ORIGINAL PAPER

Photocontrol of differential gene expression and alterationsin foliar anthocyanin accumulation: a comparative study usingred and green forma Ocimum tenuiflorum

Pritesh Vyas • Inamul Haque • Manish Kumar •

Kunal Mukhopadhyay

Received: 15 December 2013 / Revised: 31 March 2014 / Accepted: 9 May 2014

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2014

Abstract Anthocyanins impart red, purple and violet

colour to many flowers and fruits, mainly to attract poll-

inators and seed dispersers, but their function and biosyn-

thetic regulation in foliages of several plants are less

studied. The red and green forma of Ocimum tenuiflorum

differ in anthocyanin accumulation in leaves and provide

an excellent system for exploring the course of its regula-

tion. It was observed that red forma gradually changed to

green upon transfer to a particular greenhouse with limited

transmission of ultraviolet light (both UV-B and UV-A).

The sequential monitoring of anthocyanin content con-

firmed positive correlation between visible and ultraviolet

light intensity with leaf colour and antioxidant activities.

An ultra-performance liquid chromatography method of

\3.5 min was developed for rapid and precise

quantification of anthocyanidins. Expressions of PAL, CHS

and CHI were down-regulated by low light in both forma.

The F3H and F30H genes had reduced expression in both

forma and were supported by reduced levels of cyanidin in

red forma plants within greenhouse. The expression of late

biosynthetic genes, DFR and LDOX, also plummeted

within the greenhouse. The regulatory transcription factors

bHLH and WD40 were severely down-regulated within the

greenhouse suggesting that bHLH and WD40 control the

expression of F30H, DFR and LDOX to regulate the bio-

synthesis of anthocyanin pigments in leaves of O. tenui-

florum, whereas the expression of Myb remained almost

unaffected.

Keywords Anthocyanin biosynthesis � Ocimum

tenuiflorum (Indian Holy Basil) � Forma-specific gene

regulation � Quantitative real-time PCR � Ultra-

performance liquid chromatography � Ultraviolet light

Abbreviations

bHLH Basic helix loop helix

CHI Chalcone isomerase

CHS Chalcone synthase

CIE Commission Internationale d’Eclairage

DFR Dihydro flavonol reductase

F3H Flavonone 3 hydroxylase

F30H Flavonone 30 hydroxylase

FRAP Ferric reducing antioxidant power assay

LATAMOS Land surface atmosphere and

micrometeorological observational system

LDOX Leucoanthocyanidin dioxygenase

MBW Myb-bHLH-WD40

MSA Multiple sequence alignment

PAL Phenylalanine ammonia lyase

Communicated by J. Kovacik.

P. Vyas and I. Haque have contributed equally to this work.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11738-014-1586-9) contains supplementarymaterial, which is available to authorized users.

P. Vyas � I. Haque � M. Kumar � K. Mukhopadhyay (&)

Department of Biotechnology, Birla Institute of Technology,

Mesra, Ranchi 835215, India

e-mail: kmukhopadhyay@bitmesra.ac.in

Present Address:

P. Vyas

Department of Biotechnology and Allied Sciences, Jayoti

Vidyapeeth Women’s University, Jharna, Jaipur 303007, India

Present Address:

I. Haque

Department of Botany, Derozio Memorial College, Rajarhat

Road, P.O.: Rajarhat-Gopalpur, Kolkata 700136, India

123

Acta Physiol Plant

DOI 10.1007/s11738-014-1586-9

qPCR Quantitative real-time polymerase chain

reaction

Q-Tof Quadrupole-time-of-flight

UPLC Ultra-performance liquid chromatography

Introduction

Anthocyanins belong to the diverse group of secondary

metabolites known as flavonoids, a type of polyphenols

that impart red, purple, violet and blue tones to fruits,

flowers and leaves, many of which are commonly con-

sumed. Attracting pollinators and seed dispersal being the

major function of anthocyanins in flowers and fruits,

respectively (Grotewold 2006), but their functional roles in

foliar pigmentation are not clearly understood and have

been a focus of significant research (Gould 2004). Antho-

cyanins being the most versatile of all plant pigments, their

multifarious roles in foliages against free radical scaveng-

ing, amelioration of stress responses, protection of phot-

olabile defense compounds and the photosynthetic

apparatus have been proposed (Gould 2004). Anthocyanin

pigments also interact with other phytochemicals to protect

against a myriad of human diseases and have positive

effects on human health (Vyas et al. 2009).

Anthocyanin biosynthesis has been well characterized in

several plant species and requires two classes of genes,

structural and regulatory (Holton and Cornish 1995). The

structural genes code for the enzymes responsible for the

formation and storage of anthocyanins, and their expres-

sion thus makes the plant colourful and attractive (Kayesh

et al. 2013). They are highly conserved among various

plant species. The regulatory genes control the expression

of the structural genes, primarily at the level of transcrip-

tion either by activation or by repression (Hichri et al.

