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Plant Physiology and Biochemistry 47 (2009) 895–903

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Anthocyanin production in callus cultures of Cleome rosea: Modulation by cultureconditions and characterization of pigments by means of HPLC-DAD/ESIMS

Claudia Simoes a,*, Carlos Henrique Brasil Bizarri b, Lıvia da Silva Cordeiro a,Tatiana Carvalho de Castro a, Leonardo Cesar Machado Coutada b, Antonio Jorge Ribeiro da Silva c,Norma Albarello a, Elisabeth Mansur a

a Nucleo de Biotecnologia Vegetal, Universidade do Estado do Rio de Janeiro (UERJ), Rua Sao Francisco Xavier, 524 - PHLC, sala 509, Maracana.CEP: 20550-013, Rio de Janeiro, RJ, Brazilb LACEM, Fundaçao Oswaldo Cruz, Farmanguinhos, Brazilc NPPN, Universidade Federal do Rio de Janeiro, Brazil

a r t i c l e i n f o

Article history:Received 26 December 2008Accepted 10 June 2009Available online 18 June 2009

Keywords:Acylated anthocyaninsAuxinsCyanidinsColor valueNH4þ/NO3

� ratioSucrose

Abbreviations: PIC, 4-amino-3,5,6-trichloropicolini2,4-dichlorophenoxyacetic acid; DW, dry weight; FWacetic acid; NAA, naphthaleneacetic acid.

* Corresponding author. Tel.: þ 55 21 25877361; faE-mail address: labplan_uerj@yahoo.com.br (C. Sim

0981-9428/$ – see front matter � 2009 Elsevier Masdoi:10.1016/j.plaphy.2009.06.005

a b s t r a c t

Leaf and stem explants of Cleome rosea formed calluses when cultured on MS medium supplementedwith different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-tri-chloropicolinic acid (PIC). The highest biomass accumulation was obtained in the callus cultures initiatedfrom stem explants on medium supplemented with 0.90 mM 2,4-D. Reddish-pink regions were observedon callus surface after 6–7 months in culture and these pigments were identified as anthocyanins.Anthocyanins production was enhanced by reducing temperature and increasing light irradiation. Pig-mented calluses transferred to MS1/2 with a 1:4 ratio NH4

þ/NO3�, 70 g L�1 sucrose and supplementation

with 0.90 mM 2,4-D maintained a high biomass accumulation and showed an increase of 150% onanthocyanin production as compared with the initial culture conditions. Qualitative analysis of calluseswas performed by high performance liquid chromatography coupled to diode array detector and elec-trospray ionization mass spectrometry (HPLC-DAD/ESIMS). Eleven anthocyanins were characterized andthe majority of them were identified as acylated cyanidins, although two peonidins were also detected.The major peak was composed by two anthocyanins, whose proposed identity were cyanidin 3-(p-coumaroyl) diglucoside-5-glucoside and cyanidin 3-(feruloyl) diglucoside-5-glucoside.

� 2009 Elsevier Masson SAS. All rights reserved.

1. Introduction

Despite the increasing interest on secondary metabolites, someproblems remain in the extraction of these compounds from wildplants, taking into account that natural populations are submittedto seasonal variations and environmental factors. Furthermore, thechemical synthesis of several plant-derived compounds is not aneconomically viable alternative because of their complex structure[1]. Plant tissue culture technologies offer strategies to minimizethese problems, allowing the continuous production of secondarymetabolites of commercial importance, and providing enhancedcontrol over the chemical and physical environments [2]. Moreover,these systems are being explored as controlled models for

c acid; CV, color value; 2,4-D,, fresh weight; IAA, indole-

x: þ 55 21 25877663.oes).

son SAS. All rights reserved.

production of bioactive substances from plants, once they canpotentially be manipulated in order to alter metabolite quality andquantity [3].

