Changes of phenolic compounds in Carignan merithallus (Vitis vinifera L.) during bud dormancy and end of dormancy phase: correlation with rhizogenesis

doi:10.4236/as.2011.24064

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Vol.2, No.4, 498-504 (2011)

doi:10.4236/as.2011.24064

Agricultural Scienc es

Changes of phenolic compounds in Carignan

merithallus (Vitis vinifera L.) during bud dormancy and

end of dormancy phase: correlation with rhizogenesis

Kraiem Dardour Zohra1*, Zairi Asma1, Msaada Kamel2, Hamdi Helmi3, Ezzili Béchir1

1Biotechnology Center in Borj Cedria Technopole, Hammam-lif, T unisia; *Corresponding Author: [email protected]

2Laboratory of Bioactive Substances, Biotechnology Center in Borj Cedria Technopole, Hammam-lif, T unisia;

3Water Research and Technology Center, Borj Cédria Technopole, Soliman, Tunisia.

Received 29 August 2011; revised 17 September 2011; accepted 21 October 2011.

ABSTRACT

The aim of the present study is to address the

type of correlation that may exist between phe-

nolic compounds and vine rhizogenetic poten-

tial by analyzing some phenolic compounds in

the Carignan merithallus. Phenolic compounds

were analyzed by HPLC in the young shoots (or

merithallus) of Carignan vine (Vitis vinifera L.)

and we established a correlation between the

studied compounds and the rhizogenetic po-

tential of shoots during the phases of bud dor-

mancy and end of dormancy, taking into ac-

count the position effect on shoots. This work

was carried out for the first time on this type of

vine. Among the studied phenolic compounds,

we observed a negative correlation between

coumarin and rhizogenetic potential of Carignan

vine. In contrast, positive correlations were found

with naringin and syringic acid. Obtained re-

sults confirmed the fact that the action of phe-

nolic compounds is complex and might be

qualified as cofactors th at interact with auxin on

rhizogenesis.

Keywords: Phenolic Compounds; HPLC; Carignan

Shoots; Rhizogenesis

1. INTRODUCTION

Polyphenols, a group of substances with a broad spec-

trum of physiological activities, are spread in plants used

in traditional and modern medical systems. Phenolics

have been shown to have a role in tissue browning, fla-

vor, and color characteristics of the derived products [1].

An understanding of phenolic composition in fresh fruit

and the factors that affect phenolic compounds are criti-

cal in the design of products and storage conditions [2].

Many studies have been undertaken on the phenolic acid

compositions of various fruits and their related cultivars

such as apples and pears [3,4], Pyrus [5,6], pome and stone

fruit [7], Diospyros [8], carrots [9], and Prunus [10].

Phenolic compounds are active biological molecules

having one or more benzene rings with one or more hy-

droxyl functions [11]. At the plant level, phenolic com-

pounds contribute to development, cell multiplication,

reproduction, differen tiation, flowering, and lignification.

Their content in plants depends on many genetic, phy-

siological and environmental parameters [12].

Phenolic compounds accumulate mainly in the cell

membrane (lignin and some flavonoids) and in vacuoles

where soluble phenols such as chlorogenic acid, antho-

cyanins, flavonols and tannins are stocked [13]. These

compounds can participate also in the phenomenon of

rhizogenesis. Some authors reported that such contribu-

tion might be positive [14-17]. However, other studies

showed no or negative effects of these compounds on

rhizogen esi s [1 8 - 2 1].

The purpose of this work was to establish the correla-

tion type that may exist between phenolic compounds

and vine rhizogenetic potential by analyzing some phe-

nolic compounds in the Carignan merithallus.

2. EXPERIMENT

2.1. Plant Material

The influence of the sampling date on the rhizogenetic

potentiality of vine merithallus has been previously

studied [22], Carignan vines grown in double Guyot at

the experimental station of the Obligatory Grouping of

Wine Growers and Fruit Producers (GOVPF) in Baddar

(Northeast Tunisia). In this study, vine shoots of various

Carignan genotypes were sampled monthly on five spe-

cific dates that coincide with the phenomenon of bud

dormancy and end of dormancy [23]. The first sampling

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took place on September 13, 2005, the second on Octo-

ber 13, 2005, the th ird on November 13 , 2005, th e fourth

on December 13, 2005 and the last on January 13, 2006.

Thirty merithallus of Carignan vine were sampled

from various rows and positions according the conven-

tion of friml [24]. Samples were dried and reduced to a

fine powder prior to phenolic compound analysis.