2011). Two classes of transcription factors, Myb com-

prising of a conserved R2R3-type DNA-binding domain

and bHLH (basic Helix Loop Helix) domain, regulate the

structural genes. Both these transcription factors can

interact among themselves and also with WD40 protein-

binding domain to constitute the MBW ternary transcrip-

tion complex that binds to the promoters of the structural

genes to regulate their expression (Ramsay and Glower

2005). Myb transcription factors were found in these

studies to be the primary determinants for colour devel-

opment, but required bHLH transcription factors and/or

WD40 proteins as co-regulators besides UV irradiation.

Comprehensive study on regulation of anthocyanin bio-

synthesis was carried out in leaves of Perilla frutescence, a

medicinal plant of the family Lamiaceae that also occur in

two forma, i.e. red and green (Gong et al. 1997, 1999a, b;

Saito and Yamazaki 2002; Yamazaki et al. 2003a, b). But

in Indian Holy Basil, this kind of study is lacking.

Indian Holy Basil, Ocimum tenuiflorum, a diploid plant

of the family Lamiaceae, commonly known as ‘Tulsi’, is

considered as a premier divine herb in the Indian traditional

Ayurvedic system of medicine for thousands of years.

Extracts of O. tenuiflorum are widely used for various

health promoting purposes in tonics as antioxidants, im-

munostimulators, neuromodulators, to sharpen memory

and for treatments of common cold and cough (WHO

monograph: Folium Ocimi sancti). The plant occurs in two

strikingly different forma that differ in anthocyanin accu-

mulation. ‘Krishna Tulsi’ exhibits purple to dark red col-

ouration on leaves and stems at all stages of development,

while the leaves and stems of ‘Sri/Lakshmi Tulsi’ are green

and accumulate only trace amounts of anthocyanins. In

spite of the economical and medicinal importance of O.

tenuiflorum, not much is known about the regulation and

biosynthesis of anthocyanins at the molecular level in this

plant.

During an earlier study on the phenylpropanoid (meth-

yleugenol) biosynthesis in O. tenuiflorum, it was observed

that the red forma plants gradually lost the purple-red

colour of the leaves and transformed to green upon transfer

to a particular greenhouse (Renu et al. 2014). This

prompted us to take up the present investigation to study

the relationship between the expression of the different

regulatory and structural genes of anthocyanin biosynthetic

pathway and its accumulation under different conditions of

sunlight exposure.

Materials and methods

Plant material, growth conditions, agro-meteorological

data and sample collection

Seeds of red and green forma O. tenuiflorum Linn. f. (syn.

O. sanctum L.) were obtained from the Indian Botanic

Garden, Howrah, India, soaked overnight in tap water and

sown on a nursery bed at the Indigenous Medicinal Plant

Garden of BIT-Mesra (23�240N, 85�260E, 619 m asl) dur-

ing October 2012.

After four weeks, the seedlings were replanted on pots

(30 cm Ø) containing commercial potting mix and were

allowed to grow for another six weeks under natural

environment. The plants were watered every alternate day,

and axillary buds were removed occasionally to prevent

development of bushy architecture. Five pots containing

red leaf plants and another five containing green leaf plants

were transferred to a greenhouse constructed of Lexan

Thermoclear (LT2UV) multiwall polycarbonate sheets

(Sabic Innovative Plastics). Both sides of the sheets have

Acta Physiol Plant

123

UV protective surface that prevent transmission of UV-B

(280–315 nm) and UV-A (315–380 nm) but transmit visi-

ble and near infrared (400–1,200 nm) radiation. Similar

numbers of pots containing red and green leaf plants were

maintained in the field under natural environmental con-

ditions as control.

The agro-meteorological data for the dates of leaf

sampling for RNA and anthocyanin extraction for quali-

tative studies spanning 14 December 2012 to 25 January

2013 were acquired from the fast and slow response da-

talogger of the land surface atmosphere and micrometeo-

rological observational system (LATAMOS) of the

Department of Applied Mathematics, BIT-Mesra.

Leaf samples for anthocyanin content and RNA

extraction were collected every fourth day at 13:00 h from

all green and red forma plants maintained in the field and

greenhouse. Leaves were immediately frozen in liquid

nitrogen, pulverized to a fine powder and stored at -70 �C

until further use.

Light microscopy and leaf colour measurements

Digital images of hand prepared transverse sections of

leaves from red and green plants maintained in natural

environment and in the greenhouse were visualized on a

Leica DM1L inverted microscope (Leica Microsystems

CMS GmbH). As anthocyanins accumulate in the sub-

vacuolar compartments of vacuoles, so it was stained with

neutral red to visualize anthocyanin-containing cells

(Poustka et al. 2007). For this purpose, peeled epidermis

were soaked in 0.6 mg mL-1 of neutral red solution

(Sigma Aldrich Co. Ltd.,) for 20 min at room temperature,

rinsed with MilliQ water to remove unbound stains and

visualized under the same microscope. The peeled

unstained epidermis were also checked for autofluores-

cence properties of anthocyanin in a Leica DM LB2 fluo-

rescent microscope (Leica Microsystem CMS GmbH),

using an excitation filter of 596 nm and emission filter of

620 nm (Poustka et al. 2007). All images were captured

with a Leica DFC300 FX CCD camera and processed using

the Leica FW4000 software.