Anthocyanins are water-soluble plant flavonoids providingscarlet to blue colors in flowers, fruits, leaves and storage organs. Inaddition to being present in the human diet at relatively highconcentrations, anthocyanins are used to color food as the substi-tute of synthetic red dyes, once natural food additives are increas-ingly preferred over synthetic ones, due to safety considerations [4].Recently, great attention has been focused on the anthocyaninsmultifaceted pharmacological potential [5,6], including specificbiological activities as antioxidant [7], anti-inflammatory [8] andantitumor [9]. Anthocyanins production by in vitro cell cultures hasbeen obtained from several plant species [10–13]. However,research on regulation of anthocyanins biosynthesis under in vitroconditions is still limited, restricting the commercial application oftheir production by plant cell cultures [14].

In vitro production of secondary metabolites by species fromgenus Cleome was only reported in Cleome chelidonii [15] and

Table 1Callus biomass accumulation from stem and leaf explants of Cleome rosea inoculatedon MS medium supplemented with 2,4-D or PIC, after 60 days in culture. Datarepresent mean � standard deviation. Same letters on each column are not signif-icantly different by Tukey test at 5%.

Growth regulator(mM)

Type of explant

Leaf Stem

Fresh weight Dry weight Fresh weight Dry weight

2,4-D (0.45) 8.03 � 1.83 bc 0.28 � 0.03 bc 10.19 � 1.04 ab 0.33 � 0.02 b2,4-D (0.90) 11.46 � 0.78 a 0.43 � 0.03 a 12.92 � 0.55 a 0.45 � 0.01 aPIC (0.41) 6.59 � 1.54 c 0.25 � 0.05 c 10.69 � 1.88 ab 0.25 � 0.09 cPIC (0.82) 9.56 � 0.98 bc 0.35 � 0.02 b 8.33 � 1.32 bc 0.34 � 0.02 b

C. Simoes et al. / Plant Physiology and Biochemistry 47 (2009) 895–903896

Cleome spinosa [16]. Cleome rosea is a Brazilian garden ornamentalspecies frequently found in sandy coastal plains, which areecosystems submitted to an intense anthropic pressure [17]. Thisspecies has been recently investigated through plant cell culturestrategies [18] and with relation to its medicinal potential [19].

In the present work, we established fast growing anthocyanin-producing callus lines of C. rosea and analyzed the influence ofchemical and physical factors on pigment production. In addition,pigments were characterized by means of HPLC-DAD/ESIMS.

2. Material and methods

2.1. Plant material and callus induction

In vitro plants of C. rosea [18] were used as source of stem (0.5 cm)and leaf (0.5 cm2) explants. Cultures were initiated on MS medium[20] with 30 g L�1 sucrose and supplemented with naphthalene-acetic acid (0.54; 1.07 mM), indoleacetic acid (0.57; 1.14 mM), 4-amino-3,5,6-trichloropicolinic acid (0.41; 0.82 mM) or 2,4-dichlor-ophenoxyacetic acid (0.45; 0.90 mM). Media were adjusted to pH 5.8prior to adding agar (8 g L�1, Merck), autoclaved (121 �C,104 KPa) for15 min and dispensed into 8� 7 cm flasks (30 mL of culture mediumper flask) closed with polypropylene caps. Five flasks containingfour explants each were cultured per treatment and each experi-ment was repeated twice. Cultures were incubated in a growthchamber at 26 � 2 �C under 16 h photoperiod provided by cool-white fluorescent tubes (45 mmol m�2 s�1). Subcultures to mediawith the same composition were performed after 30 days of culture.Callus biomass accumulation was estimated after 60 days of culturebased on fresh (FW) and dry (DW) weight measurements. Dry masswas obtained after drying at 45 �C to constant weight. Stock calluscultures were maintained under the same physical conditionsdescribed above with subcultures at 20-day intervals.

2.2. Anthocyanin identification, extraction and quantification

Initial identification of the pigments was performed bysubmitting samples of pigmented calluses to an atmosphere satu-rated with vapors of ammonium hydroxide (alkaline pH) orhydrochloric acid (acid pH). This methodology allows the identifi-cation of anthocyanic pigments through color changes inducedby pH variation. Furthermore, samples of pigmented and non-pigmented calluses were extracted for 24 h at 4 �C using acidifiedmethanol prepared with 1% (v/v) HCl (MeOH–HCl). The extractswere centrifuged (1000 � g for 10 min) and submitted to spec-trophotometric analysis (200–800 nm) using a UV–Vis spectro-photometer (Shimadzu UV – 160).

