresh

weight basis (nmol/gfw).

For estimation of total glutathione (GSH?GSSG) 20 ll

of extract was added in a mixture of 860 ll 120 mM

phosphate buffer (pH 7.6; containing 6 mM EDTA),

100 ll of 6 mM DTNB and 10 ll of 50 mM NADPH.

Reference cuvette contained everything except extract.

Reaction was started by adding 10 ll of glutathione

reductase (GR; 0.1 unit/ll). Absorbance was measured for

5 min at 412 nm, continuously. For estimation of oxidized

form (GSSG), 200 ll extract was added to 5 ll of

2-vinylpyridine, mixed well and incubated at room tem-

perature for 20 min. The reaction mix was centrifuged at

12,0009g for 15 min at 4 �C. 160 ll supernatant was

transferred to fresh tube. 20 ll was assayed as for total

glutathione. Concentration of reduced glutathione was

arrived at by deducting oxidized glutathione content from

the total glutathione. The concentration of glutathione was

expressed in nmol/gram fresh weight basis (nmol/gfw).

Cloning of cDNAs of pkapx, pkmdhar and pkgr

RNA was isolated from leaf tissue using the method of

Ghawana et al. [17] and digested with DNase 1 (RNase

free) (Fermentas Inc, USA). Complementary DNA (cDNA)

was synthesized as described by Singh et al. [18]. Degen-

erate primers for apx, mdhar and gr were designed from the

conserved regions of corresponding genes reported from

different plant sources and the partial gene sequences were

amplified by PCR as detailed in Table 1. The amplicons

were cloned in pGEM�-T Easy Vector (Promega, USA),

plasmids were isolated using Fermentas GeneJET Plasmid

Miniprep Kit (Fermentas Inc, USA), and sequencing was

performed using BigDye terminator cycle sequencing mix

(Version 3.1; Applied Biosystems, USA) using an auto-

mated DNA sequencer (ABI 3130 xl Genetic Analyzer,

Applied Biosystems, USA). Protocols were followed

essentially as described by the respective manufacturers.

Full-length cDNAs were cloned by performing rapid

amplification of cDNA Ends (RACE; SMARTerTM RACE

cDNA Amplification Kit; Clontech, USA) as per the manu-

facturer’s instructions using the gene specific primers (pkgr-

50RACE-R1, pkgr-30RACE-L1, pkapx-50RACE-R1, pkapx-

30RACE-L1, pkmdhar-50RACE-R1, pkmdhar-30RACE-L1

Table 1). These primers were designed based upon the par-

tial sequences of the genes as cloned above. After aligning

the sequences obtained by 50 and 30 RACE, full-length cDNA

was amplified using the end sequences, cloned in pGEM�-T

Easy Vector (Promega, USA) and confirmed by sequencing.

Sequence alignments were performed according to the

method of Higgins and Sharp using Clustal program [19].

Similarity scores between (pkapx, pkmdhar and pkgr) and

other (apx, mdhar and gr) were calculated using the online

BLASTN, BLASTX, and BLASTP programs of National

Center for Biotechnology Information (NCBI) with default

parameters (http://www.ncbi.nlm.nih.gov/). Secondary

structure of the deduced amino acid sequence was analyzed

using self-optimized prediction method with alignment

(SOPMA; http://www.npsa-pbil.ibcp.fr/). CDD (http://

www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) was used

to identify the conserved domains in the particular deduced

amino acid sequence.

Semi-quantitative expression analysis by RT-PCR

Semi-quantitative RT-PCR based expression analysis was

performed to study the expression level of genes (1) in

different tissue, and (2) during different time points of the

day. For tissue specific expression study, leaves, roots and

rhizomes samples were harvested from plants grown in

experimental farm. For diurnal variation study, 4th leaf

position (with respect to the top apical leaf designated as

first leaf) of plant of 45 DAT were used. Sampling was

performed at 6 h interval for continuous 2 days. Young

leaves (100 mg) were harvested from plants at different

time points [6:00 a.m., 12:00 p.m., 6:00 p.m., 12:00 a.m.