To evaluate the visual colour changes in leaves, the tri-

stimulus colorimetry CIEL*a*b* scale of the Commission

Internationale d’Eclairage, Vienna, was followed. L* mea-

sures lightness from black (L* = 0) to white (L* = 100); a*

is positive on red and negative on green, whereas b* is

positive on yellow and negative on blue. Colours of fresh

leaves of each plant included in the experiment were mea-

sured using a colorimeter (Colorflex, HunterLab) under the

condition C (400–700 nm, 7,400 K), and data were obtained

at 2� viewing angle and D65 illumination. The instrument

operation and data acquisition were done using EasyMatch

QC software. Five measurements each for five leaves of each

plant were taken, and the averages were used for data com-

putation. Leaf colours were further analysed by calculating

chroma (quantitative attributes of colour intensity)

C* = (a*2 ? b*2)0.5 and hue angle (qualitative attributes of

colour) hab = tan-1 (b*/a*).

Extraction, quantification, identification and analysis

of anthocyanins

Anthocyanins were extracted from 1 g finely chopped leaves

with 5 mL acidified methanol (1 % HCl v/v) at 4 �C over-

night in dark. Samples were centrifuged at 11,000g for

5 min, and supernatants were used for spectrophotometric

determination of total anthocyanin content, using the mod-

ified pH differential method (An et al. 2012). The results

were expressed as mg of cyanidin (the main anthocyanin of

O. tenuiflorum) per g fresh weight, based on the extinction

coefficient of 26,900 and molecular weight of 449.2.

For liquid chromatography, 100 mg of frozen powdered

leaves was extracted with 1 mL solvent (acetonitrile/0.3 %

phosphoric acid, 80/20, v/v) on a rotary shaker at 4 �C for

16 h. The cleared lysates were passed through Sep-Pak plus

short tC18 cartridges and 0.20 lm membranes. Samples

(1 mL) were acid hydrolysed by the addition of 2 M HCl at

150 �C for 30 min in a sealed ampoule to get the anthocy-

anidins. Reference standards of all six anthocyanidins (chlo-

rides of cyanidin, delphinidin, malvidin, pelargonidin,

peonidin and petunidin) (Extrasynthese SAS, Genay, France)

were prepared by dissolving 0.1 mg in 1 mL of methanol.

Analytes were separated using a Acquity ultra-perfor-

mance liquid chromatography (UPLC) system (Waters

Corporation) consisting of a systems manager, sample

manager, tunable UV (TUV) detector and an H/T column

heater containing an Acquity UPLC BEH C18 reverse

phase column (2.1 mm 9 50 mm; 1.7 lm particle size).

The binary mobile phase consisted of (A) 0.3 % phos-

phoric acid in water and (B) acetonitrile. A linear gradient

elution programme was applied as follows: initial: 90 % A,

10 % B; 0–4 min: 80 % A, 20 % B; 4–4.2 min: 90 % A,

10 % B with a flow rate of 0.5 mL min-1. The temperature

of the column and sample manager was set at 40 and 5 �C,

respectively, and injection volume was 2 lL for standards

as well as for samples. The TUV detector was set at

525 nm, and instrument operation, data acquisition and

processing were performed using EmPower2 chromato-

graphic data software. Peak identification and quantifica-

tion were performed by the comparison of retention time

and area with that of standards. To confirm the identified

major anthocyanidins in red and green forma of O. tenui-

florum, the TUV eluent was sent to an interfaced quadru-

pole-time-of-flight mass spectrometer (Q-Tof-micro) that

was operated in positive ion mode. Probe and source

conditions included capillary voltage 2.54 kV, 200 �C

Acta Physiol Plant

123

desolvation temperature, 50 L h-1 cone gas, 400 L h-1

desolvation gas and 110 �C block temperature. Anthocya-

nins were identified based on comparison of the mass

fragmentation tandem MS analysis data, with previously

reported data (Yamazaki et al. 2003b).

Ferric reducing/antioxidant power assay

Total antioxidant activity of anthocyanins extracted as

mentioned earlier from leaves of red and green forma O.

tenuiflorum plants growing in the field as well as within the

greenhouse was assessed on the day the studies were ini-

tiated (14 December 2012) and concluded (25 January

2013) using FRAP method (Benzie and Strain 1999) with

minor modifications (Yuan et al. 2009). Standard curve was

prepared using different concentrations (100–1,000 mM)

of L-ascorbic acid (Duchefa biochemie, the Netherlands).

The difference in the increase in the absorbance of the

samples with respect to the standard was determined and

used to calculate the FRAP values that were expressed as

lmol Fe2? g-1 fresh weight of samples. All measurements

were performed in triplicates.

RNA extraction, cDNA synthesis and qPCR

Total RNA was isolated from 100-mg powdered leaf

samples using Nucleospin RNA Plant Kit (Macherey–Na-

gel GmbH) according to the manufacturer’s recommenda-

tions that included an on-column rDNase digestion step to

remove contaminating genomic DNAs. Equal amount of

RNA (1 lg) was used to synthesize cDNA using the

Blueprint First-Strand cDNA Synthesis Kit (Takara Bio

Inc.) following the manufacturer’s instruction.