Anthocyanin quantification was based on the color value index(CV), which is an indicator of total anthocyanins [11,21,22], with smallmodifications. Briefly, samples of pigmented callus (100 mg) wereextracted as described above and the absorbance was measured at525 nm (average of maximum absorbance of callus extracts). The CVindex was calculated with the following equation: 10%Absorbance� dilution rate¼ 0.1� OD 525� 40/1 g FW, where OD525

is the absorbance measured at 525 nm and 40 is the level of dilution(100 mg of callus extracted in 4 mL of MeOH–HCl). The CV index wasselected to quantify the total anthocyanin content instead of theextinction coefficient of one particular type of anthocyanin, once theextracts from callus cultures are mixtures of various anthocyanins,which can vary under different culture conditions [14].

2.3. Modulation of anthocyanin production

Four grams of pigmented calluses were cultured at differenttemperatures (24� 2 �C and 32� 2 �C) and light intensities (67 and

80 mmol m�2 s�1). The cultures were maintained at these physicalconditions for 40 days with a subculture after day 20. Calluses werealso transferred to growth regulators-free MS (MS0) medium orto MS medium supplemented with 0.90 mM 2,4-D containing:a) different sucrose concentrations (30; 50; 70; 90 g L�1);b) different total nitrogen concentrations (50; 60; 70; 80 mM);c) different ratios of NH4

þ to NO3� (1:1; 1:2; 1:4; 1:6) or d) different

mineral salt concentrations, either by diluting MS medium (MS1/2;MS1/4) or by using White medium [23]. Callus biomass accumu-lation and anthocyanin content were determined in calluses grownunder the best physical conditions previously established foranthocyanin production (24� 2 �C under 80 mmol m�2 s�1). Fifteenflasks containing four grams of callus were cultured per treatmentand each experiment was repeated three times.

2.4. HPLC-DAD/ESIMS analysis of anthocyanins

Samples from pigmented calluses as well as from stems of field-grown plant were extracted using MeOH–HCl as described above.All extracts were prepared at the final ratio of 1 g of fresh weight/2 mL of solvent. Anthocyanin analysis was performed with a Shi-madzu chromatographic system (MassLynx software, LC-10Advpbinary gradient pump, SIL-10Advp autosampler, SPD-M10Avpdiode-array detector and SCL-10Avp system controller). Sampleswere analyzed under a gradient using a Supelco C18 column(250 � 4.6 mm i.d., 5 mm particle size) with injection volume of20 mL. The flow rate was 1 mL min�1 and detection was at 525 nm.Mobile phase A was 7.5% formic acid in acetonitrile (v/v) and mobilephase B was 7.5% formic acid in water (v/v). The gradient used wasas follows: 3% A for 1 min, 3–15% A for 11 min, 15–25% A for 12 min,25–30% A for 4 min, and 30% A for 7 min before returning to theinitial conditions. DAD UV–Vis absorption spectra were recordedon-line during HPLC analysis. The diode-array detector was set toan acquisition range of 200–600 nm at a spectral acquisition rate of1.56 scans s�1 (peak width 0.2 min). The diode-array detector wascoupled to a Waters ZQ single quadrupole mass spectrometer. Massspectra were achieved by electrospray ionization in positive modescanning from m/z 100 to m/z 1500. The capillary temperature andvoltage used were 100 �C and 3 kV, respectively. In order to obtaina better spectra profile, three voltages were applied to the cone(30 V, 50 V and 70 V). Nitrogen was used as sheath gas at a flow rateof 400 L h�1 at 250 �C.

2.5. Statistical treatment

Data are presented as mean � standard deviation and wereanalyzed using one-way analysis of variance (ANOVA). The differ-ences among means were tested by Tukey test at 5% level ofsignificance. The analyses were carried out with the statisticalsoftware MSTAT-C (version 2.1, Michigan State University, MI, USA).

Fig. 1. Callus cultures of Cleome rosea. A) Non-anthocyanic callus. Bar ¼ 1.0 cm; B) Anthocyanic callus. Bar ¼ 1.1 cm; C) pH-dependent changes in callus coloration: 1 – control, 2 –acid pH, 3 – alcaline pH. Bar ¼ 0.8 cm; D) Anthocyanic callus after transfer to MS0. Bar ¼ 0.8 cm; E) Anthocyanic callus after transfer to AC medium (MS1/2; 1:4 ratio of NH4

þ to NO3�;

70 g L�1 sucrose and supplementation with 0.9 mM 2,4-D). Bar ¼ 0.8 cm.