(first day), 6:00 a.m., 12:00 p.m., 6:00 p.m., 12:00 a.m.

(second day), designated as 6, 12, 18, 24, 30, 36, 42, and

48 h, respectively] for continuous 2 days. The harvested

samples were immediately frozen in liquid nitrogen and

stored at -80 �C. Primers for expression analyses were

designed from the sequences of cloned cDNAs and are

given in Table 1.

Mol Biol Rep (2013) 40:6593–6603 6595

123

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6596 Mol Biol Rep (2013) 40:6593–6603

123

To analyse the expression levels of the cloned genes,

Total RNA was isolated from leaf tissue (100 mg) using

the method of Ghawana et al. [17]. cDNA was synthesized

from DNA-free RNA using SuperScript� III Reverse

Transcriptase (Invitrogen, USA) This cDNA was used as

template for Reverse transcription-PCR reaction using

gene specific primers (pkgrF1, pkgrR1, pkmdharF1, pkm-

dharR1, pkapxF1, pkapxR1) as mentioned in Table 1.

Cycling conditions were optimized to obtain amplification

under the exponential phase. 26S rRNA based primer pair

was used as internal control for expression studies [18].

Amplicons were analyzed and quantified using the Alpha

DigiDoc Gel Documentation and Image analysis system

(Alpha Innotech, USA). Each experiment was repeated at

least twice with three biological replicates each time andTa

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14 28 42 56 70 84 98 112 126 140

14 28 42 56 70 84 98 112 126 140

asco

rbic

aci

d (n

mol

/gfw

)

days

totalreduced

Fig. 1 Spatio-temporal status of total and reduced ascorbic acid

content (nmol/gfw) in leaves (a), rhizomes (b) and roots (c) of

picrorhiza with respect to the different developmental stages. Each

value is the mean of three independent samples. Black bars represent

total content of ascorbic acid, white bars represent reduced content of

ascorbic acid. Error bars represent standard error of mean (n = 3)

Mol Biol Rep (2013) 40:6593–6603 6597

123

the representative figure of one experiment is shown in the

manuscript.

Results

Spatio temporal status of ascorbic acid and glutathione

contents in picrorhiza

Ascorbic acid and glutathione were estimated during different

stages (as mentioned under ‘‘Materials and methods’’ section)

of plant development, in three tissues of picrorhiza viz; leaf,

rhizome and root. During different development stages, both

ascorbic acid and glutathione contents varied in all the three

tissues. Also, the spatial variation was observed for both glu-

tathione and ascorbic acid contents. Leaves contained maxi-

mum ascorbic acid followed by rhizomes and roots (Fig. 1). In

leaves ascorbic acid content varied from 2,999 ± 111 nmol/

gfw (day 14 DAT) to 4,390 ± 151 nmol/gfw (126 DAT). In

case of rhizome, the ascorbic acid content varied from

1,972 ± 62 to 3,063 ± 171 (day 14) nmol/gfw (126 DAT).

The ascorbic acid contents varied from 1,396 ± 126 (day 14)

to 2,171 ± 74 nmol/gfw (126 DAT) in roots. The data indi-

cated continuous increase in ascorbic acid content with the age

of plants except the observed last stage which was marked by

onset of leaf senescence in picrorhiza. The percentage of the

reduced ascorbic acid varied from 78.4–90.1, 70.4–93.3 and

74.0–90.8 % in leaf, rhizome and root, respectively.