Since sequences of anthocyanin biosynthetic and regu-

latory genes as well as the reference gene Actin were not

available for O. tenuiflorum, sequences of those genes,

mostly belonging to the related Lamiaceae plant Perilla

frutescens, were downloaded from NCBI (www.ncbi.nlm.

nih.gov). These sequences were used to design primers

(Primer Express Version 2, Applied Biosystems) to

amplify *400 bp from O. tenuiflorum cDNA (Supple-

mentary Table 1). The amplicons were sequenced com-

mercially. These *400 bp O. tenuiflorum sequences were

imported in the Universal Probe Library (UPL) assay

design centre (www.universalprobelibrary.com) and short

hydrolysis probes as well as forward and reverse primers

for quantitative real-time PCR were designed (Supple-

mentary Fig. 1; Supplementary Table 2) following Singh

et al. (2012). The qRT-PCR experiments were performed

on a 7500 Real-Time PCR system (Applied Biosystems),

and reaction was carried out in a 20-lL reaction volume

comprising of 1 9 FastStart TaqMan Probe Master [Rox]

(Roche Diagnostics GmbH) and 2 lL cDNA. The probe

and primer concentration for most efficient amplification

were optimized for all the selected genes as well as for the

reference gene Actin (Supplementary Table 1). The

96-well optical reaction plates containing reaction mixture

were incubated at 50 �C for 2 min, 95 �C for 10 min fol-

lowed by 45 cycles of 95 �C for 15 s and 60 �C for 1 min.

All qRT-PCR experiments were run with three technical

replicates. Instrument operation, data acquisition and pro-

cessing were performed using Sequence Detection System

version 1.2.2 software (Applied Biosystems). Fluorescence

signals were collected at each polymerization step, and a

threshold constant (CT) value was calculated from the

amplification curve by selecting the optimal DRn in the

exponential region of the amplification plot. Gene expres-

sion levels were computed relative to the expression of the

reference gene Actin using the 2-DDCT method (Rieu and

Powers 2009). A heat map was prepared with R package

(v3.0.3) to display the expression profiles of all the genes

on different days during progression of the experiment (R

Core Team 2014). After completion of the real-time PCR

reactions, the amplified products were cloned, sequenced

and analysed using BLAST.

Since many of the anthocyanin regulatory and biosyn-

thetic genes as well as PAL gene exist in several isoforms

in plant cells and perform different functions, the *400 bp

O. tenuiflorum partial cDNA sequences of all eleven genes

included in the study were subjected to multiple sequence

alignment (MSA) and phylogenic analysis using ClustalX

2.1 (Larkin et al. 2007) and PHYLIP version 3.68 (Fel-

senstein, 2002) to confirm these sequences with known and

characterized same genes from other plants.

Statistical analysis

Pearson’s correlation coefficients were examined to con-

firm correlations between anthocyanin accumulation in O.

tenuiflorum red forma leaves and visible light intensities

within and outside greenhouse, UV light intensities, FRAP

values and different chromatic parameters using MS Excel

2007. The open source software GRETL 1.9.1 was used to

conduct Engle–Granger time series co-integration test on

the LATAMOS dataset to find significant relationships

among the different parameters considered in this study

with anthocyanin contents.

Results

Environmental factors modulating anthocyanin

biosynthesis and leaf phenotypes

The UV intensities that were present in the natural envi-

ronment were totally absent within the greenhouse. The

Acta Physiol Plant

123

details of environmental variables on the days of sampling

for RNA and anthocyanin extraction are shown in Table 1.

The Engle–Granger time series co-integration test revealed

statistically significant relationship (p = 0.050;

t ratio = -2.306) between anthocyanin content and visible

light intensity of red forma plants.

During the first few weeks of growth in the field under

natural environment, young seedlings of the red forma O.

tenuiflorum showed intense dark purple-coloured leaves

and stems (Fig. 1a). Upon transfer of the 10-week-old

plants to the greenhouse, the red forma plants started to

develop a greenish tinge from 8 days (Fig. 1b) onwards

and became completely green after 20 days (Fig. 1c). The

green forma plants in the field had a faint red colour only

on the mid-veins (Fig. 1d), while within the greenhouse,

they lost the reddish tinge and developed complete green

leaves within 12 days (Fig. 1e).

Localization of anthocyanin in leaves and measurement

of colour variation

Transverse sections of leaves of the red forma plants

maintained in the field revealed the presence of red-col-

oured cells only in the adaxial epidermis and trichomes of

the lamina and bundle sheath cells of the main vein

(Supplementary Fig. 2a). When such plants were trans-

ferred to the greenhouse, the epidermis and trichomes

became colourless (Supplementary Fig. 2c). The green

forma plants growing in the field had faint red-coloured

cells only in bundle sheath of the main vein (Supplemen-

tary Fig. 2b) that became colourless upon transfer to the

greenhouse (Supplementary Fig. 2d). The photosyntheti-

cally active mesophyll cells of both red and green forma

plants were devoid of anthocyanins. Neutral red-stained

cells and autofluorescence properties were observed in the

epidermal peels of only the red forma plants maintained in

field (Supplementary Fig. 3a, e), indicating that all major

anthocyanins of O. tenuiflorum red forma plants fluoresce

red. Epidermis of green forma plants from the field as well

as both red and green forma plants kept in the greenhouse,

neither stained with neutral red nor displayed autofluores-

cence (Supplementary Fig. 3b, c, d, f).