Fig. 2. Time course of anthocyanin production in callus cultures of C. rosea maintainedon MS medium supplemented with 30 g L�1 sucrose and 0.9 mM 2,4-D, during 25 daysin culture.

C. Simoes et al. / Plant Physiology and Biochemistry 47 (2009) 895–903 897

3. Results

3.1. Calogenesis and anthocyanin production

The calogenic process was induced in response to 2,4-D and PIC.Leaf explants formed calluses at the cut ends and over the midrib,while stem explants initially developed calluses at the cut enddirectly in contact with the culture medium. The presence of NAAonly caused direct rhizogenesis in both explant types, while IAA

Table 2Anthocyanin content in callus cultures of Cleome rosea submitted to differentconditions of temperature and light intensity, after 20 days in culture.

Physical condition Anthocyanin content(CV/g FW)

Temperature (�C) Light intensity (mmol m�2 s�1)

24 � 2 80 15.10 � 1.11a26 � 2 67 13.02 � 1.10ab26 � 2 45 10.04 � 1.85b32 � 2 67 5.88 � 2.15c32 � 2 45 6.22 � 1.20c

C. Simoes et al. / Plant Physiology and Biochemistry 47 (2009) 895–903898

induced shoot proliferation on stem explants, but did not cause anymorphogenic response in leaf tissues.

Calluses produced on media with 2,4-D or PIC were light beige,friable and fast growing. Calluses from stem explants formed onmedia supplemented with 0.90 mM 2,4-D presented the highestbiomass production (Table 1) and were maintained as stockcultures. Due to their rapid growth, subcultures were performedeach 20 days. After 6–7 months of culture, reddish-pink spotsappeared in the callus surface. The pigmented parts were isolatedmechanically and subcultured under the same culture conditions.This selective procedure allowed the establishment of non-pig-mented (Fig. 1A) and pigmented (Fig. 1B) callus lines. Both linespresented a similar growth rate, achieving more than 5-foldincrease on biomass accumulation after 20 days in culture (data notshown).

Pigmented callus exhibited a green-bluish coloration whensubmitted to an alkaline atmosphere and became red under an acidatmosphere (Fig. 1C). Moreover, the spectrophotometric analysis of

Fig. 3. Anthocyanin content and biomass accumulation (Fresh weight , Dry weight ,) in2,4-D, varying the following parameters: a) Sucrose concentration; b) Total nitrogen; c) NH

extracts obtained from these calluses showed maximum absorp-tion bands around 280 nm and 525 nm. Taking into account thatanthocyanins display two distinct absorption bands, one in theUV-region (260–280 nm) and another in the visible region(490–550 nm), these results provided further evidences of theanthocyanic nature of the pigments produced in the callus cultures.

To determine the best period of culture for anthocyanin quan-tification, callus samples were collected at 5-day intervalsthroughout 25 days. The anthocyanin content increased until aboutday 15 (CV ¼ 10.04 � 1.84/g FW), after which the levels remainedconstant until day 25 (Fig. 2). After this period, calluses becamebrown when maintained on the same medium without subculture,probably due to pigment degradation. Therefore, further quantifi-cations of anthocyanins were performed at day 20.

In order to evaluate the influence of environmental parametersin anthocyanin production, pigmented calluses were transferred todifferent conditions of light irradiation and temperature. Temper-atures of 32 � 2 �C were deleterious to pigment accumulation,

callus of C. rosea, after 40 days in culture on MS medium supplemented with 0.9 mM

4þ : NO3

� ratio; d) Reduction in salt concentration.

Table 3Anthocyanin content and biomass accumulation in calluses of C. rosea cultivated onmedium supplemented with 0.9 mM 2,4-D, after 40 days in culture. The AC mediumis composed of MS1/2 containing 1:4 ratio of NH4

þ to NO3� þ 70 g L�1 sucrose.