redu

ced

form

(%

)

a

b

0

10

20

30

40

50

60

70

80

90

100

asco

rbic

aci

d (n

mol

/gfw

)

days

0

10

20

30

40

50

60

70

80

90

100

14 28 42 56 70 84 98 112 126 140

14 28 42 56 70 84 98 112 126 140

glut

athi

one

(nm

ol/g

fw)

days

Leaf rhizome root

Leaf rhizome root

Fig. 3 Spatio- temporal status of total reduced form of ascorbic acid

and glutathione in picrorhiza with respect to the different develop-

mental stages. a represent reduced form of ascorbic acid (nmol/gfw)

in leaf, rhizome and root.b represent reduced form of glutathione

(nmol/gfw) in leaf, rhizome and root. Each value is the mean of three

independent samples. Black bars represent per cent ascorbic acid/

glutathione in leaf, gray bars represent percent ascorbic acid/

glutathione in rhizome, white bars represent percent ascorbic acid/

glutathione in root

c

a

b

glut

athi

one

(nm

ol/g

fw)

0

200

400

600

800

1000

1200

0

200

400

600

800

1000

1200

0

200

400

600

800

1000

1200

14 28 42 56 70 84 98 112 126 140

14 28 42 56 70 84 98 112 126 140

14 28 42 56 70 84 98 112 126 140

totalreduced

days

Fig. 2 Spatio- temporal status of total and reduced glutathione

content (nmol/gfw) in leaves (a), rhizomes (b) and roots (c) of

picrorhiza with respect to different developmental stages. Each value

is the mean of three independent samples. Black bars represent total

glutathione content, white bars represent reduced glutathione. Error

bars represent standard error of mean (n = 3)

6598 Mol Biol Rep (2013) 40:6593–6603

123

Glutathione content was recorded maximum in case of

roots, followed by rhizome and leaves (Fig. 2). In roots

glutathione content varied from 792 ± 30 (14 DAT) to

939 ± 124 nmol/gfw (126 DAT). In rhizomes the glutathi-

one content varied from 460 ± 46 to790 ± 31 nmol/gfw.

In leaves the content varied from 361 ± 32 (14 DAT) to

507 ± 72 nmol/gfw (126 DOT). The amount of reduced

glutathione varied from 85.2 ± 5.8–89.4 ± 8.4, 85.0 ±

5.7–89.3 ± 8.4, and 87.1 ± 6.7–90.6 ± 9.7 % in leaf,

rhizome and root, respectively (Fig. 3).

Cloning of pkapx, pkgr and pkmdhar

Degenerate primers (Table 1) were designed according to

the highly conserved amino acid sequences of plant APXS,

MDHARS and GRS. Partial cDNA fragments composed of

532, 388 and 710 nucleotides of pkapx, pkmdhar, pkgr

respectively, were isolated by RT-PCR. Blast analysis

showed that these amplicons exhibit strong homology with

the other plants reported genes in NCBI database. Based on

these sequences, a RACE strategy was used to identify the

open reading frames of apx (753 bp), mdhar (1,461 bp)