Colorimetric values of intact leaves measured in the

present study showed the chromatic parameter a* values

clearly separated the red forma plants growing in field from

those that turned green within the greenhouse along with

the green forma plants growing either in the field or

transferred to the greenhouse (Table 2). The CIE Labora-

tory parameters (L*, a*, b*, C* and hab) and anthocyanin

contents determined by pH differential method of similar

plants are also provided in Table 2. The anthocyanin

content of the red forma plants decreased strikingly upon

transfer to the greenhouse. A statistically significant Ta

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Acta Physiol Plant

123

positive Pearson’s correlation coefficient of r = 0.991633

(p \ 0.01) between the chromatic parameter a* and

anthocyanin content indicates that anthocyanins are solely

responsible for the red colouration in O. tenuiflorum red

forma plants. The 2D spectral plot showing Lab scale

suggests the change of colour of red forma leaf from red to

green within the greenhouse (Supplementary fig. 4).

Targeted profiling of anthocyanins using UPLC tandem

mass spectrometry

UPLC, a fast and effective method to separate and analyse

plant metabolites, was performed to identify the different

anthocyanins present in the leaves of O. tenuiflorum.

Optimization of the UPLC conditions resulted in better

separation and simultaneous peak detection of all the six

anthocyanin standards (Fig. 2a). The method identified

cyanidin and peonidin simultaneously from both red

(Fig. 2b) and green (Fig. 2c) forma field plants within a

total run time of 4.5 min, including 1 min for system

equilibration. The target compound cyanidin (RT:

1.652 min) had m/z 449 and MS/MS fragment m/z 285.7545

values (left inset in Fig. 2b), whereas peonidin (RT:

2.648 min) had m/z 463.1 and MS/MS fragment

m/z 299.7162 values (right inset in Fig. 2b). Cyanidin and

peonidin were found to be 47.15- and 16.8-fold less in green

forma as compared to the red forma plants. The optimized

method was used to precisely quantify cyanidin, the major

anthocyanin in red forma plants on different days after

transfer to the greenhouse (Table 3). The data show the

gradual decrease of the target compound in the red forma

plants, which became almost equal to the green forma plants

after 35 days within greenhouse.

Antioxidant activity of anthocyanins

Anthocyanins isolated from plants, red or green, growing in

the field showed higher antioxidant activity than plants

transferred to the greenhouse (Fig. 3). Concomitant with

high anthocyanin levels of red forma O. tenuiflorum

growing in the field, the same plants had the highest anti-

oxidant activity (2.68 ± 0.05 lmol g-1 fresh weight) than

those of green forma plants (1.26 ± 0.05 lmol g-1 fresh

weight). The reduction in antioxidant activity upon transfer

to the greenhouse was found to be much higher in red

forma plants (3.57-fold) as compared to green forma plants

(1.15-fold). A statistically significant positive Pearson’s

correlation coefficient (r = 0.96737, p \ 0.01) between

FRAP values and anthocyanin content from similar sam-

ples indicates that anthocyanins have the major contribu-

tion towards the antioxidant property of O. tenuiflorum

leaves.

Fig. 1 Phenotypes of red and green forma O. tenuiflorum at different

days and conditions of sunlight exposure. a Red forma plants growing

in natural environment; b after transfer to the greenhouse on 8 days

and c on 20 days; d green forma plants growing in natural

environment and e on 12 days after transfer to the greenhouse.

Chlorotic leaves can be seen on plants in b, c, e

Table 2 Commission Internationale d’Eclairage L*a*b* parameters and anthocyanin contents determined by pH differential method

Sample Colour value Chroma (C*) Hue angle (hab) Anthocyanin content

(mg. 100 g-1 FW)L* a* b*

Green field 39.69 (0.021) -8.22 (0.15) 5.43 (0.24) 9.85 86.22 0.251

Green Gh 35.46 (0.047) -8.37 (0.12) 5.37 (0.21) 9.94 89.52 0.242

Red field 22.73 (0.064) 5.58 (0.13) 13.78 (0.18) 14.8 23.8 15.14

Red Gh 40.29 (0.078) -6.24 (0.32) 5.43 (0.43) 8.2 65.85 0.239

Values within parenthesis represents SD, n = 5 one leaf of each plant from each set of experiment; five measurement for each sample

Gh greenhouse

Acta Physiol Plant

123

Changes in gene expression pattern

Since differences were observed in the anthocyanin

profiles between the two forma, differential forma-spe-

cific gene expression of the anthocyanin biosynthetic

structural and regulatory genes was expected. The tran-

script levels were compared using short hydrolysis

probe-based qPCR. The relative quantity of each gene is

expressed as fold change relative to field grown red

forma plants on the day of transfer to the greenhouse

that was used as calibrator and set to the nominal value

of 1. A comprehensive expression profiling of all the 11

genes on different days of the experiment is represented

in a heat map (Fig. 4).