Culture medium Anthocyanincontent (CV/gFW)

Biomass accumulation (g)

Fresh weight Dry weight

MS þ 30 g L�1 sucrose 15.10 � 1.11c 24.34 � 0.93a 0.57 � 0.05cMS þ 70 g L�1 sucrose 32.26 � 2.81b 24.70 � 3.35a 1.40 � 0.18aMS (1:4 ratio of NH4

þ to NO3�)

þ 30 g L�1 sucrose25.46 � 5.68b 20.92 � 1.80ab 0.56 � 0.07c

MS1/2 þ 30 g L�1 sucrose 28.06 � 3.65b 20.55 � 0.94b 0.47 � 0.06cAC medium 38.67 � 2.74a 22.06 � 3.27ab 1.08 � 0.06b

Fig. 5. DAD UV–Vis spectrum of anthocyanin produced by callus cultures of C. rosea,showing maximum absorption bands around 280 nm and 525 nm (asterisks) anda third band around 315 nm (arrow), indicating the presence of acylated groups.Spectrum referred to peak 2 (22.10 min) as listed on Table 4.

C. Simoes et al. / Plant Physiology and Biochemistry 47 (2009) 895–903 899

causing callus browning irrespective of the light irradiation applied.On the other hand, increasing on light irradiation had a positiveeffect in anthocyanin production (Table 2). Based on these results,another assay was performed by transferring callus cultures to24 � 2 �C and 80 mmol m�2 s�1. Cultures maintained under theseconditions achieved the highest anthocyanin content(CV ¼ 15.10 � 1.11/g FW) and biomass accumulation (24.32 � 0.93FW/0.57 � 0.05 DW), showing that low temperatures associated tohigh light irradiation are the best physical conditions to pigmentproduction. These conditions were used in further assays.

Calluses cultivated on MS0 (Fig. 1D) showed a 2-fold reductionin biomass accumulation and a 4-fold reduction in the anthocyanin

Fig. 4. HPLC anthocyanin profiles (525 nm) of C. rosea. A) Calluses maintained on MSmedium supplemented with 30 g L�1 sucrose and 0.9 mM 2,4-D; B) Calluses transferredto modified MS medium (MS1/2 with a 1:4 ratio NH4

þ/NO3�) supplemented with

70 g L�1 sucrose and 0.9 mM 2,4-D; C) Stem from field-grown plants. Peaks numbersrefer to Tables 4 and 5.

content (4.22� 1.26 CV/g FW) when compared to those maintainedon medium supplemented with 2,4-D. When media containing2,4-D were supplemented with 70 g L�1 sucrose, anthocyanincontent was increased in 110%, in addition to high biomassproduction (Fig. 3A). However, the values of both parametersdecreased significantly in the presence of 90 g L�1 sucrose.

Changes in the total nitrogen concentration caused a lowanthocyanin production when compared with the nitrogen content(60 mM) traditionally used in MS medium (Fig. 3B), although thebiomass accumulation was not significantly altered. On the otherhand, a 1:4 ratio of NH4

þ to NO3� increased the anthocyanin content

in 68% when compared to MS medium ratio (1:2) and also did notinfluence biomass accumulation (Fig. 3C).

Pigmented calluses cultured on half-strength MS medium (MS1/2)showed an increase of 85% on pigment content. On the other hand,calluses transferred to MS1/4 as well as to White basal mediumpresented a reduction on biomass accumulation (Fig. 3D).

Based on the results of the above experiments, an optimizedmedium formulation, denominated anthocyanin medium (ACmedium), was established by combining MS1/2 with a 1:4 ratio ofNH4þ to NO3

�, 70 g L�1 sucrose and supplementation with 0.90 mM2,4-D. Pigmented calluses transferred to this formulation main-tained a high biomass accumulation and presented 150% increase inthe anthocyanin content (Table 3). This increase could be clearlyobserved by visual examination (Fig. 1E). Taking into account thatindividual calluses reached around 24 g after 20 days in culture, theanthocyanin production can be estimated in 850 CV/callus.