and gr (1,482 bp). Two pairs of gene specific primers were

designed for the 30 RACE and 50 RACE based on partial

sequences. Using the RACE method, full length cDNAs of

pkapx, pkmdhar and pkgr were cloned and subsequently

confirmed by sequencing. The full length pkapx cDNA was

1,049 bp long with an open reading frame (ORF) of

753 bp. The ORF was flanked by a 99 bp 50 untranslated

region (UTR) and a 197 bp 30 UTR. In case of pkmdhar,

full length cDNA of 2,016 bp was obtained that contained

an ORF of 1,461 bp. The ORF was flanked by 325 bp 50

APX

MDHAR

832

Picrorhiza kurrooa

0.05

Lycopersicon esculentum

Arabidopsis thaliana

Brassica oleracea

Spinacia oleracea

Zantedeschia aethiopica

876

1000

505

648

Vigna unguiculata

Phaseolus vulgaris

Arabidopsis thaliana

Picrorhiza kurrooa

Mesembryanthemum crystallinum

Zinnia elegans

1000

0.02

Nicotiana tabacum

Capsicum annum

Lycopersicon esculentum

Picrorhiza kurrooa

Rehmannia glutinosaAcanthus ebracteatus499

951

0.02

321

GR

Fig. 4 Dendrogram illustrating

the phylogenetic relationship of

pkapx, pkmdhar and pkgr from

picrorhiza and those of other

plant species. Phylogenetic tree

was constructed using tree

explorer program (http://

evolgen.biol.metro-u.ac.jp/TE/

TE_man.html) and bootstar

values are indicated above the

lines. Phylogenetic tree analysis

showed close relationship of

picrorhiza with Rehmannia apx,

Mesembryanthemum gr and

Lycopersicon mdhar using tree

explorer program

Mol Biol Rep (2013) 40:6593–6603 6599

123

UTR and 230 bp 30 UTR. The full length pkgr cDNA was

found to be 1,664 bp long with an ORF of 1,482 bp. The

ORF was flanked by 99 bp 50 UTR and 197 bp 30 UTR.

Sequence data from this study has been deposited in the

GenBank database under the accession nos. EU870515,

EU870516 and EU870517 for pkapx, pkgr and pkmdhar,

respectively.

The deduced PKAPX protein contained a total of 250

amino acids with molecular weight of 27.384 kDa and

isoelectric point (pI) of 5.53. While there were 486 amino

acids in the deduced protein sequence of PKMDHAR

having total molecular weight of 52.733 kDa and pI 6.87.

GR was composed of 493 amino acids with a molecular

weight of 53.4 kDa and a pI of 6.2. The PKAPX, PKM-

DHAR and PKGR proteins showed high degree of simi-

larity with sequences reported from other plant species in

the database using ClustalW (http://www.ebi.ac.uk/

clustalw). The alignment of the deduced amino acid

sequence of PKAPX with other plant APXS revealed more

than 90 % identity with APX of Rehmannia glutinosa

(AAS19934) and Acanthus ebracteatus (ABK32072),

respectively While, PKMDHAR showed 80–82 % identity

with MDHAR of Lycopersicon esculentum (ABG57052)

and Arabidopsis thaliana (BAA12349), respectively.

PKGR showed the highest 83 % similarity with Mesem-

bryanthemum crystallinum GR (CAC13956).

Phylogenetic analysis showed close relationship of

PKAPX with that from Rehmannia APX, PKMDHAR with

that of Lycopersicon MDHAR, and PKGR with that of

Mesembryanthemum GR (Fig. 4). In silico analysis showed

the presence of corresponding conserved domains in all the

three deduced proteins (Electronic Supplementary Fig. S1).

In PKAPX peroxidase domain was detected between amino

acid (aa) positions 26 to 244. In PKMDHAR, pyridine

nucleotide-disulphide oxidoreductase domain was detected

between aa positions 159 and 246. Pyridine nucleotide-

disulphide oxidoreductase (between aa positions 142 and

234) and pyridine nucleotide-disulphide oxidoreductase,

dimerisation domain (between aa positions 310 and 420)

were present in PKGR.

In PKAPX, SOPMA analysis revealed 39.60 % a-heli-

ces, 5.60 % b-turns, 10.00 % extended strands and

44.80 % random coils. In case of PKMDHAR, a-helices,

b-turns, extended strands, and random coils were 30.25,

9.05, 20.99, 39.71 %, respectively. In PKGR a-helices,

b-turns, extended strands, and random coils were 32.86,

7.51, 23.33, 36.31 %, respectively (Electronic Supple-

mentary Table S1), Deduced secondary structures were in

agreement with those reported for functional genes in other

plant systems. Various in silico analyses suggested the

cloned genes to be functional as has been reported by the

other studies.

Spatial expression analysis of pkapx, pkmdhar and pkgr

Semi-quantitative RT-PCR based expression analysis was

performed to study the expression level of the cloned

genes. Transcripts of all the three genes i.e. pkapx, pkm-

dhar and pkgr were observed in all the three tissues of

picrorhiza though their levels were found to be varying

(Fig. 5). All the three genes pkapx, pkmdhar and pkgr were

found to express highest in roots as compared to rhizomes

and leaves. Lowest amount of pkapx, pkmdhar and pkgr

transcripts were observed in leaves, rhizomes, and leaves,

respectively (Fig. 5).