Fig. 2 UPL chromatograms of

anthocyanin standards and

samples at 525 nm and MS/MS

fragmentation patterns.

a Separation of mixture

containing six anthocyanin

standards. b Anthocyanins from

red forma field plants; cyanidin

with retention time 1.65 min

and peonidin with retention time

2.65 min. Inset on left showing

daughter ion spectra of cyanidin

(287.7545) and inset on right

showing daughter ion spectra of

peonidin (299.7162).

c Anthocyanins from green

forma field plants. Y-axis

represents absorption intensity

(AU), and X-axis represents

retention time (min) in the

chromatograms, whereas X-axis

in the insets represents m/

z values

Acta Physiol Plant

123

The expression of PAL, the anthocyanin biosynthetic

pathway upstream gene, was almost twofold higher in the

green forma as compared to the red forma field plants. Its

expression got reduced, in both forma, upon transfer to the

greenhouse. The early biosynthetic genes, CHS, CHI, F3H

and F30H, had elevated levels of expression in the red forma

than the green forma field plants. The expression levels of

CHS and CHI in both the forma, particularly in the red forma,

got reduced upon transfer to the greenhouse. The F3H and

F30H genes, which play key role in determining the antho-

cyanin patterns, also expressed differentially between the

two forma. Their transcript levels were twofold higher in the

red forma field plants relative to green forma that reduced

markedly upon transfer to the greenhouse and were almost

unperceivable after 16 days. The expression of the late

biosynthetic genes, DFR and LDOX, was 10- and 2-fold

higher, respectively, in field grown red plants compared to

the green. The expression levels of both the genes in both

forma severely diminished upon transfer to the greenhouse.

The DFR transcripts were undetectable after 12 days of

transfer in the red forma, whereas LDOX continued to

express at a minimum level.

The expression of all the three genes in the MBW complex

was 1.7-fold to 4-fold higher in field grown red forma plants

compared to the same green forma plants. The expression of

all these genes was down-regulated in both forma, specifi-

cally in the red forma. The relative transcript abundance of

the three regulatory genes presented quite unrelated

expression profiles upon withdrawal of direct sunlight. Upon

transfer to the greenhouse, bHLH expression levels gradually

reduced in both forma, with severe reduction in the red forma

plants. The Myb gene expressed at a higher level in red forma

field plants compared to the green ones. Within the green-

house, expression of Myb gene was more repressed in red

forma plants than in green forma plants. The response of WD

repeat protein genes to withdrawal of high-light intensities

and UV was more astounding. Their expression levels

decreased eightfold in red forma plants by 4 days within the

greenhouse. The transcripts absolutely disappeared from

8 days onwards indicating low-light intensities within the

greenhouse did not favour expression of WD40 repeat genes

in O. tenuiflorum.

Fig. 3 Total antioxidant

activity as determined by FRAP

assay of anthocyanins isolated

from green and red forma O.

tenuiflorum leaves from plants

growing in the field and within

the greenhouse. Samples were

collected on the initial and

concluding days of the

experiment from all red and

green plants maintained either

in the field or within the

greenhouse (five each). The

FRAP experiments were

performed in triplicates with

each sample, and standard

deviation is presented as error

bars

Table 3 Data of UPLC profiles of the samples

Days Green field Green Gh Red field Red Gh

0 0.252 (0.17) 0.253 (0.32) 15.117 (0.13) 15.114 (0.19)

4 0.249 (0.12) 0.251 (0.31) 15.085 (0.23) 11.781 (0.20)

8 0.248 (0.19) 0.249 (0.07) 15.087 (0.21) 8.902 (0.14)

12 0.244 (0.12) 0.245 (0.10) 15.032 (0.19) 4.312 (0.21)

16 0.251 (0.18) 0.244 (0.12) 15.131 (0.12) 1.322 (0.22)

20 0.248 (0.13) 0.244 (0.06) 15.115 (0.24) 0.761 (0.19)

24 0.251 (0.19) 0.242 (0.11) 15.101 (0.22) 0.363 (0.13)

35 0.252 (0.17) 0.241 (0.15) 15.091 (0.21) 0.256 (0.16)

Anthocyanins were extracted on every fourth day from 100-mg

powdered leaf samples of red and green forma plants growing in field

as well as within the greenhouse. Data are averages of three UPLC

injections from three different plants under similar experimental

conditions and represent lg of cyanidin 100 mg-1 frozen leaf sam-

ples. Peonidin was not considered as its values were in very minute

quantities

Values in parenthesis represent SD, n = 3

Gh greenhouse

Acta Physiol Plant

123

Multiple alignments and phylogenetic relationships of the

*400 bp O. tenuiflorum sequences to PAL and anthocyanin

biosynthetic structural and regulatory genes from other

plants with known roles in anthocyanin biosynthesis

revealed high homology and thereby indicating proper

selection of the genes [PAL (Supplementary Fig. 5a, b), CHS

(Supplementary Fig. 6a, b), CHI (Supplementary Fig. 7a, b),

F3H (Supplementary Fig. 8a, b), F30H (Supplementary

Fig. 9a, b), DFR (Supplementary Fig. 10a, b), LDOX (Sup-

plementary Fig. 11a, b), bHLH (Supplementary Fig. 12a, b),

Myb (Supplementary Fig. 13a, b), WD40 [(Supplementary

Fig. 14a, b), Actin (Supplementary Fig. 15a, b)]. This was

further supported by BLAST results of the sequences

amplified through real-time PCR.