3.2. HPLC-DAD/ESIMS analysis of anthocyanins

The chromatograms of extracts obtained both from callusesmaintained on MS medium (Fig. 4A) and on AC medium presentedthe same profile (Fig. 4B), but with different anthocyanin concen-trations. Cultures inoculated on AC medium presented a 2-foldincrement in anthocyanin content, corroborating the color valuedata. Similarities on the chromatographic profiles were alsoobserved between extracts from calluses and those from stems offield-grown plant (Fig. 4C).

In addition to the two characteristic bands of anthocyanins(260–280 nm and 490–560 nm), a third absorption band rangingfrom 310 to 320 nm was observed indicating the presence ofacylated groups in the molecules (Fig. 5). The acylation wasconfirmed by mass spectrometric analysis with a prevalence ofr-coumaroyl, caffeoyl and feruloyl fragments.

The HPLC-DAD/ESIMS analysis of callus extracts revealed thepresence of eleven anthocyanins. Fragment ions at m/z 287 and m/z

C. Simoes et al. / Plant Physiology and Biochemistry 47 (2009) 895–903900

301 allowed the identification of the aglycones cyanidin and peo-nidin, respectively (Table 4). On the other hand, MS fragmentationpattern of the compounds found in stem extracts revealed only thepresence of cyanidins (Table 5). Some isobaric anthocyanins dis-played in Tables 4 and 5 (e.g. peak 2 and 5a in Table 4) wereidentified with different numbering. In these cases, the compoundswere identified by their retention times.

The major peak in the chromatogram obtained from calluses iscomposed by two cyanidins. One of them showed fragment ionsat m/z 919; 757; 449; 287 (Fig. 6A) and was identified as cyanidin

Table 5Characterization of anthocyanins from stems from field-grown plants of Cleome rosea us

Peak tR (min) [M]þ (m/z) Fragments ions (m

1 13.33 773 611/449/287A 19.06 979 817/449/2872 21.83 919 757/449/2873 22.21 935 773/449/287B 22.73 979 449/287C 24.30 1125 963/449/2875a 24.84 919 757/449/287D 24.84 1125 963/449/2876 25.48 1095 933/449/2877 25.78 1065 903/449/287E 29.57 1273 1111/449/287

Table 4Characterization of anthocyanins from callus of Cleome rosea using HPLC-DAD/ESIMS.

Peak tR (min) [M]þ (m/z) Fragments ions (m

1 13.55 773 611/449/2872 22.10 919 757/449/2873 22.44 935 773/449/2874 24.43 1081 919/449/2875a 24.94 919 757/449/2875b 24.94 949 787/449/2876 25.73 1095 933/449/2877 26.06 1065 903/449/2878 26.33 1095 933/449/2879 27.16 933 771/463/30110 28.62 1079 917/463/301

Fig. 6. Electrospray (þ) mass spectra of anthocyanins present in peaks 5a (A) and 5b (B) inFig. 4.

3-(p-coumaroyl) diglucoside-5-glucoside. The other one showedfragment ions at m/z 949; 787; 449; 287 (Fig. 6B) and was identifiedas cyanidin 3-(feruloyl) diglucoside-5-glucoside. The contributionof each on peak area was estimated as 83% and 17%, respectively.

Extracts obtained from stems of field-grown plants displayeda major peak composed by two cyanidins. The first one showedfragment ions at m/z 919; 757; 449; 287 (Fig. 7B) and was identifiedas cyanidin 3-(p-coumaroyl) diglucoside-5-glucoside, similarly tothe observed in callus extracts. The other showed fragment ions atm/z 1125; 963; 449; 287 (Fig. 7A) and was identified as cyanidin

ing HPLC-DAD/ESIMS.

/z) Proposed identity

cyanidin 3-diglucoside-5-glucosidecyanidin 3-(sinapoyl)diglucoside-5-glucosidecyanidin 3-(p-coumaroyl)diglucoside-5-glucosidecyanidin 3-(caffeoyl)diglucoside-5-glucosidecyanidin 3-(sinapoyl)diglucoside-5-glucosidecyanidin 3-(feruloyl)(feruloyl)diglucoside-5-glucosidecyanidin 3-(p-coumaroyl)diglucoside-5-glucosidecyanidin 3-(feruloyl)(feruloyl)diglucoside-5-glucosidecyanidin 3-(p-coumaroyl)(feruloyl)diglucoside-5-glucosidecyanidin 3-(p-coumaroyl)(p-coumaroyl)diglucoside-5-glucosidecyanidin 3-(caffeoyl)(feruloyl)diglucoside-5-glucoside