Diurnal expression analysis of pkapx, pkmdhar

and pkgr

Temporal expression analysis of the cloned genes (pkapx,

pkmdhar and pkgr) revealed differential transcript levels.

In case of pkapx, expression was higher during dark stages

(24, 30 and 48 h) as compared to that during the light

period (12 and 36 h) (Fig. 6). Transcript of pkgr was higher

during light period (12 and 36 h) as compared to that of

rhiz

ome

leaf

root

26S rRNA

pkmdhar

pkgr

pkapx

rRNA

0

50

100

150

200

250

pkapx pkmdhar pkgr 26S rRNA

IDV

(X

103

)

gene

leaf

root

rhizome

a

b

Fig. 5 Expression analysis of pkapx, pkmdhar and pkgr in different

tissues of picrorhiza. Total RNA was isolated from leaf, root and

rhizome and subjected to semi-quantitative RT-PCR analysis (a). A

constitutive 26S rRNA gene was used as an internal control to

normalize differences in template concentrations. b integrated density

value (IDV) of each amplicon as obtained in a

6600 Mol Biol Rep (2013) 40:6593–6603

123

dark period (24 and 48 h). Transcripts of pkmdhar exhib-

ited zigzag pattern of expression (Fig. 6).

Discussion

The antioxidant potentials varied in different parts (leaves,

root and rhizome) of picrorhiza plants. Of the three tissues,

ascorbic acid was found to be highest in leaves and lowest

in roots. These findings were supported by the observation

of Vijaykumar et al. [20] who reported higher level of

ascorbic acid content in mature leaves as compared to

tender leaves. However, just opposite to it, glutathione was

highest in roots and lowest in leaves. Our results concurred

with the finding of Jaleel [21]. The percentage of the

reduced ascorbic acid varied from 80.9–90.1, 70.4–93.3

and 74.0–90.8 %, in leaf, rhizome and root, respectively.

The amount of reduced glutathione varied from 85.2–89.4,

85.0–89.4, and 87.1–90.6 %, in leaf, rhizome and root,

respectively. These results concurred with the findings of

Mahan and Wanjura [22], who reported the amount of

ascorbic acid and glutathione in leaves of cotton grown at

three water levels. They also summarized that in general,

the majority of glutathione in an unstressed plant cell was

found in the reduced form (GSH) typically comprising 70

to 90 % of the total [23]. The sudden decrease in ascorbic

acid content at senescence stage was also reported by Chen

et al. [24] in case of tobacco in which highest amount of

ascorbic acid was found at expanding leaf stage and least at

senescence stage.

For the first time, we report in this paper the cloning and

characterization of complete cDNAs encoding apx mdhar

and gr from picrorhiza. Previously, apx sequences have been

reported from Gossypium hirsutum [25], Triticum aestivum

[26], Manihot esculenta [27], Spinacia oleracea [28] and L.

esculentum [29]. mdhar have been isolated from Pisum

sativum [30, 31], S. oleracea [32], Brassica rapa subsp.

Pekinensis [33] and L. esculentum [34]. The specific domains

like plant peroxidase domain and pyridine nucleotide-

disulphide oxidoreductase domain and pyridine nucleotide-

disulphide oxidoreductase dimerisation domain, pyridine

nucleotide-disulphide oxidoreductase domain were found in

the amino acids sequences of pkapx, pkgr and pkmdhar,

respectively. These domains are responsible for the specific

enzymatic activities of the pkapx, pkmdhar and pkgr.