Discussion

Understanding the regulation of anthocyanin biosynthetic

pathway is important to generate and select plants enriched

in anthocyanins with desirable dietary and medicinal prop-

erties (Hichri et al. 2011). Most studies on deciphering the

underlying mechanism regulating anthocyanin production

and accumulation had been focused on fruits (Allan et al.

2008; Niu et al. 2010; An et al. 2012; Kayesh et al. 2013;

Zhang et al. 2013), flowers (Laitinen et al. 2008), heads of red

cabbage (Yuan et al. 2009), cauliflower (Chiu et al. 2010)

and leaves of Arabidopsis, Petunia and lettuce (Rowan et al.

2009; Albert et al. 2009). Tissue-specific expression of the

different anthocyanin biosynthetic genes in Tartary Buck-

wheat (Fagopyrum tataricum) revealed differential expres-

sion and anthocyanin accumulation pattern (Park et al.

2011). Substantial research on the structure and expression

of the regulatory and structural genes of the red and green

chemotypes of the Lamiaceae plant P. frutescens, that is

widely used as food colourant and in traditional medicines of

Japan and other eastern Asian countries, provided important

insights on regulation of foliar biosynthesis of anthocyanin

(Gong et al. 1997, 1999a, b; Saito and Yamazaki 2002;

Sompornpailin et al. 2002; Yamazaki et al. 2003a, b).

Fig. 4 Heat map depicting relative expression profiles of structural and

regulatory anthocyanin biosynthetic genes in red and green forma O.

tenuiflorum plants growing in the field as well as upon transfer to the

greenhouse. Gene names are mentioned on the right: PAL phenylal-

anine ammonia lyase, CHS chalcone synthase, CHI chalcone isomer-

ase, F3H flavonone 3 hydroxylase, F30H flavonone 30 hydroxylase, DFR

dihydro flavonol reductase, LDOX leaucoanthocyanidin dioxygenase,

Myb Myb transcription factor, bHLH basic helix loop helix transcription

factor, WD40 WD40 repeat protein. Days of data collection are

mentioned at the bottom. The relative expression is expressed as fold

change relative to red forma field plants on 0 day. Data represent means

of three replicate reactions. Changes in expression levels are displayed

from red (down-regulated) to green (up-regulated) as shown in the

colour gradient at the top left corner (colour figure online)

Acta Physiol Plant

123

The field grown green plants had higher expression of

PAL, the upstream gene of the anthocyanin biosynthetic

pathway, which also shares common steps for the biosyn-

thesis of other phenylpropanoids and flavonoids. This might

reflect redirection of metabolites towards other branches of

the phenylpropanoid pathway in the green forma. The results

also demonstrate that expression levels of all the structural

genes decreased to various extents upon transfer to the

greenhouse in both red and green forma. Since decrease in

cyanidin-based anthocyanins was identified in the present

study, so the low expression levels of F30H gene within the

greenhouse supported the fact (Kobayashi et al. 2009). The

expression levels of the late biosynthetic genes DFR and

LDOX reduced sharply within the greenhouse in both forma

suggesting their pivotal roles in the formation of anthocya-

nins. This low level of expression of DFR and LDOX is likely

to be the bottleneck for the sharp reduction of anthocyanin

biosynthesis in red forma leaves of O. tenuiflorum plants

within the greenhouse. Similar to results obtained in this

study, expression of LDOX was weak, whereas expression of

DFR was less than level of detection under weak light in P.

frutescens (Gong et al. 1997). White light and UV-A-medi-

ated regulation of DFR and LDOX genes were also reported

in red grapes (Gollop et al. 2001, 2002). These results show

that all the structural genes examined in the present study are

expressed in a forma-specific manner and their expression

levels are repressed to various extents by removal of direct

sunlight.

The transcription factors bHLH, Myb and WD40 repeat

protein-coding gene were selected as probable candidates

responsible for determination of leaf colour in O. tenuiflo-

rum. The role of Myb and bHLH transcription factors in

stimulating anthocyanin biosynthetic structural genes varies

among plant species. In apple, higher expression of Myb was

responsible for red colouration (Allan et al. 2008), but in red

cabbage, lower expression levels of BoMYB3 were observed

in red seedlings and young leaf (Yuan et al. 2009). The

transcripts of WD40 protein-coding gene rapidly declined, as

was also observed with TTG1 and EGL3 in Arabidopsis

thaliana (Rowan et al. 2009). Recently, An et al. (2012)

showed that the WD40 proteins interact with only bHLH and

not with MYB to regulate anthocyanin accumulation in

apples. The gradual down-expression of bHLH in both the

forma of O. tenuiflorum under diminished light suggests the

absolute requirement of UV light for its induction. The

expression levels of Myb were also reduced, but were very

stable under low light conditions in both forma during the

course of the experiment. The reduced expression of bHLH

and WD40 transcription factors may explain the low

expression of the structural genes and the involvement of

specific regulatory factor(s) for forma-specific gene

expression (Fig. 5). A comparison of the differential

expression of anthocyanin biosynthetic structural and regu-

latory genes on different days after transfer to greenhouse

with respect to the red forma O. tenuiflorum field plants is

shown in Supplementary Fig. 16.