/z) Proposed identity

cyanidin 3-diglucoside-5-glucosidecyanidin 3-(p-coumaroyl)diglucoside-5-glucosidecyanidin 3-(caffeoyl)diglucoside-5-glucosidecyanidin 3-(caffeoyl)(p-coumaroyl)diglucoside-5-glucosidecyanidin 3-(p-coumaroyl)diglucoside-5-glucosidecyanidin 3-(feruloyl)diglucoside-5-glucosidecyanidin 3-(p-coumaroyl)(feruloyl)diglucoside-5-glucosidecyanidin 3-(p-coumaroyl)(p-coumaroyl)diglucoside-5-glucosidecyanidin 3-(feruloyl)(p-coumaroyl)diglucoside-5-glucosidepeonidin 3-(p-coumaroyl)diglucoside-5-glucosidepeonidin 3-(p-coumaroyl)(p-coumaroyl)diglucoside-5-glucoside

callus cultures of C. rosea. Peaks are listed on Table 4 and the HPLC profile is shown on

Fig. 7. Electrospray (þ) mass spectra of anthocyanins present in peaks D (A) and 5a (B) in stems of field-grown plant of C. rosea. Peaks are listed on Table 5 and the HPLC profile isshown on Fig. 4.

C. Simoes et al. / Plant Physiology and Biochemistry 47 (2009) 895–903 901

3-(feruloyl)(feruloyl) diglucoside-5-glucoside. The contribution ofeach one on peak area was estimated as 41% and 59%, respectively.

4. Discussion

The culture conditions established in the present work inducedthe formation of friable fast-growing calluses from stem explants ofC. rosea. Two distinct callus lines were established after repeatedsubcultures of non-pigmented and pigmented regions. Theseresults corroborate the idea that callus cultures are a mixture ofsubpopulations of cells differing in morphology, gene expression,morphogenetic capacity, and thus, in the ability to producedifferent metabolites [24].

Pigmented callus began to turn brown if maintained on thesame medium for more than 4 weeks without subculturing. Thiswas also observed in anthocyanin-producing calluses from Prunusincisa and can result from nutrient depletion or oxidative stress dueto toxic accumulation of spent materials in the culture medium orsecondary metabolites synthesized by stressed cells [25].

Although temperature is not frequently considered whiledeveloping protocols for callus cultures, this parameter was shownto be an important environmental factor regulating anthocyaninproduction in calluses of C. rosea. The highest production wasobtained at 24 � 2 �C, while higher temperatures resulted in callusbrowning. This effect can be a consequence of the hydrolysis ofglycosidic bonds by glucosidases, resulting in anthocyanin degra-dation and formation of brown condensation products [26]. Theinfluence of temperature on in vitro anthocyanin production seemsto be species-dependent once in callus cultures of Daucus carota thehighest anthocyanin production was achieved at 30 �C whencompared to lower temperatures [27].

Light has also been shown to be an important environmentalfactor influencing anthocyanin biosynthesis in plants [28]. In thepresent work, a positive correlation between anthocyaninproduction and light irradiation was observed in callus cultures.Light supplied to the cells may affect the activity of key enzymesinvolved in anthocyanin biosynthesis, such as phenylalanine

ammonia-lyase and chalcone synthase, whose expression isincreased in response to high irradiation [14]. The majority of cellculture systems described before required illumination for antho-cyanin accumulation [10,12,13], although its production in the darkhas also been reported [22,29].

In addition to physical factors, various parameters have beenstudied in order to increase the production of anthocyanins incallus and cell cultures, such as osmotic pressure, hormones andnutrient stress. The effects of plant growth regulators on in vitroanthocyanin induction are apparently variable. Although 2,4-D hasbeen shown to inhibit the production of a wide range of secondarymetabolites, including anthocyanins [27], in the present work thesupplementation with 2,4-D proved to be essential to supportbiomass increase as well as high anthocyanin production. Similarresults were reported for other species [22,30].