The tissue-specific expression of pkapx, pkmdhar and

pkgr genes was investigated by RT-PCR analysis with

respect to 26S rRNA. Transcripts of all the three genes i.e.

pkapx, pkmdhar and pkgr were observed in all the three

tissue of picrorhiza though quantitative analysis revealed

their differential levels. All the three genes were found to

express highest in roots as compared to rhizomes and

leaves. Lowest amount of pkapx, pkmdhar and pkgr tran-

scripts were observed in leaves, rhizomes, and leaves,

26S rRNA

pkmdhar

pkgr

pkapx

rRNA

4 51 2 3 6 7 8

0

50

100

150

200

250

IDV

(X

103

)

time (hours)

pkapx

0

50

100

150

IDV

(X

103

)

time (hours)

pkmdhar

0

50

100

150

200

250

300

350

IDV

(X

103

)

time (hours)

pkgr

0

50

100

150

200

6 12 18 24 30 36 42 48

6 12 18 24 30 36 42 48

6 12 18 24 30 36 42 48

6 12 18 24 30 36 42 48

IDV

(X

103

)

time (hours)

26SrRNA

a

b

Fig. 6 Diurnal expression

pattern of pkapx, pkmdhar and

pkgr in picrorhiza. Total RNA

was isolated from leaves at

different time points (6, 12, 18,

24, 30, 36, 42, 48 h) for

continuous 2 days and subjected

to semi-quantitative RT-PCR

analysis. a gene expression

wherein 26S rRNA was used as

an internal control for equal

loading. The lanes 1, 2, 3, 4, 5,

6, 7, 8 represent the samples at

6, 12, 18, 24, 30, 36, 42, 48 h

time points, respectively.

b integrated density value (IDV)

of each amplicon as obtained

in a

Mol Biol Rep (2013) 40:6593–6603 6601

123

respectively. The expression pattern of pkapx was in con-

currence with the result obtained by Jaleel [21]. The dif-

ferential expression of pkapx is in agreement to the apx of

Pimpinella brachycarpa [35]. pbapx transcript was found

to express in all tissues, with the higher levels in

embryogenic calli and roots but lower levels in leaves and

petioles. Likewise tissue specific expression of pkmdhar

was also studied. Expression of pkmdhar was found higher

in roots followed by leaves and rhizomes. The similar

differential expression of mdhar gene has also been

reported in P. sativum [31]. Expression of pkgr was found

higher in roots as compared to rhizome and leaves. The

antioxidant system genes of picrorhiza differed in their

expression when analysed for diurnal expression pattern.

The present study revealed higher expression of pkapx

during dark stages (24, 30 and 48 h) i.e. during low photon

flux density as compared to that during light period indi-

cating its negative correlation with the light intensity.

Interestingly, for pkgr the expression pattern was found to

be just opposite to that of pkapx which showed higher

expression during light period as compared to the dark

period suggesting its positive correlation with light inten-

sity. Expression pattern of pkmdhar revealed its light

independent expression behavior as evident from zigzag

pattern of its expression. The expression pattern for pkapx

was also supported by the work of Peltzer and Polle [36]

who analyzed the diurnal fluctuations of antioxidative

systems in leaves of field-grown beech trees (Fagus sylv-

atica) in response to light and temperature.

From this study, it is concluded that nonenzymatic compo-

nents viz; ascorbic acid and glutathione in picrorhiza exhibit

differential spatio-temporal status whereby leaves contain

comparatively higher amount of ascorbic acid and roots contain

comparatively higher glutathione. Also, the genes encoding

enzymes of antioxidant system exhibited differential expres-

sion behaviour. The finding may be useful in devising strategies

for engineering picrorhiza for better adaptation.

Acknowledgments We greatly acknowledge Council of Scientific

and Industrial Research (CSIR), India, for providing infrastructural

support and funding the projects; OLP-0035, OLP-0036, BSC-0111

and BSC-0109. J. P. thanks University Grant Commission (UGC),

India, for award of Junior Research Fellowship. The manuscript

represents IHBT publication number 3454.

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