Quantitative anthocyanin estimation and qualitative

composition are important for determining health benefits

of traditional medicines and tonics, prepared from O. ten-

uiflorum leaf extracts. As anthocyanins in vivo absorb

green and yellow light in the waveband of 500–600 nm, the

amount of red light reflected from red pigmented leaves

does not always correlate with the anthocyanin content, as

the amounts of chlorophylls are stronger determinants of

red reflectance (Neill and Gould 1999). Hence, anthocya-

nins were extracted from leaves and used for precise

quantification in the present study using pH differential

method and liquid chromatography. Though HPLC had

been in use for a long time to identify and quantify

anthocyanins from a variety of plant sources (Allan et al.

2008), UPLC had also been used, but the time required for

good separations was 26 min (Hosseinian et al. 2008).

UPLC method developed in the present study can separate

all six major anthocyanins within a reasonable time of

3.5 min. The process could as well identify a 59.03-fold

reduction in cyanidin contents over a period of 35 days.

Fig. 5 Effect of bHLH and WD40 regulatory genes on up- and down-

regulation of specific structural genes in red forma O. tenuiflorum

within greenhouse. Downward arrows within parenthesis represent

the down-regulation of the gene

Acta Physiol Plant

123

The experiments were designed in mid-winter (14

December 2012–25 January 2013) to take advantage of

clear skies, low temperature fluctuations and absence of

rainfall for better assessment of the environmental effects

on foliar anthocyanin biosynthesis in O. tenuiflorum. The

Engle–Granger time series co-integration test significantly

correlated anthocyanin contents in leaves only with visible

light intensity among the different LATAMOS parameters,

thus reducing the possibilities of influence of other envi-

ronmental factors on accumulation of anthocyanin in O.

tenuiflorum leaves. The anthocyanin localization data

indicate the tissue- and forma-specific accumulation of

anthocyanins in O. tenuiflorum. In contrast to P. frutescens

where anthocyanins were found in both upper and lower

epidermis (Saito and Yamazaki 2002) in the present study,

anthocyanins were found localized only in the upper epi-

dermis. The hue angle for field grown red forma plants was

of 23.8� which fall well within the typical red colour for

anthocyanins (Hurtado et al. 2009).

Since synthetic antioxidants commonly used in the

pharmaceutical industry are associated with health risk

and toxicity, they need to be replaced (Dudonne et al.

2009) and efforts are ongoing to explore water-extractible

hydrophilic antioxidants from plant sources for better

formulation of nutraceuticals. The antioxidant activity of

anthocyanins from different plant sources is well known

(Yuan et al. 2009; Dudonne et al. 2009). FRAP assay was

employed in the present study to evaluate the total anti-

oxidant capacity of anthocyanins isolated from leaves of

red and green forma plants of O. tenuiflorum growing in

field and in the greenhouse. The decrease in FRAP values

in both red and green forma plants correlated with the

decrease in anthocyanin content. Earlier studies with

purple Asparagus officinalis (Sakaguchi et al. 2008) and

red Elatostema rugosum (Neill et al. 2002) leaves showed

enhanced antioxidant properties as compared to extracts

of green leaves of the same plant species.

In conclusion, we suggest that UV light modulate two

members of the MBW complex, bHLH and WD40, which

in turn regulate the late biosynthetic genes F30H, DFR and

LDOX, whose transcript levels decreased simultaneously

with reduction in anthocyanin contents implying their

critical connection in anthocyanin biosynthesis in O. ten-

uiflorum leaves.

Author contribution PV and IH performed the experi-

ments and prepared the initial draft manuscript, MK ana-

lysed data and KM conceived the idea, designed

experiments and finalized the manuscript.

Acknowledgments We gratefully acknowledge Dr. Manoj Kumar

of Department of Applied Mathematics, BIT-Mesra for providing

the LATAMOS data, Mr. Sanjay Swain, Mr. Ashwani Singh and

Mr. Dharmendra Singh of BIT-Mesra for excellent technical

assistance. The work was supported, in part, by University Grants

Commission of India [34-275\2008 SR], Ministry of Food Pro-

cessing Industries, India [47/MFPI/R&D/2006/517], and Infra-

structure Development Fund by Department of Agriculture,

Government of Jharkhand [5/B.K.V/Misc/12/2001]. Fellowships

were provided to PV by BIT-Mesra and IH by CSIR [9/554 (13)

2007-EMR-I].

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