2.2. Polyphenols Analysis

2.2.1. Determination of Total Phenolic

Compounds

Analysis of total polyphenols in Carignan merithallus

samples was carried out using Folin-Ciocalteu reagent

[25]. In alkaline medium, this reagent is reduced to

tungsten and molybdenum oxides, giving a blue color in

the presence of polyphenols. Briefly, 125 µL of diluted

extract (methanol 80%) was mixed with distilled water

(500 µL) and 125 µL of Folin-Ciocalteu reagent. Then,

the mixture was agitated, paused for 3 min before the

addition of 1250 µL of CO3(Na)2 at 7%. Finally, the

mixture’s volume was adjusted by distilled water to 3

mL. After 90 min in darkness, absorbance was deter-

mined at a wavelength of 760 nm. The standard curve

was prepared using gallic acid at 50, 100, 200, 3 00, 400,

and 500 mg/L. Total polyphenol content was expressed

as mg of gallic acid equivalent p er g dw.

2.2.2. Solvent Extraction

Solvent extraction was performed according to the

method described by [26]. Merithallus sample (2.5 g dw)

was mixed with 15 mL water, agitated for 30 min and

kept for 24 h at 4˚C in total darkness. The mixture was

then filtered on ash-free filter paper (Whatman n 4) be-

fore storage at 4˚C for subsequent analyses.

2.2.3. HPLC Analysis

Phenolic compounds were identified and quantified

using RP-HPLC coupled to a UV-visible detector and

equipped with a specific C18 column: Hypersil ODS (250

× 4.6 mm, 4 µm). The mobile phase consisted of ace-

tonitrile (solvent A); and a mixture of HPLC-grade water

and sulphuric acid at 0.2% (solvent B). The gradient

program was set up for the mixture (A)-(B) as follows:

15% - 85% for 0 - 12 min, 40% - 60% for 12 - 14 min,

60% - 40% for 14 - 18 min, 80% - 20% for 18 - 20 min,

90% - 10% for 20 - 24 min, and 100% - 0% for the last

24 - 28 min. Injected volume was 20 µL and peaks were

examined at 280 nm. Peaks were characterized by their

retention times and corresponding compounds were id-

entified as compared to retention times of pure stan-

dards. All chemical analyses were conducted in tripli-

cate.

2.3. Statistical Analysis

All determinations were performed in triplicate and

the results are expressed as means values ± standard

deviations (SD). The data were subjected to statistical

analysis using statistical program package STATISTICA

[27]. The one-way analysis of variance (ANOVA) fol-

lowed by Duncan multiple range test were employed and

the differences between individual means were deemed

to be significant at P < 0.05. Correlation coefficients were

calculated based on coumarin, naringin and syringic acid

percentages at different rhizogenetic potential levels.

3. RESULTS

3.1. Total Polyphenols

The total mean content of polyphenols in merithallus

as function of sampling date is presented in Table 1. The

total polyphenol content is expressed as mg of gallic

acid equivalent per g of dry weight (mg GAE·g–1 dw).

Total content of phenolic compounds in Carignan meri-

thallus was the high est in November, but sho wed almost

a constant variation for the rest of sampling period (Ta-

ble 1). By considering the merithallus position on Carig-

nan shoots (apical, central and basal), results showed a

great variation in total polyphenol content (Table 2).

This variation was accentuated in November and char-

acterized by a greatest concentration of polyphenols in

the apical position , which was three-fold high er than that

observed in central and in basal positions.

Table 1. Va riation of total phenolic compounds (T) in Carignan merithallu s as function of sampling date (mg GAE·g–1 dw).

September October November December January

T (mg EAG·g–1 dw) 3.77 ± 0.76b 3.30 ± 0.64d 7.63 ± 0.17a 1.93 ± 0.94e 3.55 ± 0.26c

Values are the means of triplicates ±SD. Values in the same row with different superscript, a-eare significantly different at P < 0.05.

Table 2. Variation of total phenolic content in Carignan merithallus (mg GAE·g–1 dw) as function of their position on vine shoots.

Month of the year

Position September October November December January

Apical 1.76 ± 0.49d 4.18 ± 0.60c 14.73 ± 1.46a 1.21 ± 0.42e 5.22 ± 0.04b

Central 6.06 ± 0.36a 3. 06 ± 0 .01d 5.64 ± 0.84b 1.32 ± 0.68e 3.27 ± 0. 84c

Basal 3.5 ± 0.01a 2.65 ± 0.01c 2.52 ± 0.53cd 3.27 ± 0.52b 2.17 ± 0.26d

Values are the means of triplicates ±SD. Values in the same row with different superscript, a-eare significantly different at P < 0.05.

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3.2. Variation of Individual Phenolic

Compounds

Figure 1 indicates on chromatographic profile (reten-

tion time) of phenolic compounds found in Carignan

merithallus. Nine phenolic compounds (out of 12) were

identified according to their respective retention time as

follows: 1) gallic acid, 2) caffeic acid, 3) dihydroxy-

phenylacetic acid, 4) chlorogenic acid, 5) syringic acid,

6) ferulic acid, 7) naringin, 8) quercetin, 9) coumarin.