Sucrose concentration also proved to be an important factor formodulating anthocyanin accumulation in calluses of C. rosea.Supplementation of the culture medium with 70 g L�1 sucroseincreased anthocyanin production and supported a high biomassaccumulation. However, a decrease of callus biomass was observedon media containing 90 g L�1 sucrose, probably caused by inhibi-tion of nutrient uptake due to an increase in the osmotic potentialof the medium. Although sucrose is used as a nutrient in culturemedia, it also acts as an osmotic agent when present at highconcentrations. Furthermore, in plants, sugars represent not onlyenergy sources and structural components, but also physiologicalsignals regulating the expression of a variety of genes [31]. Vitracet al. [32] reported that sucrose signal transduction leading toanthocyanin accumulation involves the phosphorylation of hexosesby hexokinase. The effect of high levels of sucrose on both cellgrowth and anthocyanin production was reported for other in vitrocultures [30,33].

The nitrogen source is another important factor affectinganthocyanin production by plant cell cultures. In addition, theoverall levels of total nitrogen as well as the ratio of NH4þ to NO3�

have been shown to markedly affect the production of plantsecondary metabolites [34]. In callus cultures of C. rosea the total

C. Simoes et al. / Plant Physiology and Biochemistry 47 (2009) 895–903902

nitrogen content used on MS medium (60 mM) was shown to bethe most suitable for anthocyanin production. Nevertheless, themodification in the ratio of NH4

þ to NO3� of MS medium to 1:4

significantly increased pigment accumulation. These results are inaccordance with those described by Narayan and Venkataraman[33] in callus cultures of D. carota.

In general, the depletion of some nutrients leads to enhance-ment of secondary metabolites, but with growth limitations [27].This behavior was also observed in pigmented calluses of C. roseatransferred to MS medium with reduction on total salt concentra-tion. The increase in anthocyanin content, probably due to a nutri-tional stress condition, was associated to a decrease in biomassproductivity mainly when salts were reduced to 1/4. On the otherhand, transfer of anthocyanin-producing calluses to a mediumformulation containing 70 g L�1 sucrose and a 1:4 ratio of NH4

þ toNO3� resulted in 150% increase in pigment content associated to an

efficient callus growth. These results support the notion thatmodifications on the nutrient composition of the plant cell culturemedium are an efficient strategy to induce secondary metabolitesproduction under in vitro conditions [35].

HPLC coupled to diode array detector and electrospray ioniza-tion mass spectrometry is a powerful technique for the character-ization of anthocyanins from various sources, involving minimalsample preparation. The majority of the anthocyanins produced byC. rosea, in addition to presenting the maximum absorption bandsaround 280 nm and 525 nm, also displayed a third band in the 310–320 nm range, which characterizes the presence of acylating groupsin the molecule [36]. The acylated anthocyanins are more stable inneutral aqueous solutions and thus more suitable for application asfood and beverage colorants [37].

Despite the lack of information on anthocyanin characterizationin Capparaceae species, studies with species from the related familyCrucifereae (Brassicaceae) reported the occurrence of polyacylatedanthocyanins with prevalence of the same aromatic acids pre-sented on C. rosea [38,39]. The presence of peonidin in extractsfrom callus cultures of C. rosea, but not from field-grown plants,could be related to bioconversion of cyanidin to peonidin bymethylation of the B-ring of the aglycon. In strawberry cell cultures,reduction in 2,4-D concentration enhanced the methylation ofanthocyanins and increased the level of peonidin-3-glucoside [40].

In most in vitro culture systems, biomass accumulation andsecondary metabolite production require different media condi-tions to induce a shift from the growth state to the metaboliteproduction state, thereby limiting the efficiency of these systems tobe used commercially. Therefore, the success obtained with calluscultures of C. rosea, where anthocyanin production was associatedto high growth rates on the same medium, makes this protocola suitable and reliable system for in vitro anthocyanin production.

Acknowledgements

The authors are grateful to Gisele de Oliveira (UniversidadeFederal do Rio de Janeiro) and Maria Francisca Santoro Assunçao(Universidade do Estado do Rio de Janeiro) for the valuable tech-nical assistance. This work was supported by Fundaçao CarlosChagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro(FAPERJ) and Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq).

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