Percent distribution of analyzed phenolic compounds

within shoots of Carignan merithallus as function of

sampling date is presented in Table 3. We noted that the

content of some phenolic compounds (ferulic acid and

coumarin) was proportional to the v ariation of total con-

tent in merithallus. On the other hand, quercetin and

dihydroxyphenylacetic acid contents increased with time

in contrast to chlorogenic acid. Gallic acid percentage in

the central part of shoots increased steadily to reach

highest values in December before decreasing. For the

same position, caffeic acid was highly expressed in No-

vember while the concentrations of naringin and syringic

acid showed a sawtooth-shaped variation (Ta bl e 3). As

the variation of non-id entified compounds present in the

chromatogram was not stable over time, only phenolic

compounds that have significant correlations with the

rhizogenetic potential were co nsidered for fu rther invest-

tigation.

3.3. Correlation between Phenolic

Compounds and Rhizogenetic

Potential

When addressing the variation of % coumarin and

carignan rhizogenetic potential with respect to sampling

date [28], we noticed that there was a strictly negative

correlation for merithallus in basal position (Figure 2).

Our investigation showed a moderately positive correla-

tion between syringic acid and the rhizogenetic potential

(Figure 3). There is also a positive correlation between

naringin and rhizogenetic potential but not significant

(Figure 4).

Figure 1. Chromatographic profile of phenolic compounds found in Carignan shoots. 1. Gallic acid; 2. Caffeic acid; 3. Dihydroxy-

phenylacetic acid; 4. Chlorogenic acid; 5. Syringic acid; 6. Feulic acid; 7. Naringin; 8. Coumarin; NI. Non identified.

(a) (b)

Figure 2. Evolution of the coumarin in the merithallus (a) and correlation between these percentages in basal position and the

rhizogenetic potential (b).

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(a) (b)

Figure 3. Evolution of syringic acid in the merithallus (a) and correlation between these percentages and the rhizogenetic potential (b).

(a) (b)

Figure 4. Evolution of the naringin of the merithallus (a) and correlation between these percentages and the potential rhizogenetic

according to the date of taking away (b).

4. DISCUSSION

Interactions between phenolic compounds, organo-

genesis and plant growth have been studied frequently as

mentioned in the introduction section. The regulation of

the endogenous rate of auxin as part of the enzymatic

activity of auxin-oxydase, and auxin transport are two

important events.

The polarized transport of auxin has been studied by

several authors [29-32]. However, this transport can be

disturbed or controlled by some phenolic compounds. In

terms of regulation of polarized transport, the role of

some specific phenolic compounds (phytotropins) is the

most evident as they compete with efflux transporters of

auxin according [29].

The variation of percent coumarin in Carignan and the

rhizogenetic potential as function of sampling dates [22 ]

showed a significant negative correlation for the basal

position of the merithallus on vine shoots. Syringic acid

and naringin can also co rrelate positively with the rhizo-

genetic potential. We noticed also that the quercetin

content increased during the phase of dormancy of buds

and reached highest levels by January (Ta bl e 3). Darne

and Atalay [33] reported that there was a direct correla-

tion between the rhizogenetic capacity and the synthesis

of phenolic compounds in vine shoots.

By studying the adventive rhizogenesis in Pinus con-

torta, Brinker et al. [32] noticed that the active transport

of auxin is reduced during the first phase of the rhizo-

genesis. They also observed a reduction in the transcript-

tion of the gene which codes for the kinase-like protein,

involved in the control of auxin transport. Intermediate

products resulting from the biosynthesis of phenolic

compounds may accumulate and create a barrier that

inhibits auxin transport. Brinker et al. [32] indicated that

treatments with auxin activate the cellular division. This

hormone has a major role in the initiation and the de-

velopment of adventive roots. The mechanism utilizes

phenolic compounds as a barrier that prevents auxin

transport. This natural barrier consists of a protein of

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alue

Table 3. Percent distribution (%, w/w) of analyzed phenolic compounds within shoots of Carignan merithallus as function of sampling date.

Vs are the means of triplicates ±SD. Values in the same row with different superscript, a-kare significantly different at P < 0.05. Co mp ou nd s : 1: Gallic acid, 2: cafeic ac i d, 3: NI, 4: dihy droxypheny l ac et i c acid, 5:

I, 6: chlorogenic acid, 7: NI, 8: syringic acid, 9: ferrulic acid, 10: Naringin, 11: Coumarin.

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having a flavonolic origin [30,32] noticed that the tran-

scription of the gene coding for this enzyme is affected

during the pha se of radial primordial formation.

5. CONCLUSIONS

The evolution of phenolic compounds was studied on

Carignan merithallus during the phases of bud dormancy

and end of dormancy. Our outcomes indicate that rhizo-

genesis coincides with quercetin synthesis. This phenolic

compound belongs to the class of phytotropins according

to Brunn et al. [29]. We observed also a negative corre-

lation between coumarin and rhizogenetic potential of

Carignan vine. In contrast, positive correlations were

found with naringin and syringic acid.

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